Progress in Retinal and Eye Research 26 (2007) 263–301 Ion channels in the RPE So¨nke Wimmers, Mike O. Karl, Olaf Strauss Experimentelle Ophthalmologie, Klinik und Poliklinik fu ¨ r Augenheilkunde, Universita ¨ tsklinikum Hamburg-Eppendorf, MartinistraX e 52, 20246, Hamburg, Germany Abstract In close interaction with photoreceptors, the retinal pigment epithelium (RPE) plays an essential role for visual function. The analysis of RPE functions, specifically ion channel functions, provides a basis to understand many degenerative diseases of the retina. The invention of the patch-clamp technique significantly improved the knowledge of ion channel structure and function, which enabled a new understanding of cell physiology and patho-physiology of many diseases. In this review, ion channels identified in the RPE will be described in terms of their specific functional role in RPE physiology. The RPE expresses voltage- and ligand-gated K + , Cl , and Ca 2+ -conducting channels. K + and Cl channels are involved in transepithelial ion transport and volume regulation. Voltage-dependent Ca 2+ channels act as regulators of secretory activity, and ligand-gated cation channels contribute to RPE function by providing driving forces for ion transport or by influencing intracellular Ca 2+ homoeostasis. Collectively, activity of these ion channels determines the physiology of the RPE and its interaction with photoreceptors. Furthermore, changes in ion channel function, such as mutations in ion channel genes or a changed regulation of ion channel activity, have been shown to lead to degenerative diseases of the retina. Increasing knowledge about the properties of RPE ion channels has not only provided a new understanding of RPE function but has also provided greater understanding of RPE function in health and disease. r 2007 Elsevier Ltd. All rights reserved. Contents 1. Introduction ............................................................................... 264 2. The function of the RPE ...................................................................... 264 3. K + ..................................................................................... 266 3.1. K + channels and cell function .............................................................. 266 3.2. K + ions in RPE function ................................................................. 266 3.3. K + channels of the RPE .................................................................. 267 3.3.1. Inward rectifier K + channels ......................................................... 267 3.3.2. Voltage-gated K + channels .......................................................... 269 3.3.3. Ca 2+ -activated K + channels ......................................................... 270 3.3.4. Two-pore K + channels ............................................................. 271 3.4. Summary of K + channel function ........................................................... 272 4. Chloride .................................................................................. 273 4.1. The role of Cl and Cl channels in cell function ................................................ 273 4.2. Cl ions in RPE function ................................................................. 273 4.2.1. Fluid transport ................................................................... 273 4.2.2. Volume control .................................................................. 274 4.2.3. pH regulation .................................................................... 274 4.2.4. Intracellular organelles ............................................................. 275 ARTICLE IN PRESS www.elsevier.com/locate/prer 1350-9462/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.preteyeres.2006.12.002 Corresponding author. Tel.: +49 40 42803 9469; fax: +49 40 42803 5017. E-mail address: [email protected] (O. Strauss).
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ARTICLE IN PRESS
1350-9462/$ - se
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Progress in Retinal and Eye Research 26 (2007) 263–301
www.elsevier.com/locate/prer
Ion channels in the RPE
Sonke Wimmers, Mike O. Karl, Olaf Strauss�
Experimentelle Ophthalmologie, Klinik und Poliklinik fur Augenheilkunde, Universitatsklinikum Hamburg-Eppendorf,
MartinistraX e 52, 20246, Hamburg, Germany
Abstract
In close interaction with photoreceptors, the retinal pigment epithelium (RPE) plays an essential role for visual function. The analysis
of RPE functions, specifically ion channel functions, provides a basis to understand many degenerative diseases of the retina. The
invention of the patch-clamp technique significantly improved the knowledge of ion channel structure and function, which enabled a
new understanding of cell physiology and patho-physiology of many diseases. In this review, ion channels identified in the RPE will
be described in terms of their specific functional role in RPE physiology. The RPE expresses voltage- and ligand-gated K+, Cl�, and
Ca2+-conducting channels. K+ and Cl� channels are involved in transepithelial ion transport and volume regulation. Voltage-dependent
Ca2+ channels act as regulators of secretory activity, and ligand-gated cation channels contribute to RPE function by providing driving
forces for ion transport or by influencing intracellular Ca2+ homoeostasis. Collectively, activity of these ion channels determines the
physiology of the RPE and its interaction with photoreceptors. Furthermore, changes in ion channel function, such as mutations in ion
channel genes or a changed regulation of ion channel activity, have been shown to lead to degenerative diseases of the retina. Increasing
knowledge about the properties of RPE ion channels has not only provided a new understanding of RPE function but has also provided
greater understanding of RPE function in health and disease.
With the invention of the patch-clamp technique,understanding of the structure and function of ion channelssubstantially improved and changed (Ackerman andClapham, 1997; Colquhoun, 1991; Hamill et al., 1981;Jurkat-Rott and Lehmann-Horn, 2004; Neher andSakmann, 1976, 1992; Sakmann and Neher, 1984; Sig-worth, 1986). General ion channel characteristics weredescribed in detail and the understanding of their cell-specific behaviour in different tissues improved. Thisknowledge shed new light on many different cell functionsand also opened new doors toward an understanding ofpatho-physiologic changes in cell behaviour. The latterstudies coined the term ‘‘channelopathies’’ for ion channel-related diseases (Ackerman and Clapham, 1997; Lehmann-Horn and Jurkat-Rott, 1999).
The retinal pigment epithelium (RPE) fulfills manytasks, which are essential for visual function (Bok, 1993;Steinberg, 1985; Strauss, 2005). The characterizationof ion channels in the RPE is, therefore, a necessity tounderstand these important functions in retinal healthand disease. The first patch-clamp study using cells ofRPE was published in 1988 by Fox et al. (1988). Sincethen, the investigation of RPE ion channels has beenundertaken by several different research groups whohave now described a large number of different ionchannels in the RPE. In this review we have endeavouredto summarize existing knowledge about ion channelsof the RPE in relation to RPE function and its patho-physiology.
2. The function of the RPE
The RPE is a monolayer of pigmented cells covering theinner wall of the eye bulb (Bok, 1993). The apicalmembrane of the RPE faces the light-sensitive outer-segments of photoreceptors and the basolateral membraneof the RPE is enface with the fenestrated capillaries of thechoroid (Bok, 1993; Boulton and Dayhaw-Barker, 2001;Marmorstein, 2001; Marmorstein et al., 1998). Theinteraction with the adjacent tissues relies on extracellularmatrices on both sides of the RPE. For a close interactionwith photoreceptors, the RPE has long apical microvillithat surround the outer-segments of photoreceptors(Boulton, 1991). The space in-between the RPE andphotoreceptors is filled with the interphotoreceptor matrix(IPM). The IPM is essential for the interaction betweenRPE and photoreceptors (Acharya et al., 1998; Gonzales-Fernandez, 2003; Hageman and Johnson, 1991; Hollyfield,1999; Pepperberg et al., 1993; Uehara et al., 1990). In thedifferentiated eye, the IPM facilitates the interactionbetween RPE and photoreceptors enabling the exchangeof nutrients, signalling molecules, and metabolic endproducts (Acharya et al., 1998; Gonzales-Fernandez,2003; Hageman and Johnson, 1991; Hollyfield, 1999;Pepperberg et al., 1993; Uehara et al., 1990). At thebasolateral side, the RPE is separated from the chorioca-pillaris by Bruch’s membrane, a multilayered extracellularmatrix structure (Garron, 1963; Guymer et al., 1999;Lerche, 1963; Marshall et al., 1998; Sumita, 1961). Bruch’smembrane represents an interface for exchange of nutrientsand signalling molecules, in this case between RPE and the
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Fig. 1. Summary of RPE function to support vision. The figure is from Strauss (2005). MV ¼ apical microvilli; OS ¼ photoreceptor outer segments;
S. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 265
blood stream and between the RPE and the choroidalendothelial cells (Fig. 1).
Electrophysiological studies have characterized the RPEas a tight epithelium with a paracellular resistance 10 timeshigher than the transcellular resistance (Miller and Stein-berg, 1977a, b). This is achieved by a specific subset oftight-junction proteins and defines the RPE as a part of theblood/retina barrier (Ban and Rizzolo, 2000a, b; Knieseland Wolburg, 1993; Kojima et al., 2002; Konari et al.,1995; Nishiyama et al., 2002; Williams and Rizzolo, 1997).With its location and characteristics, the RPE is able tofulfil a multitude of functions that are essential for visualfunction (Bok, 1993; Steinberg, 1985; Strauss, 2005).
With its pigmentation, the RPE absorbs light energy,which is focussed onto the macula by the lens (Boulton,1991, 1998; Boulton and Dayhaw-Barker, 2001). As part ofthe blood/retina barrier, it transports nutrients such asglucose or vitamin A from the blood to the photoreceptors(Baehr et al., 2003; Ban and Rizzolo, 2000b; Marmorstein,2001; Sugasawa et al., 1994). On the other hand, the RPEeliminates water from the subretinal space. Water accu-mulates in the subretinal space from metabolic activity ofphotoreceptors and from the vitreous humour driven byintraocular pressure (Hamann, 2002; Marmor, 1990;Moseley et al., 1984). The transport of water is mainlydriven by a transepithelial transport of Cl� from thesubretinal space to the blood (Bialek et al., 1996; Blauget al., 2003; Edelman et al., 1994a, b; Edelman and Miller,1991; Hamann, 2002; Hughes et al., 1984; Maminishkiset al., 2002; Miller et al., 1982; Peterson et al., 1997; Rymeret al., 2001; Stamer et al., 2003; Steinberg, 1985).Furthermore, the RPE is essential for establishing andmaintaining the immune privilege of the eye. This is notonly established by its barrier function, but also byinterfering with the signalling pathways which coordinatethe immune system and has immune suppressive functionin the healthy eye (Ishida et al., 2003; Streilein et al., 2002;
Wenkel and Streilein, 2000). The RPE not only controls theamount of fluid in the subretinal space, it helps to establisha constant ion composition in the subretinal space bycompensating for light-induced changes (Dornonville de laCour, 1993; Gallemore et al., 1997; Hughes et al., 1998;Steinberg, 1985; Steinberg et al., 1983; Steinberg andMiller, 1973). For example, illumination of photoreceptorsresults in a decrease of the K+ concentration in thesubretinal space from 5 to 2mM (Dornonville de la Cour,1993; Gallemore et al., 1997; Miller and Steinberg, 1977b;Steinberg et al., 1983). To maintain constant excitabilityof photoreceptors this change in the K+ concentrationis compensated for by the RPE, which releases K+ into thesubretinal space (Dornonville de la Cour, 1993; Gallemoreet al., 1997; Miller and Steinberg, 1977b; Steinberg et al.,1983).The transport and metabolism of vitamin A is coupled to
another function essential for visual function, a metabolicpathway named the visual cycle of retinal (Baehr et al.,2003). The process of vision starts with absorption of lightenergy by 11-cis retinal in the pigment of rhodopsin (Baehret al., 2003; Baylor, 1996; Bok, 1993). The light absorptionleads to the isomeric change of retinal from the 11-cis to theall-trans isomer. Photoreceptors are unable to re-isomeriseall-trans retinal into 11-cis retinal. For re-isomerisation,retinal is transported to the RPE where it is re-isomerisedinto the 11-cis isomer (Arshavsky, 2002; Mata et al., 2002;Thompson and Gal, 2003). After re-delivery to thephotoreceptors it serves to regenerate rhodopsin.Light exposure of photoreceptors is accompanied by
photo-oxidative damage of proteins and phospholipids ofthe outer segments (Beatty et al., 2000). To maintainthe excitability of photoreceptors, the outer segments haveto be regenerated (Bok, 1993; Bok and Hall, 1971;Finnemann, 2003; LaVail, 1976; Steinberg, 1985). In thisregeneration process, new photoreceptor outer-segmentsare constantly assembled from the connecting cilium. The
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tips of the outer segments are constantly shed from thephotoreceptors and phagocytized by the RPE. Properfunction of photoreceptors depends on the maintenance ofthe right size of outer segments. This is achieved by thecoordinated shedding and phagocytosis of the tips andrequires a tight regulation in communication between RPEand photoreceptors. The regulation takes place in acircadian manner with a peak after onset of light (LaVail,1976). In humans, the photoreceptor outer segments arecompletely renewed every 11 days.
Transport, phagocytosis, and immune suppression re-quire communication with adjacent tissues. For thispurpose, the RPE expresses a large variety of receptorsand is also able to secrete a variety of factors (Campo-chiaro, 1993; Strauss, 2005). The secretory function of theRPE tissue enables not only a functional coordination withphotoreceptors and endothelium, but also immune sup-pression and stabilization of the functional integrity ofphotoreceptors and endothelial cells. For the latterpurpose, the RPE secretes growth factors, such as thepigment epithelium derived factor (PEDF) to the apical orthe photoreceptor side (Becerra et al., 2004; King andSuzuma, 2000; Steele et al., 1992) and the vascularendothelial growth factor (VEGF) to the basolateral orendothelial side (Blaauwgeers et al., 1999; Slomiany andRosenzweig, 2004a; Witmer et al., 2003).
These collective functions of the RPE are tightly linkedto alterations in ion channel activity and underscore theimportance of the study of ion channels in the RPE inorder to shed new light on the cellular mechanisms bywhich the RPE supports visual function.
3. K+
3.1. K+ channels and cell function
The largest group of ion channels in the human genomerepresents the family of K+ channels consisting of at least78 pore-forming a subunits (Yu et al., 2005). These includethe 15 members of the inwardly rectifying K+ channelsubfamily with two transmembrane domains, 40 mainlyoutwardly rectifying voltage-gated K+ channels with sixtransmembrane domains, 9 Ca2+-activated K+ channelswith six or seven transmembrane domains, and 15 two-pore or leak K+ channels with four transmembranedomains and two pores.
The term rectification refers to the characteristicconductance changes of ion channels upon membranepotential changes. The inwardly rectifying ion channelshave larger ion conductivity in response to hyperpolarizingmembrane potential changes than in response to depolar-ization. Nevertheless, the current mediated by inwardlyrectifying K+ channels is predominantly a K+ efflux sincethe direction of K+ flux is controlled by the reversalpotential or equilibrium potential for this ion, which istypically around �80mV. Accordingly, at potentialspositive to �80mV, K+ ions flow out of the cell and at
potentials negative to �80mV, K+ flows into the cell. Theoutwardly rectifying channels have a larger ion conduc-tance in response to depolarization of the membranepotential. The subsequent outflow of K+ ions repolarizesthe membrane potential back towards the K+ equilibriumpotential. All K+ channels are highly selective for K+ ions.Why this diversity in K+ channels? K+ as a charge
carrier and an osmotically active molecule is involved inmany cell functions. One task of K+ channels in excitablecells is to control the resting membrane potential of thecells shifting it towards the reversal potential of K+.Additionally, K+ channels are involved in the shaping/duration of action potentials and in adaptive mechanismsby repetitive excitation of neurons (Hille, 2001).In non-excitable cells, K+ channels are responsible for
the maintenance of the hyperpolarized resting membranepotential. In addition to this, these channels play essentialroles in (1) transepithelial transport processes, (2) thecontrol of cell volume and intracellular pH, (3) therecycling and secretion of K+, (4) the cell cycle andapoptosis, and (5) in the regulation of hormone and growthfactor secretion (Ashcroft and Gribble, 1999; Bauer et al.,1999; MacDonald and Wheeler, 2003; Masi et al., 2005;Warth, 2003).
3.2. K+ ions in RPE function
As stated above, (Section 2) one function of the RPE isto control the ion composition in the subretinal space. TheK+ concentration of the subretinal space in the absence oflight is approximately 5mM. Under these conditions K+
enters the RPE cells through the apically located Na+/K+
ATPase and leaves the cells via the basolateral membrane,leading to a net absorption of K+ (la Cour et al., 1986;Miller and Steinberg, 1982). When exposed to light,photoreceptors hyperpolarize due to the closure of cyclicnucleotide-gated Na+-conducting cation channels, whichare located in the membrane of the light sensitive outersegments (Liebman et al., 1987). In the darkness, the K+
ions entering the photoreceptors and the RPE cells throughtheir Na+/K+-ATPases and the K+ ions leaving thephotoreceptors as counterion to Na+ are in equilibrium(Baylor, 1996; Strauss, 2005). Due to the hyperpolarizationinduced by light exposure, less K+ ions leave thephotoreceptors. Consequently, the K+ concentration inthe subretinal space decreases in the light from 5 to 2mM(Oakley and Steinberg, 1982; Steinberg et al., 1980).Because of the large K+ conductivity of the apicalmembrane, the decrease of the extracellular K+ concentra-tion leads to a hyperpolarization of the apical membrane ofthe RPE (Griff and Steinberg, 1984; Miller and Steinberg,1977b; Steinberg et al., 1970). Additionally, the lowextracellular K+ concentration inhibits the Na+/K+/2Cl� co-transporter in the apical membrane of the RPEcells (Bialek et al., 1995; Joseph and Miller, 1991) resultingin a decrease of the intracellular Cl� concentration (Josephand Miller, 1991), and as a consequence to an extrusion of
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cytosolic water and shrinkage of the RPE cells andacidification of the cytosol (Adorante, 1995; Adoranteand Miller, 1990; Bialek and Miller, 1994; Huang andKarwoski, 1992; Kennedy, 1994; Li et al., 1994a; Lin andMiller, 1991). The extrusion of water together with cellshrinkage leads to an increase of the subretinal space andto a further decrease of the subretinal K+ concentration.The large apical K+ conductance helps to restore thenormal subretinal K+ concentration.
Another function of K+ channels is to counteract Ca2+
influx. As Ca2+ influx leads to an increase of intracellularfree Ca2+ and to depolarization of the cell, and Ca2+ ionsare involved in many RPE functions (see Section 5) theremust be a counterbalance for Ca2+ flux in RPE cells. K+
channels are good candidates because they are activated bydepolarization (voltage-gated K+ channels) and by Ca2+
itself (Ca2+-activated K+ channels).Additionally, they may support functions as described in
other epithelial cell types such as wound healing, regulationof nutrient transport, and cell proliferation/differentiation(Warth, 2003).
3.3. K+ channels of the RPE
In Fig. 2, patch-clamp data from two representative RPEcells are shown. Both cells have an inwardly rectifying currentbut with very different kinetics. Cell 2 has an additionaloutwardly rectifying current showing the heterogeneity ofK+ conductances in these cells. In the next chapters, thedifferent K+ conductances identified in RPE cells will beintroduced in relation to their particular K+ channel classesand discussed in their possible roles for RPE function.
3.3.1. Inward rectifier K+ channels
3.3.1.1. Inward rectifier K+ (Kir) channels in general. Kirchannels are activated upon hyperpolarization. Though
100 pA
10 ms
100 pA
10 ms
30 mV
-60 mV
-150 mV
Cell 1 Cell 2
RPE
K+
K+
Fig. 2. Patch-clamp measurements of two mouse RPE cells exemplifying
the heterogeneity of membrane K+ currents in these cells. The cells were
held at �60mV and stimulated with voltage-steps of 10mV, first 10
depolarizing steps then 10 hyperpolarizing steps.
their name suggests that they maintain K+ inward current,their main function in most cells is to bring the membranepotential towards the K+ equilibrium potential. As in mostcells, in RPE cells the equilibrium potential is negative tothe resting membrane potential; thus Kir channels mainlyconduct outward K+ currents. Kir channels are composedof four pore-forming subunits with two transmembranedomains, a pore loop and cytoplasmic N- and C-termini(Bichet et al., 2003). Their inward rectification is caused bya block of the channel pore by internal Mg2+ and bycytoplasmic polyamines such as spermine or spermidine atpositive voltages (Lu, 2004; Nichols and Lopatin, 1997).Based on their biophysical properties and their sensitivityto intracellular signals the Kir channels are subdivided intoseven subfamilies (Kubo et al., 2005). The Kir1 subfamilyconsists of only one member with weak inwardly rectifyingproperties (Ho et al., 1993). The four Kir2 subfamilymembers are strong inward rectifiers (Kubo et al., 1993a;Morishige et al., 1994; Takahashi et al., 1994; Topert et al.,1998). The members of the Kir3 or GIRK subfamily areG-protein-coupled inward rectifiers being activated by theGbg subunits of G proteins (Dissmann et al., 1996; Kofujiet al., 1995; Kubo et al., 1993b; Spauschus et al., 1996).Members of the Kir4 and 5 subfamilies are able to formheteromultimers with each other (Pearson et al., 1999;Pessia et al., 1996). While Kir4 members are able to formfunctional channels alone (Pessia et al., 1996), the Kir5subunit must be co-expressed with Kir4 subunits (Bond etal., 1994). Both homomeric Kir4 channels and heteromericKir4/5 channels are weakly inwardly rectifying (Pessiaet al., 1996). The Kir6 subfamily members co-assemblewith sulfonylurea receptor SUR1 and 2 to form K+
channels that are sensitive to the intracellular ATP/ADPratio (Aguilar-Bryan et al., 1995; Inagaki et al., 1995a, b,1996; Sakura et al., 1995). Finally, the Kir7.1 subunit formsa weak inward rectifier that has higher conductance withdecreasing extracellular K+ concentrations (Doring et al.,1998; Krapivinsky et al., 1998). Though, most Kir channelsare blocked by extracellular Ba2+, the subfamilies can befurther characterized by their different sensitivity to Ba2+.Kir7.1 has, for example, a very low Ba2+ sensitivity withan IC50 of 1mM (Doring et al., 1998; Krapivinsky et al.,1998) while Kir4.1 is much more sensitive with an IC50 inthe micromolar range (Bond et al., 1994).
3.3.1.2. Inward rectifier K+ (Kir) channels in the RPE. Dif-ferent subtypes of inwardly rectifying K+ channel subunitshave been identified in the RPE (Ettaiche et al., 2001;Ishii et al., 1997; Kusaka et al., 1999, 2001; Shimura et al.,2001; Yang et al., 2003). In the libraries of the NationalEye Institute only KCNJ13 (or Kir7.1) is listed in boththe human and the mouse library of expressed sequencetags (EST; http://neibank.nei.nih.gov/). RT-PCR andimmunohistological investigations have identified theadditional expression of Kir4.1 (Kusaka et al., 1999)and Kir6.2 together with its auxiliary subunit SUR1 inthe RPE of rats (Ettaiche et al., 2001). Further, several
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electrophysiological investigations using Ussing chambersas well as the patch-clamp technique, have shown that RPEcells contain K+ currents with inwardly rectifying proper-ties (Gallemore et al., 1993; Hernandez et al., 1995; Hugheset al., 1995a; Hughes and Steinberg, 1990; Hughes andTakahira, 1996, 1998; Immel and Steinberg, 1986; Kusakaet al., 1999, 2001; Poyer et al., 1996; Segawa and Hughes,1994; Shimura et al., 2001; Strauss et al., 1993, 1994;Takahira and Hughes, 1997; Tao et al., 1994; Wen et al.,1993; Wollmann et al., 2006; Yuan et al., 2003). The Ussingchamber investigations allowed only their indirect identi-fication by the application of the unspecific blockers Ba2+
and Cs+, which also block other ion channels includingvoltage-gated, Ca2+-activated, and two-pore K+ channels(Goldstein et al., 2005; Grissmer et al., 1993; Ho et al.,1999; Hurst et al., 1996). Nevertheless, it has been shownby these studies that there is a large Ba2+-sensitive K+
conductance in both the apical and the basolateralmembrane (Gallemore et al., 1993; Hughes et al., 1995a).Using the patch-clamp technique, single freshly isolated aswell as cultivated RPE cells of different species have beenstudied. In most of these studies, inwardly rectifyingK+ currents were identified. The inwardly rectifying K+
currents appeared in all species investigated: frog, monkey,human, rat, toad, rabbit, bovine, and mouse. In rat andbovine RPE cells, combined patch-clamp and molecularbiological studies revealed that these inwardly rectifyingcurrents are carried either by Kir7.1 channels alone or byKir7.1 and Kir4.1 channels (Kusaka et al., 1999, 2001;Shimura et al., 2001; Yang et al., 2003). Both of thesechannels were shown to be located in the apical membraneof the RPE cells by immunohistochemistry (Kusaka et al.,1999, 2001; Yang et al., 2003). The currents measured inthese studies showed some features typical for Kir7.1channels: (i) weak inward rectification, (ii) an increase inthe conductance with reduced K+ concentration in thebath (all other inward rectifiers show a reverse K+
dependence) and (iii) a relative low Ba2+ sensitivity. Whilesome of the observations seem to fit very well with thepresence of Kir7.1 channels, some data suggest that theremight be different types of inward rectifiers expressed in theRPE, even in the same species (Strauss et al., 1993; Wen etal., 1993). In bovine RPE cells an inward rectifier has beendescribed that exhibits a pronounced rundown of currentwhen measured without intracellular ATP (Hughes andTakahira, 1998). Although it has not been investigated indetail, the Kir7.1 channels seem to be ATP-independentwhile Kir4.2 is known to be enhanced by ATP (Yang et al.,2000). This finding supports the study of Kusaka et al.(1999) who showed by single channel recordings from theapical side of RPE cells and by immunohistochemistry thatKir4.1 channels are also localized in the apical membrane.Additionally, in cultured RPE cells, currents have beenmeasured with a much more pronounced inward rectifica-tion than that of Kir7.1 and Kir4.1 channels which bothare weak inward rectifiers (Hughes and Takahira, 1996).Thus, there might also be channels of the Kir2 or the Kir3
subfamilies expressed. Since comparison of inwardlyrectifying currents between freshly isolated and culturedhuman or monkey RPE cells revealed some noticeabledifferences in respect to inactivation, Ba2+-sensitivity, anddependence on extracellular K+, the expression of inwardrectifiers with strong inward rectification may be anexpression of the potential of RPE cells to de- and trans-differentiate in culture (Zhao et al., 1997). Accordingly, forthe investigation of the in vivo situation, the use of freshlyisolated RPE cells should be favoured.
3.3.1.3. Role of inwardly rectifying K+ (Kir) channels for
RPE function. The RPE is responsible for the homo-eostasis of K+ in the subretinal space (Dornonville de laCour, 1993; Strauss, 2005). Since K+ continuously entersthe RPE cells through the apical membrane via theelectrogenic Na+/K+-ATPase and a Na+-K+-2Cl� co-transporter and since very little K+ is transcellularlytransported at least with low subretinal K+ concentrationsof 2mM as occurs in the light (Dornonville de la Cour,1993), the RPE needs a way to recycle the K+ at the apicalmembrane, e.g. for the activity of the apical Na+/K+
ATPase (la Cour et al., 1986). Kir7.1 channels, perhapstogether with Kir4.1 channels, are expressed in the apicalmembrane. Due to their weak inwardly rectifying proper-ties, these channels are well suited for K+ recyclingthrough the apical membrane. In the dark, this K+
recycling through the inward rectifier and the Na+
recycling through the Na+/K+-ATPase on the apicalmembrane supports Cl� transport through the cell (Bialekand Miller, 1994; La Cour, 1992). This Cl� transportacross the cell supports fluid transport through the RPE inthe retina to choroid direction (DiMattio et al., 1983;Frambach and Misfeldt, 1983; Miller and Edelman, 1990;Tsuboi et al., 1986). Thus, the inward rectifier in the apicalmembrane supports the absorption of water across theRPE. The consequent reduction of the subretinal spaceholds the retina in the proximity of the RPE, which isnecessary for the maintenance of retinal function and helpsto prevent retinal detachment. Upon illumination, the K+
concentration in the subretinal space decreases from 5 to2mM (Huang and Karwoski, 1992; Li et al., 1994a, b;Oakley and Steinberg, 1982; Steinberg et al., 1980) and thevolume of the subretinal space increases. This volumeincrease seems to be caused by an increased water effluxfrom the RPE (Huang and Karwoski, 1992). The drivingforce for this water efflux is believed to be a decreasedintracellular K+ concentration in RPE cells. Part of thisK+ decrease is likely driven by K+ efflux through inwardlyrectifying K+ channels. The atypical behaviour of Kir7.1channels in their dependence to the extracellular K+
concentration, whereby at lower K+ concentrations theypossess a higher conductivity (Doring et al., 1998;Krapivinsky et al., 1998), makes them good candidates togenerate the underlying increased K+ efflux.Besides this volume regulation of the subretinal space,
there are some reports that a Ba2+-sensitive K+ channel is
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involved in the regulation of the RPE cell volume itself(Adorante, 1995; Kennedy, 1994). Since inwardly rectifyingK+ channels are blocked by the Ba2+ concentrations usedin these studies, the participation of inward rectifiers in theregulatory volume decrease (RVD) induced by hypoosmo-tic stress seems very likely. Nevertheless, Ba2+ is a non-specific blocker of voltage-gated and Ca2+-activatedK+ channels (Bello and Magleby, 1998; Grissmer et al.,1993; Park et al., 2003). As the direct activation ofCa2+-activated K+ channels has been demonstrated (Sheuet al., 2004), the participation of inward rectifiers in volumeregulation remains to be proven.
In other cell types a participation of inwardly rectifyingchannels on hormone secretion has been reported (e.g. inpancreatic b cells the intracellular ATP/ADP ratio iscoupled to the activity of inwardly rectifying K+ channels).The increase of this ratio leads to the closure of ATP-sensitive inward rectifiers composed of the same subunitsfound in the RPE, Kir6.2 and SUR1 (Ashcroft andGribble, 1999; Ettaiche et al., 2001). This closure leads todepolarization of the cells, to Ca2+ entry through voltage-gated Ca2+ channels, and to increased insulin secretion. Asthe secretion of a variety of cytokines is controlled byintracellular Ca2+, which enters the cell via voltage-gatedCa2+ channels (see Section 5), a participation of theseATP-sensitive inward rectifiers on the regulation ofsecretion in RPE cells may not be excluded. Nevertheless,functional data showing the physiological relevance ofthese channels in the RPE are still missing.
3.3.2. Voltage-gated K+ channels
3.3.2.1. Voltage-gated K+ channels in general. Like in-wardly rectifying K+ channels, voltage-gated K+ channelsare composed of four pore-forming a subunits (Yellen,2002). The channels can be formed of four of the samesubunits forming homomeric channels or of differentsubunits forming heteromeric channels (Jan and Jan,1997). As the channels open upon membrane depolariza-tion, most channels are outwardly rectifying (Gutmanet al., 2005). Homology sequence analysis was used tosubdivide the voltage-gated K+ channels into 11 sub-groups with up to eight subfamily members. Though, themembers of the Kv5, 6, 8 and 9 subfamilies do not formfunctional K+ channels when expressed alone in hetero-logous expression systems, they form part of the channelpore when expressed together with members of differentsubfamilies (Gutman et al., 2005). The different a subunitsare further characterized by different biophysical andpharmacological properties. While some channels inacti-vate very fast at depolarizing voltages (so-called Acurrents), others show only slow or no inactivation at all.Additionally, the channels differ in their voltage-sensitivity,and for most channels highly selective blockers have beendescribed.
In non-excitable cells the role of voltage-gated K+
channels is difficult to define because most of these cellsshow only small membrane potential changes and some of
them stay in hyperpolarized states in which the expressedchannels should not be active. Nevertheless, voltage-gatedK+ channels in non-excitable cells have been shown to beinvolved in the following functions: (i) transport ofnutrients and electrolytes, (ii) recycling and secretionof K+, (iii) regulation of cell volume and pH, (iv) controlof cell cycle progression, and (v) regulation of growthfactor secretion (O’Grady and Lee, 2005; Warth, 2003).
3.3.2.2. Voltage-gated K+ channels in the RPE. Accord-ing to the databases of EST of the National Eye Institute,six different pore-forming subunits of voltage-gated K+
channels are expressed in the RPE/choroid (Kv1.2, 2.1, 7.1-3and 8.2). Additionally, two ESTs of auxiliary subunits havebeen identified in these preparations (Kvb2, KCNE4 ¼MiRP3). By RT-PCR the expression of Kv1.2 and 2.1 hasbeen confirmed (Pinto and Klumpp, 1998). Further,transcripts of Kv1.3, 1.4 and 4.2 have been identified byRT-PCR. Western blot and immunohistochemcical datashow that Kv1.3 and 1.4 subunits are located in the apicalmembrane of RPE cells (Pinto and Klumpp, 1998; Strausset al., 2002; Wollmann et al., 2006). As voltage-gated K+
channels are composed of four pore-forming subunits andthe different members of one subfamily are able to formfunctional channels together, the Kv1.2, 1.3 and 1.4subunits may form heteromeric channels. The Kv4.2subunits were localized to the basolateral membrane (Pintoand Klumpp, 1998). The expression of Kv7 subunits hasrecently been confirmed in human and monkey RPE cells(Hughes et al., 2006) in a combined molecular biologicaland electrophysiological study. The data confirmed theexpression of Kv7.1 but instead of Kv7.2 and 7.3 theyfound transcripts of Kv7.4 and 7.5. Patch-clamp experi-ments with different blockers suggest that these currentsare mainly mediated by Kv7.5 channels. Additionally, inpreparations from bovine RPE, K+ currents have beenmeasured that resemble the M current, which is known tobe carried by Kv7 channels (Takahira and Hughes, 1997).In all species investigated, in addition to these M currents,other voltage-gated outwardly rectifying K+ currentshave been characterized electrophysiologically (Fox andSteinberg, 1992; Hughes and Steinberg, 1990; Hughes andTakahira, 1996; Hughes et al., 1995b; Poyer et al., 1996;Strauss et al., 1993, 1994, 2002; Takahira and Hughes,1997; Tao et al., 1994; Wen et al., 1993; Wollmann et al.,2006). But the currents display very heterogeneousbiophysical properties. Even in the same species outwardlyrectifying K+ currents with different properties weredescribed (Hughes and Takahira, 1996; Hughes et al.,1995b; Strauss et al., 1993; Wen et al., 1993). For example,in freshly isolated human RPE cells a non-inactivatingcurrent with an activation threshold of �30mV wasobserved (Wen et al., 1993) while in another preparationnon-inactivating currents with a threshold of �60mV werefound (Hughes et al., 1995b). Despite these differences allidentified currents have a relatively low sensitivity totetraethylammonium (TEA) in common—an unspecific
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blocker of voltage-gated K+ channels. Additionally, withone exception, all currents exhibit slow or no inactivation.These findings suggest that members of the Kv4.xsubfamily are not functionally expressed—though Pintoand Klumpp found Kv4.2 in the basolateral membrane ofrat RPE cells (Pinto and Klumpp, 1998)—since thesechannels display a very fast inactivation (Gutman et al.,2005). Such a fast inactivation was only found in onepreparation of foetal human RPE cells (Wen et al., 1993)and not in the rat cells which were studied by Pinto andKlumpp. So far, only one attempt has been made toidentify the molecular basis of outwardly rectifying K+
currents in RPE cells (Strauss et al., 2002). The currentinvestigated in that study was inhibited by the K+ channelblocker agitoxin-2, which is highly specific for Kv1.1, 1.3and 1.6 channels (Garcia et al., 1994) while blockersspecific for other K+ channels had no effect on the current.Furthermore, the expression of Kv1.3 was confirmed bywestern blots. However, it cannot be excluded that theseKv1.3 subunits form heteromeric channels with othersubunits of the same subfamily.
3.3.2.3. Role of voltage-gated K+ channels for RPE
function. Most voltage-gated K+ currents described inRPE cells activate at potentials positive to �40 to �30mV(Fox and Steinberg, 1992; Hughes and Steinberg, 1990;Strauss et al., 1993, 1994, 2002; Takahira and Hughes,1997; Tao et al., 1994; Wen et al., 1993), some of them ateven more positive potentials (Hughes and Takahira, 1996;Wollmann et al., 2006). As RPE cells have restingmembrane potentials between �50 and �40mV with onlysmall changes to more positive potentials (Dornonville dela Cour, 1993; Fujii et al., 1992; Gallemore et al., 1997;Hughes and Steinberg, 1990; Joseph and Miller, 1991;Miller and Steinberg, 1977b; Quinn and Miller, 1992;Steinberg et al., 1978; Wen et al., 1993), it remains unclearhow these channels might contribute to RPE cell function.One possible mechanism that may lead to their opening hasbeen shown by Strauss et al. (2002). This study demon-strated that the outwardly rectifying K+ channel investi-gated in rat RPE cells is completely abolished by theinhibition of a tyrosine kinase and increased by theaddition of an activated pp60c�src tyrosine kinase. As thisactivation might be attributed to a shift in the activationthreshold, this provides a way to activate voltage-gatedchannels without changing the membrane potential. Thesechannels are probably composed of Kv1.3 subunits. Kv1.3channels are widely expressed in cells of the immune system(Cahalan et al., 2001; George Chandy et al., 2004). Inmicroglia Kv1.3 channels are necessary for their immunereaction (Fordyce et al., 2005). As RPE cells are part of theimmune system in the posterior part of the eye (Streilein,2003; Streilein et al., 2002), the delayed rectifier in the RPEcells may be part of their immune response.
The M current identified in freshly isolated bovine RPEcells activated at potentials positive to �80mV. Accord-ingly, these channels may contribute to the maintenance of
the resting membrane potential. M currents, carried byKv7 channels, are inhibited by Gq/11 coupled membranereceptors (Delmas and Brown, 2005). Gq proteins activatephospholipase Cb which hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate(InsP3) and diacylglycerol (DAG). The breakdown of PIP2
leads to the closure of M channels. RPE cells have beenshown to express different Gq/11 protein coupled receptors:epinephrine receptors a1B and a1D (Moroi-Fetters et al.,1995), serotonin receptor 5HT2 (Nash et al., 1999; Osborneet al., 1993) and purinergic receptors P2Y1 and P2Y2(Maminishkis et al., 2002; Reigada et al., 2005; Sullivanet al., 1997). Stimulation of purinergic and epinephrinereceptors leads to elevated intracellular free Ca2+ in RPEcells (Maminishkis et al., 2002; Quinn et al., 2001). Further,these stimulations lead to the depolarization of apical andbasolateral membranes. While depolarization of thebasolateral membrane may be attributed to the activationof a Ca2+-activated Cl� conductance, the apical depolar-ization may be due to the decrease of a K+ conductance.As M currents are inhibited by the activation of thesereceptors, they are good candidates for this purposepresuming that they are expressed in the apical membraneof RPE cells. Furthermore, stimulation of both purinergicand epinephrine receptors is involved in reattachment ofthe retina to the RPE by elevated fluid transport from thesubretinal space to the choroid (Maminishkis et al., 2002;Rymer et al., 2001). When these M channels are expressedin the apical membrane and Ca2+-activated K+ channelsin the basolateral membrane this would provide apossibility for the regulation of directed K+ transportthrough the RPE, as both channels are reciprocallyregulated by Gq/11-coupled receptors. One member of thissubfamily (Kv7.1) is involved in ion transport in varioustissues. This includes Cl� secretion in airway (Leroy et al.,2004) and gastrointestinal epithelia (Kunzelmann et al.,2001a, b), K+ secretion in the stria vascularis (Marcuset al., 1997, 1998; Shen and Marcus, 1998; Sunose et al.,1997) and K+ recycling in gastric parietal cells (Warth andBarhanin, 2003). Thus, in RPE, M channels may partici-pate in the ion homoeostasis of the subretinal space.
3.3.3. Ca2+-activated K+ channels
3.3.3.1. Ca2+-activated K+ channels in general. Manyprocesses in the RPE are coupled to changes in theintracellular free Ca2+ concentration (see Section 5). AsCa2+-activated K+ channels are gated by changes inintracellular free Ca2+, they are capable of fulfilling avariety of cellular functions (Vergara et al., 1998; Weigeret al., 2002). Based on single channel conductance,sequence homologies and pharmacology, Ca2+-activatedK+ channels are subdivided into three subgroups (Weiet al., 2005): (i) SK channels with a small K+ conductance(4–14 pS), (ii) IK channels with an intermediate K+
conductance (20–80 pS), and (iii) BK or maxi K channelswith a large conductance (200–300 pS). While SK and IKchannels are gated solely by intracellular Ca2+, BK
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 271
channels are gated by both intracellular Ca2+ and voltage.Like voltage-gated K+ channels they are formed of foursubunits with six transmembrane domains. The BKchannel has an additional N-terminal transmembranedomain (S0) (Bond et al., 1999; Ghatta et al., 2006).
Ca2+-activated K+ channels are found in close vicinityto voltage-gated Ca2+ channels in neurons (Gola andCrest, 1993; Grunnet and Kaufmann, 2004; Marrionand Tavalin, 1998) and in cochlear hair cells (Issa andHudspeth, 1994; Roberts et al., 1990) proposing a role asfeedback modulators for voltage-gated Ca2+ channelsthat provide the Ca2+ needed for the activation ofCa2+-activated K+ channels. In non-neuronal tissuesCa2+-activated K+ channels were found to be involvedin volume regulation, and in transepithelial ion secretion orabsorption, and cell cycle regulation (Begenisich et al.,2004; Feranchak et al., 2004; Huang et al., 2002; Joineret al., 2003; Liu et al., 2002; Turnheim et al., 2002).
3.3.3.2. Ca2+-activated K+ channels in the RPE. So far,Ca2+-activated K+ channel subunits have not beenidentified in the RPE, either by molecular biological orby protein biochemical methods. However, patch-clampmeasurements with RPE cells from different species haveprovided evidence that an outwardly rectifying K+ channelis activated by elevated intracellular Ca2+ concentrations(Ryan et al., 1999; Sheu and Wu, 2003; Sheu et al., 2004,2005; Tao and Kelly, 1996). These currents were completelyblocked by the specific BK channel inhibitor iberiotoxinand had a unitary conductance of �150pS (Sheu and Wu,2003; Tao and Kelly, 1996). As charybdotoxin, a blocker ofBK and IK channels, and apamin, a blocker of SKchannels, had no additional blocking effect on thesecurrents, it seems likely that the K+ channels activated byraising intracellular Ca2+ are of the BK subtype (Sheu andWu, 2003). Since BK channels have not yet been studied byeither immunohistochemistry or electrophysiological mea-surements using intact RPE tissue, it is not known whetherthese channels are in the apical or basolateral membrane.
3.3.3.3. Role of Ca2+-activated K+ channels for RPE
function. Different physiological stimuli have been usedto activate Ca2+-activated K+ channels in the RPE.Application of ATP has been shown to increase intracel-lular Ca2+ concentrations in RPE cells through theactivation of different purinergic receptors (see Section 5).This increase in intracellular free Ca2+ in turn stimulatesthe apical to basolateral fluid transport through the RPE(Peterson et al., 1997). The fluid transport is driven byCa2+-dependent Cl� currents in the basolateral membrane(see Section 4) and may be supported by Ca2+-activatedK+ channels. Ca2+-activated K+ channels were shown tobe involved in volume regulation in RPE cells in responseto hypotonic stress (Sheu et al., 2004). In that study, BKcurrents were increased by hypoosmotic stress. Theinduced K+ efflux together with a yet unidentified anionefflux is thought to be responsible for the RVD (Kennedy,
1994). Furthermore, intracellular Ca2+ signalling regulatesa variety of RPE cell functions (e.g. photoreceptorphagocytosis, growth factor secretion, immune responses,differentiation). To control these processes the cells needmechanisms to terminate the Ca2+ signals. One possibilityis simply to extrude the Ca2+ from the cytosol via differentmembrane transporters and pumps (Kennedy and Mangi-ni, 1996; Loeffler and Mangini, 1998; Mangini et al., 1997).The second possibility is to terminate the signal bystopping the Ca2+ influx. As in other cell types, theparticipation of Ca2+-activated K+ channels in the latterprocess has been proven (Ghatta et al., 2006) and it istempting to speculate that these channels may serve thesame purpose in the RPE. Furthermore, BK channels inthe RPE are inhibited by oxidizing agents (Sheu and Wu,2003). Mutational analysis has revealed a protectivemechanism against oxidative stress that involves theparticipation of heterologously expressed BK channels(Santarelli et al., 2006). As RPE cells are exposed to highoxidative stress (Mainster, 1987; Tanito et al., 2002; vanBest et al., 1997), the BK channels may provide part of theoxidant protective mechanisms in these cells.
3.3.4. Two-pore K+ channels
The last group of K+ channels to mention is the familyof two-pore K+ channels. In contrast to other K+
channels, which are all formed by four subunits with onepore domain each, two-pore K+ channels are formed bytwo subunits with two pore domains each (Goldstein et al.,2005; Lesage and Lazdunski, 2000). In vertebrates, 15members of this K+ channel subclass have been identifiedwhich are further divided into six subfamilies: the weaklyinwardly rectifying TWIK channels, the arachidonic acidand mechanosensitive TREK channels, the acid-sensitiveTASK channels, the alkaline-sensitive TALK channels, thehalothane-inhibited THIK channels, and the TRESKchannel (Goldstein et al., 2001, 2005). Two-pore K+
channels have a widespread tissue distribution beingexpressed in neuronal and non-neuronal tissues (Lesageand Lazdunski, 2000). They are also called leak channelsbecause they are constitutively open. The activity of thesechannels is influenced by a plethora of chemical andphysical stimuli—oxygen tension, pH, lipids, mechanicalstretch, neurotransmitters, and G-protein-coupled recep-tors (Plant et al., 2005)—and thus, may be involved inmany physiological functions relevant for RPE cellsincluding maintenance of the resting membrane potential,pH regulation, and K+ homoeostasis. As some of thesechannels are blocked by low concentrations of Ba2+
(Decher et al., 2001; Meadows et al., 2000; Patel et al.,2000) it cannot be excluded that some of the Ba2+-sensitiveeffects measured in Ussing chamber experiments must beattributed to two-pore channels and not exclusively toinwardly rectifying K+ channels as concluded in mostreports. However, as there is no direct evidence for theexpression of two-pore channels in the RPE, their possiblerole in the RPE will not be further discussed.
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3.4. Summary of K+ channel function
In Fig. 3, a scheme is presented that summarizes thepossible roles of K+ channels in RPE cells. Although it hasnot been resolved which K+ channels are expressed in theRPE and what their specific functions are, some conclu-sions can be drawn from the electrophysiological data.It is well accepted that the Na+/K+-ATPase, theNa+-K+-2Cl� cotransporter, and an inwardly rectifyingK+ channel are expressed in the apical membrane of RPEcells. They are functionally coupled via Na+ ions, whichleave the cells through the Na+/K+-ATPase and are usedby the Na+-K+-2Cl� cotransporter to uptake K+ and Cl�
into the cell. K+ is recycled into the subretinal spacethrough an inwardly rectifying K+ channel and Cl� ionsleave the cells across the basolateral membrane through anot yet identified Cl� channel. This Cl� absorption drivesapical to basolateral water transport. Kv7 channels andCa+-activated K+ channels may also play a role in K+
homoeostasis of the subretinal space. Due to the negativeactivation threshold of Kv7 channels they may also beinvolved in the K+ recycling at the apical membrane. Asthey do not inactivate at the negative membrane potentialsin RPE cells, they are capable of conducting a sustainedoutward current. The participation of two different K+
conductances allows the cells to fine-tune the K+ efflux.The inwardly rectifying Kir7.1 channels are regulated by
Ca2+
Endoplasmatic
Reticulum
PLC
Gαq/11
IP3
Epinephrine ATP
Ca2+
K+
K+K+
Cl-
M channel
(Kv7)Kv1.3
BK
channel
L-type channel CaClchannel
IP3R
apical
basolateral
Fig. 3. Schematic diagram summarizing how different K+ currents may be inv
are drawn in blue, second-messenger pathways are in yellow with r
K+-ATPase and the Na+-K+-2Cl� cotransporter are drawn in grey, G-pro
the ion through the cell and through the cell membrane, green arrows mean
AQ1 ¼ aquaporine 1; ATP ¼ adenosine triphosphate; BK channel ¼ large con
channel; Gaq/11 ¼ G protein a-subunit q/11; IP3 ¼ inositol-1,4,5-trisphosphate
extracellular K+ concentrations in that they have anincreased K+ conductance when extracellular K+ isreduced. Thus, they are activated when light falls on theretina as illumination is accompanied by a decrease insubretinal K+ concentration. By this mechanism, Kir7.1channels are involved in the K+ homoeostasis in thelight. The Kv7 channels on the other hand are regulated byGq/11-protein-coupled receptors. These receptors activatephospholipase C, which hydrolyzes PIP2 into InsP3 andDAG, leading to an inhibition of Kv7 channels and anincrease in intracellular free Ca2+, which in turn activatesCa2+-activated K+ channels. If these two K+ channels areexpressed in opposing membranes of the RPE cells, e.g. theM channels in the apical membrane and the BK channels inthe basolateral membrane, this would lead to a regulatedK+ transport through the RPE. Under normal conditions,K+ recycling across the apical membrane would be favoredwhereas upon activation of Gq/11 proteins, the absorptionof K+ ions through the basolateral membrane would befavored. This regulation of K+ transport direction couldsupport water transport through the RPE, which iselevated upon ATP stimulation, although this water fluxis mainly thought to be coupled to Cl� absorption. ThisCl� absorption is also increased by the activation ofpurinergic receptors, mediated by Ca2+-activated Cl�
channels. An additional role of the BK channels isprobably to counteract the voltage-gated Ca2+ channels
K+
K+
K+ 2 K+Na+
3 Na+
Cl-
2 Cl-
Kir7.1
Kir4.1
Kv1.3Cl channel
H2O
AQ1
AQ1
H2O
H2O
?
?
olved in RPE cell function. All ion channels as well as the water channels
ed arrows, ion transporters are drawn in light blue. The Na+/
tein-coupled receptors and their ligand in pale blue. Black arrows show
activating influence, red arrows inhibiting influence. Abbreviations are:
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in the RPE as the entering Ca2+ activates BK channels.Their activation in turn leads to the hyperpolarization ofthe cell and consequently to the closure of the voltage-gated Ca2+ channels. The role of the other voltage-gatedK+ channels including Kv1.3 in RPE cells remains obscureas these channels should be inactive at the RPE cell’sresting membrane potentials. Possibly, they are involved incell proliferation/differentiation and wound-healing pro-cesses.
4. Chloride
4.1. The role of Cl� and Cl� channels in cell function
Chloride is the most abundant permeable anion underphysiologic conditions; therefore, anion channels (hereafteronly referred to as chloride channels) mostly conductchloride. In contrast to most other ions, the Cl� restingconcentration significantly differs among various mamma-lian cell types and during cell development (Cherubiniet al., 1991; Chipperfield and Harper, 2000). Like otherions, Cl� transport is regulated across plasma membranesas well as membranes of intracellular organelles. Chloridecan be actively released out of cells, accumulated withincells, or passively distributed or co-transported to maintainelectroneutrality. Depending on Cl� ion channel localiza-tion, in the plasma membrane or intracellular organelles,Cl� channels are involved in modulating the excitability ofcells, transepithelial salt transport, cell volume regulationand homoeostasis or regulation of organelle volume, theacidification of intra- and extracellular compartments,calcium or proton gradients, the cell cycle, and apoptosis(Jentsch et al., 2005c; Nilius and Droogmans, 2003). Inmany epithelia Cl� channels are involved in the vectorial,transepithelial transport of salt and water (Begenisich andMelvin, 1998; Spring, 1998). Cl� channels also help protectepithelial cells against excessive osmotic cell swelling(Sarkadi and Parker, 1991; Spring and Ericson, 1982;Strange, 2004; Strange et al., 1996). The existence of Cl�
conductance is important for endosomal, lysosomal, andphagolysosomal vesicles and turnover along the endocyticapparatus (Faundez and Hartzell, 2004; Jentsch et al.,2005a).
Three major Cl� ion channel families (A–C) have beenwell characterized using molecular means. Other Cl�
conductances/channels (D) are described either by expres-sion studies or by functional characterization, but theirmolecular counterpart and /or their role in health anddisease remains unknown.
(A) Voltage-gated Cl� channels (CLC1–7, CLC-K1,2,CLC-0), (B) Cystic fibrosis transmembrane conductanceregulator (CFTR) channel, and (C) extracellular ligand-gated ion channels such as g-aminobutyric acid (GABA) orglycine receptors have been distinguished. Some of theseCl� channels are located primarily, or exclusively, inmembranes of intracellular organelles, but their localisa-tion is still controversial (ClC3–7). (D) Two different
classes of Cl� channels primarily defined from theirmechanism of activation and biophysical properties are:(I) calcium-activated Cl� channels (CaCC) (Evans andMarty, 1986) and (II) swelling-activated/volume-regulatedanion channels (VRAC). However, this classification is anoversimplification as many Cl� channels are regulated bymore than one mechanism and might consist of molecu-larly diverse classes. It should be mentioned that most Cl�
channel blockers are unselective, have a low potency, andhave various side effects; therefore, pharmacologicalstudies have to be interpreted with caution (d’Anglemontde Tassigny et al., 2003; Jentsch et al., 1999; Kidd andThorn, 2000). Finally, the discrepancy of functional Cl�
channels and actual cloned proteins may stem from the factthat there are no conserved gene sequences characterizingCl� channels among the known channel families. Conse-quently, entire families of Cl� channels may not have beendetermined.
4.2. Cl� ions in RPE function
4.2.1. Fluid transport
The RPE mediates water transport from the subretinalspace to the choroid. Together with Muller glial cells,which transport water from the subretinal space to thevitreous, the RPE controls the volume of the subretinalspace (Bringmann et al., 2005; Hughes et al., 1998;Marmor, 1983; Newman and Reichenbach, 1996; Steinberget al., 1983; Strauss, 2005). In fact, the primary function ofCl� channels in secretory cells is transepithelial iontransport (Begenisich and Melvin, 1998; Do and Civan,2004). The retina produces a large amount of water due toits enormous metabolic turnover and intraocular pressuredrives water movement from the vitreous body through theretina. This water is eliminated from the subretinal spaceinto the choroid plexus by the RPE and into the vitreous byMuller cells. Therefore, fluid transport from choroid toretina (secretion) and vice versa (absorption) by the RPEare essential pathways for fluid regulation in the eye. Fluidabsorption must occur without interruption for retinaladherence and interaction with the RPE to be maintained(Hamann, 2002; Marmor, 1990, 1993; Verkman, 2003). Inpathologic states like retinal detachment or macularoedema, increased fluid absorption by the RPE may leadto a recovery (Bringmann et al., 2004; Marmor, 1999).RPE transport of Cl� and K+ is thought to drive
transepithelial water transport. Under control conditions itwas estimated that the Cl� conductance is up to 70% of thetotal basolateral conductance. The transport rate of waterwas estimated between 1.4 and 11 ml cm2 h�1 (Gallemoreet al., 1997; Hamann, 2002; Strauss, 2005). Fluid absorp-tion involves different mechanisms operating in the apicaland basolateral membranes of the RPE cells (Fig. 5).Several Cl� transporters use the energy stored in trans-membrane gradients of other ions to move Cl� across theRPE cell membranes against an electrochemical gradient.The electrogenic Na+/K+-ATPase generates a Na+
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301274
gradient from the extracellular (subretinal) to the intracel-lular space, which also can be measured as the steady-stateretina-positive potential of about 10mV (Gallemore et al.,1997). This so-called standing potential depends on theseparation of apical and basolateral properties. In the dark,photoreceptors maintain K+ recycling leading to a highersubretinal K+ concentration than during illumination ofthe retina (Bialek and Miller, 1994). The increase in [K+]ostimulates the apical Na+-K+-2Cl� cotransporter, therebyincreasing the cellular uptake of K+ and Cl� (Adoranteand Miller, 1990; Hamann, 2002; Hughes et al., 1989;Joseph and Miller, 1991; Keller et al., 1988; Kennedy,1990, 1994; la Cour, 1991a, b; Lin and Miller, 1991). Thus,a high intracellular Cl� activity of up to 60mM isestablished providing a driving force for Cl� out of thecell (Bialek and Miller, 1994; Fujii et al., 1992; Quinn et al.,2001; Rymer et al., 2001). Retinal adhesion as an indicatorof functional fluid transport is lost by inhibition of theapical Na+/K+-ATPase by ouabain (Endo et al., 1988;Marmor, 1983; Marmor and Yao, 1989). Thus, Cl�
absorption in tight epithelia like the RPE is accompaniedby the transport of water to balance osmotic pressurethrough aquaporins (Bialek and Miller, 1994; Tsuboi andPederson, 1988a, b; Verkman, 2003). Evidence stronglysuggests that Cl� exits across Ca2+-activated, volume-activated, and/or cAMP-activated ion channels in thebasolateral membrane of the RPE, but its differentialcontributions are not understood and its molecularcounterparts are still controversial (Gallemore et al.,1997; Hartzell et al., 2005b; Hughes et al., 1998; Strauss,2005).
4.2.2. Volume control
Many transporting epithelia regulate their volume afterosmotic perturbations that shrink or swell the cells(Sarkadi and Parker, 1991; Spring and Ericson, 1982;Strange et al., 1996). The acute component after cellswelling is frequently mediated by conductive losses of Cl�
and K+, and related water efflux to control cell volume(RVD). Contrary, cell shrinkage results in regulatoryvolume increase (RVI) by mechanisms that decrease K+
and Cl� conductance. The specific features of cell-volumeregulation vary considerably among cell types and thephysiological significance is not always proven (la Cour,1985; Lang et al., 1993, 1998a, b), and we don’t know theexact extent of physiological osmotic changes on subretinaland choroidal sites of the RPE cell layer.
RPE cells have effective volume regulating mechanisms,as shown by volume manipulation. Cultured RPE cellspossess hypertonically-activated Na+-K+-2Cl� cotran-sporter, hypotonically-activated K+-Cl� cotransporter,and a Ba2+—inhibitable hypotonically activated K+ effluxpathway (Civan et al., 1994; Tsuboi and Pederson,1988a, b). Remarkably, Cl� channel inhibition with thebroad Cl� channel blocker NPPB reduced cell shrinkage.Thus, the RVD of RPE cells likely reflects efflux of K+ andCl� through parallel channels.
The transport situation seems to differ in the light- anddark-adapted eye. In the dark, lactate increases subretin-ally whereas it decreases in the RPE. Therefore, a gradientfrom retina to choroid is established which imposes anosmotic load for the RPE (Adler and Southwick, 1992;Hamann, 2002; Hsu and Molday, 1994; Winkler, 1995).During illumination, subretinal K+ decreases, subretinalspace volume increases and RPE cell volume decreases(Gallemore et al., 1997). Some studies suggest that changesin subretinal hydration and ion composition, RPE cellvolume regulation and fluid transport mechanisms areinterrelated (Adorante and Miller, 1990; Bialek and Miller,1994; Botchkin and Matthews, 1993; Civan et al., 1994;Hartzell and Qu, 2003; Kennedy, 1994). Moreover,phagocytosis is regulated by illumination and hypoosmoticcell swelling or application of the Cl� channel blockertamoxifen decreases RPE phagocytosis (Irschick et al.,2006; Mannerstrom et al., 2001). Therefore, it is not yetclear whether cell volume alterations due to photoreceptorphagocytic uptake, nutrient and lactate transport, orpathophysiologic disturbances in subretinal osmolarity(hydration and ion composition) require RPE cell volumecontrol capability and fluid transport across the RPE.
4.2.3. pH regulation
Controlling the ionic composition of the cytoplasm isinevitable. Most cells are more alkaline intracellularly thancalculated from electrochemical equilibrium (Chipperfieldand Harper, 2000). Light-induced changes in ion transportmaintain ion homoeostasis in the subretinal space andevoke changes in pH (Gallemore et al., 1997; Strauss,2005). The mechanism by which pH homeostasis ismaintained in RPE cells is not completely understood.RPE cells support photoreceptor function by eliminatinglactic acid thereby regulating the extracellular pH (Ha-mann, 2002). Transcellular fluid transport requires anefficient regulation of intracellular pH as well, since someCl� channels are modulated by pH changes. The subretinalpH and intracellular pH are regulated by transepithelialtransport of HCO3
�, which needs pathways for Cl�
recycling (Edelman et al., 1994a; Lin and Miller, 1991).Apically, RPE cells have a Na+/H+ exchanger and a Na+/2HCO3-cotransporter, and basolaterally a HCO3
�/Cl�
exchanger, and they are acid-loaded due to transepitheliallactate transport (Hamann, 2002; Keller et al., 1986, 1988;Strauss, 2005). Increased cytosolic HCO3
� alkalinizes thecytosol which stimulates the basolateral Cl�–HCO3
�
exchanger (Edelman et al., 1994a; Kenyon et al., 1997;Lin and Miller, 1991). The increased Cl�–HCO3
� exchangeractivity results in acidification of the cytosol by HCO3
�
extrusion and to Cl� uptake. This CI� recycled to thechoroidal site by basolateral CI� channels and facilitatesacidification by HCO3
� extrusion. Furthermore, the en-hanced CI� uptake by the Cl�–HCO3
� exchanger results inreduced Cl� and water secretion. A favoured hypothesis isthat this recycling determines the balance of fluid absorp-tion and secretion so that the net Cl� movement is toward
ARTICLE IN PRESS
d
c
b
Fig. 4. Example of voltage-dependent Cl� channel currents in freshly
isolated mouse RPE cells. (a) Schematic diagram showing the recording
configuration. Membrane currents were measured in K+-free solutions in
the whole-cell configuration. (b) Pattern of electrical stimulation: from a
holding potential of �40mV the cells were stepwise depolarized up to
a potential +50mV and then stepwise hyperpolarized to a potential
�130mV. The step duration is 50ms and the increment 10mV. (c)
Example of currents activated by electrical stimulation. (d) Effect of the
Cl� channel blocker DIDS on the voltage-dependent currents. Application
of 1mM DIDS (right panel) reduced the outwardly directed currents
observed under control conditions (left panel).
S. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 275
the subretinal space. Therefore the RPE behaves as a netsecretory epithelium.
4.2.4. Intracellular organelles
Intracellular Cl� channels have received much lessattention, but genetic evidence has revealed some of theirphysiological significance. Cl� channels in vesicles might beinvolved in endosomal, lysosomal, phagosomal, andsynaptic vesicle pathways (Jentsch et al., 2005c).
4.3. Identification and properties of Cl� channels in the RPE
Molecular counterparts of most Cl� channels in the RPEare still not known. CFTR, ClC-2, -3, -5, and -7, Ca2+
activated, and volume activated Cl� channels have beenclaimed to be expressed in RPE cells to date (Blaug et al.,2003; Bosl et al., 2001; Botchkin and Matthews, 1993;Hartzell and Qu, 2003; Hughes and Segawa, 1993; Loewenet al., 2003; Reigada and Mitchell, 2005; Strauss et al.,1996, 1998a, 1999; Ueda and Steinberg, 1994; Wenget al., 2002; Wills et al., 2000; Wollmann et al., 2006; Wuet al., 2006). Fig. 4 shows an example of CI� currents inRPE cells.
4.3.1. ClC Cl� channels
4.3.1.1. ClC Cl� channels in general. Mammalian gen-ome contain nine different genes that encode for the ClCchannels. Some comprise plasma membrane channels(ClC-1,-2, -ka, kb) and others are predominately inintracellular membranes (ClC-3, -4, -5, -6, -7). All ClCchannels examined to date are dimers. Most of thesechannels are characterized by voltage-dependent gating, alldisplay a specific permeability sequence (Cl�4Br�4I�)and are mostly regulated by extracellular anions and pH(Jentsch et al., 2005a–c, 2002; Nilius and Droogmans,2003). ClC-1 has been described as a double-barrel modelrepresented by two identical parallel pores (Middletonet al., 1996; Pusch and Jentsch, 2005). Each poreindependently opens a fast gate (10ms range) upondepolarization which displays inward rectification, and acommon slow inactivating gate (10–100 s) closes both poresat the same time. The slow gate opens upon hyperpolar-ization. Gating mechanisms and selectivity of ClC channelsare still unknown.
The functional role of ClC-1 is stabilizing the membranepotential; ClC-2 channels are inwardly rectifying afteractivation by hyperpolarization, cell swelling, or extracel-lular acidosis; and ClC-3, -4 and -5 are constitutively activeand outwardly rectifying as well as are ClC-Ka and -Kb inthe presence of the b-subunit Barttin. In a recent study,vesicular ClC-4, -5 and -7 (and possibly ClC-3 and -6) havebeen demonstrated to function as electrogenic chloride-hydrogen exchangers (also called antiporters) which extrudeprotons against their electrochemical gradient, demonstrat-ing secondary active transport (Accardi and Miller, 2004;Kasper et al., 2005; Kornak et al., 2001; Picollo and Pusch,2005; Pusch et al., 2006; Scheel et al., 2005).
4.3.1.2. ClC Cl� of the RPE. So far ClC channels -2, -3, -5,and -7 have been reported to be expressed in human RPE cell(Fig. 5) by RT-PCR, Western blotting and immunohisto-chemistry, but biophysical characterization has not beencompleted (Hartzell and Qu, 2003; Weng et al., 2002; Willset al., 2000). ClC-2 has been implicated as a potential Cl�
exit pathway in epithelia. Strikingly, disruption of ClC-2leads to degeneration of the retina (Bosl et al., 2001). Retinaldegeneration might be secondary due to RPE dysfunction,which likely would result in abnormal composition of sub-retinal fluid and secondarily impairs photoreceptor viability.RPE short-circuit currents are reduced in ClC-2 �/� mice
ARTICLE IN PRESS
Fig. 5. Schematic diagram summarizing the Cl� channel function in RPE cells. All ion channels as well as the water channels are drawn in blue, second-
messenger pathways are in yellow with red arrows, ion transporters are drawn in light blue, intracellular ion channels are drawn in light yellow. The
protein a-subunit q/11; IP3 ¼ inositol-1,4,5 trisphosphate; IP3R ¼ inositol-1,4,5 trisphosphate receptor; PLC ¼ phospholipase C; VMD2 ¼ product of
the vitelliform macular dystrophy 2 gene.
S. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301276
supporting the role in transepithelial fluid transport. But thephysiological role of ClC-2 and the pathomechanism of theRPE phenotype in ClC-2 knockout mice remain unknown.Currents resembling ClC-2 (hyperpolarization-activated,slowly activating, inwardly rectifying, acid-activated, Zn2+
-inhibited) have been described for the RPE, but definitecharacterization including a description of the anion perme-ability sequence (Cl�4I�) is missing (Hartzell and Qu,2003). In order to understand the contribution of various Cl�
ion channels to transepithelial transport the identification ofthe apical and basolateral channels is required and is, to date,unknown.
Cellular pH might be regulated by basolateral Cl�/HCO3
� exchangers and linked Cl� cycling through ClC-2.In support of this, ClC-2 is activated by an increasedintracellular Cl� concentration (Catalan et al., 2004;Niemeyer et al., 2003; Pusch et al., 1999) as well as byextracellular acidification (Edelman et al., 1994a; Jordt andJentsch, 1997).
ClC-3, -5 and -7 transcripts have been found in RPEcells, but neither has been investigated functionally (Kasperet al., 2005; Kornak et al., 2001; Stobrawa et al., 2001). Ingeneral, these channels are located on intracellular vesicles,endosomes, and lysosomes. Their disruption might impairthe acidification of intracellular compartments. Whether ornot ClC-3 channels may get to the cell membrane iscontroversial (Coca-Prados et al., 1996; Do et al., 2005;Duan et al., 1999; Hara-Chikuma et al., 2005; Jentsch
et al., 2005b, c). ClC-3, -5 and -7 channels may provide anelectric shunt for the H+-ATPase in acidic intracellularcompartments like endosomes, and ClC-7 to a large degreeto lysosomes, and therefore are likely involved in processeslike endocytosis (Hartzell et al., 2005b; Kasper et al., 2005;Kornak et al., 2001; Piwon et al., 2000; Stobrawaet al., 2001). An important RPE cell function, phagocy-tosis, depends on vesicular membrane trafficking, lysoso-mal fusion, phagolysosomal maturation, and digestion(Besharse and Defoe, 1998; Hartzell et al., 2005b).Lipofuscin granules which originate from phagocytosisaccumulate with age in the RPE, the largest increaseoccurring after the first decade of life in humans (Deloriet al., 2001). Intracellular Cl� ion channels are involved inregulating pH and ionic composition of functionallydifferent organellar compartments. Perturbation of theionic composition of cell organelles by inhibition of the Vtype ATPase or by addition of a Na+ ionophor impairsRPE functions like secretion of interphotoreceptor reti-noid-binding protein, leading to lysosomal dysfunction andinhibition of phagocytosis of photoreceptor outer segments(Deguchi et al., 1994; Edwards et al., 1987; Mahon et al.,2004; Peters et al., 2006; Sundelin et al., 1998; Sundelin andTerman, 2002; Toimela et al., 1998). Therefore, regulationof vesicular [Cl�] is important in RPE cells in dailyprocesses. Some interference might not be obviousimmediately, but might still bear some age-related patho-physiological implications.
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4.3.2. CFTR
4.3.2.1. CFTR in general. CFTR resides at the membranesurfaces and in endosomes in various epithelia, is involvedin transepithelial fluid transport. In some epithelia (e.g.colon) CFTR represents the major apical Cl� conductance(Begenisich and Melvin, 1998; Greger, 2000; Jentsch et al.,2002). A genetic defect in CFTR causes cystic fibrosis, a lifethreatening disease characterized by the clogged ducts ortubes of various organs by mucus or other secretions(Fuller and Benos, 1992; Thiagarajah and Verkman, 2003).CFTR belongs to the traffic ATPases or so called ABCtransporters, but represents the only protein in that familywhich was identified as ion channel (Anderson et al., 1991).Intracellularely, CFTR contains two nucleotide bindingdomains (NBD1 and NBD2) separated by a large polarregulatory domain with nine sites for phosphorylation byPKA and seven sites for phosphorylation by PKC(Riordan et al., 1989). ATP-driven conformational changesby binding of ATP, which in other ABC proteins fuelsuphill substrate transport across cellular membranes, inCFTR, opens and closes a gate to allow transmembraneflow of anions down their electrochemical gradient(Gadsby et al., 2006; Vergani et al., 2005). Furthermore,cAMP-dependent activation of PKA activates CFTRchannels, which are voltage independent and display apermeability sequence of Br� 4Cl�4I�4F� (Dawsonet al., 1999; Li et al., 1988; McCarty, 2000).
Besides its Cl� channel function, CFTR might beinvolved in the regulation of other ion channels, transpor-ters, or cellular functions like: electrolyte and osmolytetransport, cell volume regulation, efflux of ATP throughCFTR channels or CFTR regulated ATP release byvesicles, inhibition of epithelial sodium channel (ENaC),CaCC and VRAC.
4.3.2.2. CFTR of the RPE. Human RPE cells displayCFTR mRNA expression and CFTR protein has beenimmunolocalized to the apical and basolateral membranes.A cAMP-dependent increase in transepithelial Cl� trans-port or conductance indicates a functional role of CFTR inthe RPE (Fig. 5) (Blaug et al., 2003; Hartzell et al., 2005b;Hughes and Segawa, 1993; Loewen et al., 2003; Quinnet al., 2001; Reigada and Mitchell, 2005; Weng et al., 2002;Wills et al., 2000).
A main lead for CFTR in RPE function stems from thefact that part of the transepithelial Cl� transport and netfluid absorption is dependent on cAMP-activated Cl�
conductance (Gallemore et al., 1997). An increase of[cAMP]i in foetal human RPE induces a decrease inbasolateral membrane resistance and the activation of aCl� conductance (Weng et al., 2002). Thereby intracellularCl� decreases and generates a depolarization. Moreover,cAMP increases short circuit current, increases trans-epithelial potential, and decreases the ratio of basolateralto apical resistance (Hughes et al., 1987, 1988; Miller andFarber, 1984). Although these data suggest an increase inbasolateral conductance, the effects are complex, possibly
reflecting different transport pathways. ClC-2 is anotherpossible cAMP-activated candidate. ClC-2 is also a cAMP-activated Cl� channel with a NO3
�¼ I�4Br�4Cl�b
HCO3� permeability sequence (Hughes and Segawa,
1993). From studies of CFTR /ClC-2 double knockoutmice it was concluded that ClC-2 does not compensate forCFTR (Zdebik et al., 2004), suggesting that CFTR andClC-2 have differential roles for Cl� ion efflux regulation.Another line of observations supports a different or
additional role for CFTR in RPE cells. RPE cells releaseATP and its release mechanism has been linked to CFTRCl� channels in various epithelial cells, including RPE.cAMP-activating RPE cell manipulation led to increasedapical ATP release whereas glibenclamide and the morespecific inhibitor CFTR-172 (Ma et al., 2002) preventedhypotonically triggered ATP release. A precise pathway forthe release of Cl� and ATP in RPE cells and therelationship to CFTR remains controversial (Braunsteinet al., 2001; Prat et al., 1996; Reigada and Mitchell, 2005).
4.3.3. Ca2+-activated Cl� channels
4.3.3.1. Ca2+-activated Cl� channels in general. Chloridechannels activated by intracellular calcium (CaCC) arewidely expressed. Voltage-dependence and Cl� concentra-tion gradients allow Cl� influx or efflux that produceshyperpolarization or depolarization, respectively. CaCCconductances are implicated in diverse functions liketransepithelial fluid transport, excitability modulation,and regulation of smooth muscle cell tonus. Endogenouscalcium activated Cl� currents are voltage-dependent(outwardly rectifying), and display a SCN�4NO3
�4I�4Br�4Cl�4F� permeability sequence. The voltage-depen-dence disappears with high [Ca2+]i (Begenisich and Melvin,1998; Eggermont, 2004; Hartzell et al., 2005a; Jentschet al., 2002; Kidd and Thorn, 2000; Melvin, 1999; Morris,1999; Nilius and Droogmans, 2003; Scott et al., 1995;Suzuki et al., 2006).So far at least three potential classes of CaCC have been
suggested: (i) activated by increased [Ca2+]i, (ii) regulatedby calmodulin-dependent protein kinase II, and (iii)cGMP-dependent channel types (Jentsch et al., 2002;Matchkov et al., 2004). Putative candidate proteins (e.g.CLCA, bestrophin) have been cloned from various species(Bakall et al., 1999; Gandhi et al., 1998; Marquardt et al.,1998; Stohr et al., 2005; White et al., 2000), but thecorrelation of biophysical properties of heterologouslyexpressed channels to native calcium dependent Cl�
currents are still unsolved or controversial (Fuller et al.,2001; Jentsch et al., 2002; Loewen et al., 2002a; Nilius andDroogmans, 2003; Pauli et al., 2000). Bestrophins havebeen shown to be anion-selective channels and areactivated by physiological [Ca2+]i in heterologous expres-sion systems (Fischmeister and Hartzell, 2005; Hartzell etal., 2005b; Hartzell and Qu, 2003; Qu et al., 2003, 2004,2006b; Qu and Hartzell, 2004; Sun et al., 2002; Tsunenariet al., 2006). Four bestrophin isoforms have beendiscovered, all of which generate membrane currents in
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301278
transfected HEK293 cells and two of them with a I�4Br�4Cl� permeability sequence matching endogenousCaCC (Qu et al., 2004, 2006b; Qu and Hartzell, 2004;Sun et al., 2002; Tsunenari et al., 2006). But heterologouslyexpressed CLCAs and bestrophins showed instantaneousbut not time dependent, outwardly rectifying currents ascharacteristic for endogenous CaCC. Other studies suggestthat CLCAs and bestrophins might not only constitute aCl� channel itself, but also modulate other ion channels(Loewen et al., 2002a, b, 2003; Marmorstein et al., 2004,2006; Pusch, 2004; Rosenthal et al., 2006).
4.3.3.2. Ca2+-activated Cl� channels in the RPE. Indirectevidence from choroid-RPE preparations suggests thatCaCC might be the light-induced basolateral Cl� con-ductance important for fluid, and possibly volume regula-tion, in the RPE (Fig. 5). Extracellular epinephrine andATP activate calcium signalling, basolateral Cl� conduc-tance, and fluid absorption in RPE cells (Edelman andMiller, 1991; Joseph and Miller, 1991; Leipziger, 2003; Linand Miller, 1991; Peterson et al., 1997). ATP is currentlystill discussed as a factor activating fluid transport acrossRPE cells due to illumination (light-peak substance) andtriggering this fluid absorption by stimulating apical P2Y2receptors with a specific agonist can reduce the size of fluidblebs in the subretinal space in vivo (Maminishkis et al.,2002; Meyer et al., 2002). External application of DIDS (anunspecific Cl� channel blocker) reduced basolateral Cl�
conductance and fluid absorption and therefore CaCC aresuggested to be important for fluid and ion transportregulation across the RPE (Bultmann and Starke, 1994;Edelman et al., 1994a; Gallemore et al., 1997; Petersonet al., 1997; Quinn et al., 2001; Soltoff et al., 1993;Ziganshin et al., 1996). CaCC have been shown to bepresent in Xenopus, rat, and mice RPE cells by patch-clamp measurements (Botchkin and Matthews, 1993;Hartzell and Qu, 2003; Marmorstein et al., 2006; Strausset al., 1996, 1998a, 1999; Ueda and Steinberg, 1994;Wollmann et al., 2006). InsP3 induced calcium influx intothe cell activates a Cl� conductance, which was inhibitableby DIDS, but not flufenamic acid or Zn+. The currentsshowed outward rectification, a fast voltage-dependentactivation, and transient activation depending on [Ca2+]i.Direct application of a high intracellular Ca2+ concentra-tion activates these currents as well.
VMD2 (hBest1), was originally positionally cloned fromfamilies with Best’s vitelliform macular dystrophy (Mar-quardt et al., 2000; Petrukhin et al., 1998; Stanton et al.,2006; Stohr et al., 2002; White et al., 2000) and its geneproduct was recently suggested as a novel prospective Cl�
channel candidate expressed by the RPE (Fischmeister andHartzell, 2005; Hagen et al., 2005; Qu et al., 2004, 2006b;Qu and Hartzell, 2004; Stanton et al., 2006; Sun et al.,2002; Tsunenari et al., 2003, 2006). hBest1 and hBest2, arehighly expressed in the RPE (Bakall et al., 2003; Krameret al., 2004; Marmorstein et al., 2000; Stohr et al., 2002).Best patients have a reduced light-peak propably caused by
a reduced basolateral Cl� conductance. Heterologeousexpression of the VMD2 gene product bestrophin-1 in thehuman kidney cell line HEK293 results in an increase in themembrane conductance for Cl�, which was dependent onincreased intracellular free Ca2+ (Sun et al., 2002). Side-directed mutagenesis of the putative pore region of thechannel protein resulted in a change of the permeability forSCN� ions of the channel (Qu et al., 2004, 2006a; Qu andHartzell, 2004).
4.3.4. Volume regulated anion channels
4.3.4.1. Volume regulated anion channels in general. Vol-ume regulated anion channels (VRAC) are ubiquitouslyexpressed in mammalian cells and play an important role incell volume homoeostasis (Clapham, 1998; d’Anglemont deTassigny et al., 2003; Hoffman et al., 1998; Hoffmann,1992; Lang et al., 1998a, b, 2005; Nilius and Droogmans,2003; Nilius et al., 1997; Okada, 2004; Okada and Maeno,2001; Okada et al., 2001; Sardini et al., 2003). Small cellvolume changes might even be linked to vectorial transportof solutes and water across epithelia (Foskett, 1990). Theactivation of VRAC is believed to provide one of the initialtriggers linking cell swelling to the subsequent loss of KClthrough Cl� and K+ channels—osmolyte and water effluxresulting in a RVD. The associated current is referred to asICl,swell. In experimental setups, exposure to hypotonicsolutions is the common technique to swell cells. In mostcells this manoeuvre activates outwardly rectifying anionchannels (anion influx), which are voltage-dependent(inactivate at positive membrane potentials in many, butnot all cell types), depend on intracellular ATP, and exhibitan anion selectivity of SCN� 4I� 4NO3
�4Br�4Cl�4F�4glutamate�. This conductance is also regulated by cellshrinkage (exposure to hypertonic solution is the commonexperimental manoeuvre) and in some cells a similarconductance initiated by the hypotonic solution appearsto develop spontaneously under isotonic conditions, whichcan be suppressed by exposure to hypertonic solutions.Moreover, ICl,swell may be activated by a reduction inintracellular ion strength, shear stress, and application ofGTPgS. Some lines of evidence suggest that VRAC is alsopermeable to lactate and bicarbonate pointing to a role in pHregulation. This putative channel was named volume-stimulated osmolyte and anion channel (VSOAC) anddepends like VRAC on intracellular ATP (Jackson et al.,1994). Besides ICl,swell, other ion channels (e.g. ClC-2,bestrophin, maxi-Cl� channel) appeared to be volume-activated as well, but display discernable biophysicalcharacteristics. Besides these mentioned candidates othershave been proposed, but none of these has yet receivedacceptance (Clapham, 1998; Jentsch et al., 2002; Pusch, 2004).ICl,swell may also be important for other processes includ-
ing regulation of membrane excitability, transcellular Cl�
transport, cell proliferation, release of nucleotides (e.g.ATP), and apoptosis. VRAC may not be a single entity,but may instead stand for several different ion channels(Lang et al., 1993, 1998b; Nilius et al., 1996).
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 279
4.3.4.2. Volume regulated anion channels in the
RPE. Epithelial cells involved in transepithelial trans-port need to balance their ion transport rates tomaintain their volume within certain limits. Further-more, RPE cells phagocytose photoreceptor outer segmentsat a high rate likely challenging cell volume and maybeleading to successive osmolarity changes, which must becompensated (Hartzell et al., 2005b; Nguyen-Legros andHicks, 2000; Strauss, 2005; Wettstein et al., 2000). Hypoos-motic swelling inhibits RPE phagocytosis and suggests thatcell volume regulation and phagocytic functions are linked(Irschick et al., 2006). The RPE is likely exposed to light-dependent osmotic challenge, as photoreceptor activity altersthe solute composition in the restrictive volume of thesubretinal space (Bialek and Miller, 1994; Borgula et al.,1989; Dmitriev et al., 1999; Huang and Karwoski, 1992; Li etal., 1994b; Oakley and Steinberg, 1982; Shirao et al., 1987).And it has been demonstrated that iso-osmotic changes inRPE cell volume affect the level of RPE-retina adhesivity(Marmor, 2005) and also alter the volume of the subretinalspace (Adorante, 1995; Adorante and Edelman, 1997;Adorante and Miller, 1990; Bialek and Miller, 1994).Moreover, transepithelial salt and water transport couldplace an additional osmotic load on the RPE cell.
Hypotonic swelling of RPE cells in vitro leads to a RVD(Fig. 5) due to parallel efflux of Cl� and K+ throughrespective ion channels (Adorante, 1995; Civan et al.,1994). Inhibition of RVD by both basal Ba2+ (blockingK+ channels) and elevated basal K+ strongly suggests theRVD is mediated by K+ efflux across the basolateralmembrane (Adorante, 1995; Hughes et al., 1988; Immeland Steinberg, 1986; Kennedy, 1994). The K+-induceddepolarization also reduced the driving force for conduc-tive Cl� efflux across the basolateral membrane. Therefore,cell swelling is most likely due to a decrease in the drivingforce for K+ and Cl� efflux across the basolateralmembrane during continued solute uptake by the electro-neutral Na+-K+-2Cl� and electrogenic Na+/HCO3
� co-transporters located at the apical membrane of the RPE(Hughes et al., 1988, 1989; la Cour, 1991a, b).
RPE cell shrinkage in vitro produces RVI likely reflectingthe work of ion antiporter under baseline conditions. Na+
-K+-2Cl� cotransporter activation likely increases [Cl�]i ashas been discussed for fluid transport and, therefore, mightagain be coupled to a Cl� efflux through ion channels.
In RPE cells, application of intracellular pressure ordecrease in extracellular osmolarity induced cell swellingand activation of outwardly rectifying currents. Thesecurrents showed time-dependent inactivation and wereinhibited by DIDS, SIDS. In addition, the currents weresensitive to the potential VRAC blockers niflumic acid andanthracene-9-carboxylic acid (Botchkin and Matthews,1993; Fischmeister and Hartzell, 2005; Ueda and Steinberg,1994). Cell swelling increased Cl� conductance by as muchas 5- to 13-fold. The mechanism of activation of theswelling activated Cl� conductance is not identified yet,and could consist of, or be modulated by, Ca2+-activated
channels. The latter hypothesis stems from the observationthat hyperosmotic shrinkage of RPE cells suppressed partof the Cl� current and that heterologous expression ofhBest1 induced Cl� currents are likewise profoundlyinhibited by that manoeuvre (Fischmeister and Hartzell,2005). Among other candidate volume sensitive channelsClC-3 (CLCN3), ICln (CLNS1A) and bestrophin (VMD2)is located in RPE (NEI EST search; http://neibank.nei.nih.gov/).
4.3.5. Other Cl� channels
There are further well-characterized putative anionchannels not mentioned in this review. Searching NCBIGeo database profiles (http://www.ncbi.nlm.nih.gov/geo/)(Barrett et al., 2005) of the genes expressed in the humanRPE determined through serial analysis of gene expression(SAGE) and both in the human and in the mouse RPE/choroid library of EST (http://neibank.nei.nih.gov/)yielded nonconfirmed data on expression of CLICs,GABAA receptors, MCLC and Tweety.GABA (g-aminobutyric acid) A and C receptors are
ligand-gated Cl� channels, whereas GABA B receptors aremetabotropic receptors (Chebib and Johnston, 1999;Hevers and Luddens, 1998; Jentsch et al., 2002). GABAA
and GABAC receptors are biochemically, pharmacologi-cally, and physiologically different. Currently, there are 16human GABAA receptor subunits (a1–6, b1–4, g1–4, d, e)and two human GABAC receptor subunits (r1 and r2) thathave been cloned. GABAA receptor subunits GABRa2,GABRa6, GABRb1, GABRg2, GABRd, GABRe andGABRp appeared in NCBI Geo database for native orcultured RPE cells. Using antibodies recognizing GABAA
receptor subunit b2 and b3 showed positive immuno-reactivity for human and rat RPE in cultured cells, anddistinctive bands at two different molecular masses inWestern blot data (Wood and Osborne, 1996). And,benzodiazepine binding sites on rat, monkey, and humanRPE cells support the notion of functional GABAA
receptors on RPE cells (Robbins and Ikeda, 1989; Zarbinand Anholt, 1991), but the details still need to beelucidated.The Cl� intracellular channel (CLIC1, 4, 5, 6), MID-1-
related Cl� channel (MCLC) and TTYH1–3, humanhomologues of the Drosophila melanogaster Tweety (tty)genes suggested to represent novel maxi-Cl� channelproteins (Suzuki et al., 2006) have not been investigatedin RPE cells.
5. Ca2+
5.1. Ca2+ and cell function in general
Ca2+ ions have a high affinity to proteins (Carafoli,2005a, b; Williams, 1974, 1994). The binding of Ca2+ toproteins results in changes of their conformation andsubsequent alteration of the protein function (Carafoli,2005a, b; Williams, 1974, 1994). With these characteristics,
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301280
the functional role of Ca2+ differs from that of many otherions. It can directly act as a modifier of protein functionand, thus, regulate cell function. In many different tissues,Ca2+ was found to act in the initiation or modulation ofcontraction, secretion, gene expression, migration, apop-tosis, necrosis, cell division, endocytosis, coagulation,excitation, energy metabolism, and signal transduction(Berridge, 2005; Carafoli, 2005a; Shuttleworth, 1997;Williams, 1974). Ca2+ controls these functions either bydirectly triggering protein interaction such as in excita-tion–contraction coupling or by acting as a second-messenger, activated in response to extracellular stimuli(Berridge et al., 2003; Berridge et al., 2000). Small changesin Ca2+ concentration can have profound changes in cellfunction. Normally, cells keep the concentration ofintracellular free Ca2+ very low (at a proximal 100 nM)and maintain a concentration gradient of 1:10,000 betweenintracellular space and extracellular environment, althoughtheir total intracellular Ca2+ content can be quite high(Berridge et al., 2000, 2003; Carafoli, 2005a; Saris andCarafoli, 2005). To keep the concentration of intracellularfree Ca2+ low, cells use energy to store Ca2+ in intra-cellular compartments such as mitochondria or intra-cellular Ca2+ stores of the endoplasmic or sarcoplasmicreticulum (Berridge et al., 2000, 2003; Carafoli, 2005a;Saris and Carafoli, 2005).
Ca2+-induced changes in cell function are triggered byan increase in intracellular free Ca2+. The specificity of thissignal is encoded by the complex pattern of each Ca2+
signal which follows a specific stimulus (Berridge, 2005;Berridge et al., 2000, 2003). These patterns differ in theamplitude, and the spatial- and time-dependent distribu-tion inside the cell. The need for a tight control of a lowintracellular Ca2+ concentration at rest and the generationof complex patterns of Ca2+ increases during stimulationrequires the presence of many different Ca2+ transportingproteins, such as Ca2+ conducting ion channels, Ca2+
cotransporters, and Ca2+pumps—as well as the presenceof Ca2+ binding proteins (Berridge, 2005; Berridge et al.,2000, 2003). Thus the specificity of Ca2+ signals arisesfrom recruitment of specific sets of Ca2+ transporting orCa2+ binding proteins (Berridge, 2005; Berridge et al.,2000, 2003; Carafoli, 2005a; Saris and Carafoli, 2005;Williams, 1994).
5.2. Ca2+ homeostasis in the RPE
RPE cells contain with 15mmol/l higher amounts ofCa2+ than other cells (Bellhorn and Lewis, 1976; Fishmanet al., 1977; Hess, 1975; Salceda and Riesgo-Escovar, 1990;Ulshafer et al., 1990). Large amounts of this Ca2+ arestored in melanosomes (Boulton, 1991; Boulton andDayhaw-Barker, 2001; Drager, 1985; Hess, 1975; Ulshaferet al., 1990). To handle large amounts of Ca2+ the RPEpossesses several Ca2+ transporting proteins. A Na+/Ca2+
exchanger eliminates Ca2+ from the intracellular space inexchange with 3 Na+ ions (Fijisawa et al., 1993; Loeffler
and Mangini, 1998; Mangini et al., 1997; Salceda, 1989). Inimmunohistochemical studies, the transporter could beidentified as the cardiac subtype of Na+/Ca2+ exchanger(Loeffler and Mangini, 1998), which is also found incardiac myocytes and eliminates Ca2+ from the intracel-lular space to initiate the rhythmic relaxation of themyocardium. This Na+/Ca2+ exchanger represents a veryefficient Ca2+ transport system to extrude Ca2+ from theintracellular space. Thus, the changes in intracellular freeCa2+ in RPE cells can have the same efficient dynamics asin cardiac myocytes. Due to its stoichiometry of 1 Ca2+
exchanged with 3 Na+, the Na+/Ca2+ exchanger is anelectrogenic transporter. Accordingly, elimination of Ca2+
from the intracellular space should lead to moderate celldepolarization. On the other hand, strong depolarizationof the cell can change the transport direction in the waythat the Na+/Ca2+ exchanger now transports Ca2+fromthe extracellular space into the cell. The extrusion of Ca2+
from the cytosol by the Na+/Ca2+ exchanger is supportedby the activity of the plasma membrane Ca2+-ATPasewhich hydrolyzes ATP for an active transport of Ca2+ outof the cell against its concentration gradient into theextracellular space (Kennedy and Mangini, 1996). Further-more, like many epithelia, RPE cells are connected to eachother via gap junctions, forming a functional syncytium(Gomes et al., 2003; Himpens et al., 1999; Himpens andVereecke, 2000; Pearson et al., 2004; Stalmans andHimpens, 1997). The cell-to-cell connection via gapjunction channels enables Ca2+ signals to spread betweenthe cells within the epithelial monolayer. Thus, increases inintracellular free Ca2+ can influence the function of theentire RPE.In the past, a large number of receptors, which stimulate
an increase in intracellular free Ca2+ as a second messengerhave been identified in the RPE. Most of these receptorsact via stimulation of G proteins or tyrosine kinase activityto initiate a formation and release of InsP3 into the cytosol.InsP3 activates a release of Ca2+ from InsP3 sensitivecytosolic Ca2+ stores (Ammar et al., 1998; Berridge, 2005;Berridge et al., 2000, 2003; Crook and Polansky, 1992;Feldman et al., 1991; Fragoso and Lopez-Colome, 1999;Karihaloo et al., 1997; Kuriyama et al., 1992; Mergler andStrauss, 2002; Nakashima et al., 1989; Strauss et al., 1996).This in turn can stimulate an increase in the membranepermeability for Ca2+ and an influx of extracellularCa2+ into the cell. The latter event results in a sustainedCa2+ increase and a long-lasting change in cell function(Berridge, 2005; Berridge et al., 2000, 2003). However,some receptors, ionotropic glutamate receptors or puriner-gic P2X receptors, function as ligand-gated ion channelsand directly increase intracellular free Ca2+ (Ryan et al.,1999). Other receptors such as purinergic P2Y receptorsincrease intracellular free Ca2+ by release of Ca2+ onlyfrom cytosolic Ca2+ stores without generating an influx ofCa2+ into the cell (Collison et al., 2005; Mitchell, 2001;Peterson et al., 1997; Poyer et al., 1996; Ryan et al., 1999;Sullivan et al., 1997). In general, a sustained increase in
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 281
intracellular free Ca2+ requires an influx of extra-cellular Ca2+ into the cell and, thus, the presence ofCa2+-conducting ion channels. Thus the understanding ofthe Ca2+-dependent regulation of RPE cell function wassubstantially improved with patch-clamp studies on RPEcells uncovering the expression of different Ca2+ channels.
5.3. Ca2+ and RPE function
As stated above, the RPE has many functions which areessential to maintain structural integrity and excitability ofphotoreceptors (Bok, 1993; Steinberg, 1985; Strauss, 2005).Many of these functions are regulated by changes inintracellular free Ca2+. These are:
�
Dark adaptation of photoreceptor activity: This occurs byCa2+-dependent mechanisms inside the photoreceptorouter segments (Korenbrot, 1995; Korenbrot andRebrik, 2002). The required Ca2+ enters the photo-receptor outer segments through cGMP-gated cationchannels, which are open in the dark and conduct Ca2+.The subretinal Ca2+ concentration in the dark is largerthan in the light (Gallemore et al., 1994). A Ca2+ sourcefor this increase in subretinal Ca2+ might be melano-somes of the RPE (Drager, 1985; Hess, 1975; Lavalleeet al., 2003; Moriya et al., 1996; Salceda and Sanchez-Chavez, 2000) which is suggested by the finding thatalbino rats show slower dark adaptation than normalpigmented rats (Behn et al., 2003). � Transepithelial transport of ions and water: Increases in
intracellular free Ca2+ achieved either using a Ca2+
ionophore (Joseph and Miller, 1992) or by the stimula-tion of a-adrenergic (Edelman and Miller, 1991; Josephand Miller, 1992; Nash and Osborne, 1995; Quinn andMiller, 1992; Quinn et al., 2001; Rymer et al., 2001) orpurinergic receptors (Maminishkis et al., 2002; Mitchell,2001; Peterson et al., 1997; Ryan et al., 1999; Sullivanet al., 1997) were found to stimulate Cl� and watertransport by RPE cells. The Ca2+-dependent stimula-tion of transepithelial Cl� transport occurs by threemechanisms. One is by modulation of the electricaldriving forces for ions to move across the cell mem-brane (Edelman et al., 1994b). This is achieved byCa2+-dependent opening of non-specific cation chan-nels, K+ or Cl� channels and subsequent changes in theresting potential (Bialek et al., 1996; Fischmeister andHartzell, 2005; Hu et al., 1996; Joseph and Miller, 1992;Miller and Edelman, 1990; Quinn and Miller, 1992;Ryan et al., 1999; Ryan and Kelly, 1998; Sheu and Wu,2003; Sheu et al., 2004; Strauss et al., 1996; Sun et al.,2002; Tao and Kelly, 1996). The second mechanism isopening of Ca2+-dependent Cl� channels in thebasolateral membrane (Hartzell and Qu, 2003; Quet al., 2004; Quinn et al., 2001; Strauss et al., 1996,1999; Ueda and Steinberg, 1994). The third mechanismoccurs via the activation of apically located K+
channels (see Section 3).
�
Phagocytosis: Studies on the regulation of phagocytosisimply that the stimulation of increases in intracellularfree Ca2+ has a regulatory function (Greenberger andBesharse, 1985; Hall et al., 1991; Heth and Marescalchi,1994; Nakashima et al., 1989; Nguyen-Legros andHicks, 2000; Strauss et al., 1998b). However, the roleof the rise in intracellular free Ca2+ is not clear. Since aparticipation of the InsP3 second messenger system wasfound to play a role in the initiation of phagocytosis(Heth and Marescalchi, 1994) an increase in intracellularfree Ca2+ should represent a starting signal forphagocytosis (Berridge et al., 2003). However, inanother study an increase in intracellular free Ca2+
and subsequent activation of protein kinase C (PKC)was described as a shut-off signal for phagocytosis (Hallet al., 1991). These contradictory observations may beexplained by different patterns of Ca2+ increasesunderlying these two different regulatory effects.
� Secretion: Since in many tissues exocytosis and secretion
are known to be triggered by an increase in intracellular-free Ca2+, it is likely that these processes are triggeredby Ca2+ in the RPE (Barg, 2003; Berridge et al., 2000;Mears, 2004; Satin, 2000; Shuttleworth, 1997). Stimula-tion by ATP or various growth factors is known toinduce secretion of growth factor by the RPE (Guillon-neau et al., 1997; Mitchell, 2001; Reigada and Mitchell,2005; Rosenthal et al., 2004, 2005; Slomiany andRosenzweig, 2004a, b). Since stimulation of the RPEby growth factors (Rosenthal et al., 2004, 2005) as wellas by ATP (Mitchell, 2001; Peterson et al., 1997; Ryanet al., 1999) have been described to result in an increasein intracellular free Ca2+ it is very likely that theseincreases in cytosolic Ca2+ not only represent a signal tochange gene expression (Rosenthal et al., 2005) but alsoto trigger the release of other growth factors (Rosenthalet al., 2004, 2005).
� Differentiation: In general, growth factor-dependent
changes in gene expression were described to arise fromincreases in intracellular free Ca2+. The underlyingchanges in the gene expression are achieved by Ca2+-activated phosphorylation of transcription factors (Fieldset al., 2005). Stimulation of the RPE by basic fibroblastgrowth factor (bFGF) leads to an increase in intracellularfree Ca2+ as well as to changes in the expression of theimmediate early gene c-fos (Rosenthal et al., 2005).
In summary, a lot of RPE functions to maintain visualfunction are controlled by increases in intracellular-freeCa2+. Thus, Ca2+ channels, which provide major routesfor an influx of extracellular Ca2+ into the cell to increaseintracellular-free Ca2+ must play an important role in thecontrol of these functions.
5.4. Ca2+ channels of the RPE
So far three types of Ca2+-conducting ion channels havebeen described in the RPE: voltage-dependent Ca2+
ARTICLE IN PRESS
-70 mV
+ 20 mV
Whole-cell currents
10 mM Ba2+
Nystatin
RPE
20 ms
0.5 nA
-1600
-800
-80 -60 -40 -20 20
control
BayK 8644 (5 µM)
Pipette potential (mV)
Membrane current (pA)
a
b
c
d
Fig. 6. Example of voltage-dependent Ba2+ currents in cultured human
RPE cells indicating the presence L-type Ca2+ channels. (a) Schematic
diagram illustrating the recording technique: the patch-pipette which is
attached to the RPE cell enables a voltage-clamp of the cell membrane
potential as well as measurements of membrane currents at different
potentials. The pipette contains a solution which mimicks the ion
composition of the intracellular space. Furthermore, the pipette solution
contains an ionophore (nystatin), which perforates the membrane patch
inside the inner diameter of the pipette. With this configuration,
membrane currents could be measured without disturbance of the
intracellular milieu, which is essential to measure stable L-type channel
currents. Pipette and bath solution are K+-free to prevent the measure-
ment of K+ currents superimposed to the Ca2+ channel currents. In
addition, the bath solution contains Ba2+ as a charge carrier for Ca2+
channel currents because with Ba2+ as a charge carrier Ca2+ channels
display larger currents. (b) Electrical stimulation to activate currents
through Ca2+ channels: from the holding potential of �70mV the cells
were depolarized with nine voltage steps of 50ms duration and +10mV
increment. (c) Ba2+ currents activated by the stimulation protocol shown
in 6B in a cultured human RPE cell. (d) Current/voltage plot of the
currents shown in Fig. 6C: the maximal current amplitudes were plotted
against the potentials of the stimulation protocol. Furthermore, the effect
of the L-type channel opener BayK8644 is shown in this diagram.
S. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301282
channels, members of transient receptor potential (TRP)channel family and ligand-gated Ca2+-conducting, non-selective cation channels.
5.4.1. Voltage-dependent Ca2+ channels
5.4.1.1. Voltage-dependent Ca2+ channels in general. Vol-tage-gated Ca2+ channels are highly specific Ca2+-conducting channels which are mainly activated by changesof the membrane potential toward more positive values(i.e., by depolarization) (Catterall, 1998, 2000; Catterall etal., 2005; Striessnig, 1999; Striessnig et al., 2004). Tenvoltage-dependent Ca2+ channels have been describeddiffering in their voltage-dependence, kinetic behaviour,and pharmacology. The 10 different channels share acommon molecular architecture. They are composed of ana1-subunit, a b-subunit, an a2d subunit, and in some cases ag subunit. The a1-subunit forms the ion-conducting poreand defines the pharmacological, electrophysiological, andkinetic characteristics of the channel. The 10 differentchannel types can be subdivided into the followingsubfamilies: L-type channels (CaV1.1–1.4 or a1S, C, Dand F), N, P/Q and R-type channels (CaV2.1–2.3 or a1A, Band E) and the T-type channels (CaV3.1–3.3 or a1G–I).The known four b-subunits modify channel activity,blocker sensitivity, and targeting of the a1-subunits to theplasma membrane. In addition, nine different g subunitsand 4 different a2d subunits have been identified. Membersof both subunit types may be involved in intracellulartargeting of the a1 subunits and influence the electro-physiological properties of the Ca2+ channels.
5.4.1.2. The voltage-dependent Ca2+ channels of the
RPE. So far only one subtype of voltage-dependentCa2+ channel has been characterized in detail in RPE cells(Fig. 6). Ca2+ channels in cultured or in freshly isolatedRPE cells from various species including man werecharacterized by patch-clamp recordings of whole-cellBa2+ or Ca2+ currents (Rosenthal and Strauss, 2002;Sakai and Saito, 1997; Strauss et al., 1997, 2000; StrauXand Wienrich, 1994; Ueda and Steinberg, 1993, 1995;Wollmann et al., 2006). The usage of Ba2+ as a chargecarrier is a commonly used tool to study L-type Ca2+
channels because these channels display larger Ba2+ thanCa2+ currents. RPE cells responded to depolarization topotentials more positive than �30mV with fast activatingand much more slowly inactivating inward currents (Fig. 6)(Mergler and Strauss, 2002; Rosenthal et al., 2006; Sakaiand Saito, 1997; Ueda and Steinberg, 1993, 1995;Wollmann et al., 2006). These currents are modulated bydihydropyridine derivatives. Dihydropyridines specificallymodulate the activity of L-type channels. For exampleBayK8644 stimulates L-type channel activity whereasnifedipine or nimodipine inhibit L-type channels. In theRPE, application of the Ca2+ channel activator BayK8644resulted in larger currents whereas the Ca2+ channelblocker nifedipine decreased the inward currents(Rosenthal et al., 2001, 2006; Rosenthal and Strauss,
2002; Sakai and Saito, 1997; Ueda and Steinberg, 1993;Wollmann et al., 2006). These properties are characteristicfor L-type channels and were observed in RPE cells inprimary culture, RPE cell lines, and in freshly isolated RPEcells. Despite the fact that between the different studiesL-type currents showed some differences in inactivation,
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 283
voltage-dependence, and dihydropyridine sensitivity, allstudies concluded that L-type channels represent the majortype of voltage-dependent Ca2+ channel in the RPE(Rosenthal et al., 2006; Ueda and Steinberg, 1993;Wollmann et al., 2006). Recently, it could be shown thatsystemic application of the L-type channel blockernimodipine led to a reduction of the light-peak amplitudein the rat direct-current electro-retinogram (DC-ERG)(Rosenthal et al., 2006). Thus, these data indicate thefunctional presence of L-type channels in the RPE of anintact eye. Using Western blot techniques, the L-type a1subunit was identified as the a1D or CaV1.3 subunit or theneuroendocrine subtype of L-type channels (Rosenthalet al., 2001, 2006; Strauss et al., 2000; Wollmann et al.,2006). In the rat RPE cell line RPE-J, and in culturedmouse RPE cells, this L-type channel appeared to be themajor subtype. The conclusion that CaV1.3 subunitsmainly form the L-type Ca2+ channel pore is supportedby the fact that rather high concentrations of dihydropyr-idine derivatives were required to decrease Ba2+ currents inRPE cells (Rosenthal et al., 2006; Strauss et al., 1997). Alow sensitivity to dihydropyridines has been reported to becharacteristic for CaV1.3 when heterologousely expressed(Koschak et al., 2001, 2003; Scholze et al., 2001). Finally,RPE cells mainly express a splicing variant of CaV1.3which has not been described thus far (Rosenthal et al.,2006). It might be that properties of this splicing variantcould explain the differences between L-type currents inRPE cells and those in other tissues or heterologeousexpression systems.
5.4.1.3. Role of voltage-dependent Ca2+ channels for RPE
function. The expression of CaV1.3 Ca2+ channel sub-units, which are also termed as the neuroendocrine subtype
Ca2+
- Secreti
- Phagoc
- Transe
- Dark ad
Melanoso
Ca2+ stores
Ca2+
Ca2+
TRPC1
CaV1.3
RPE
PLC
Gαq/11
TK
IP3
FGFR2bFGF
-Adrenaline
-Growth factors
- ATP
- AtCh
Fig. 7. Schematic diagram summarizing different Ca2+-conducting ion channe
are drawn in blue. The factors, which activate these ion channels are drawn in
summarises agonists stimulating these pathways). In light blue are membrane p
homoeostasis as well. Abbreviations are: ATP ¼ adenosine triphosphate; AtC
Ca2+ channel subunit 1.3; Gaq/11 ¼ G protein a-subunit q/11; FGFR2 ¼
NMDA ¼ N-methyl-D-aspartate; NCX-1 ¼ cardiac Na+/Ca2+ exchanger; P
of L-type channels imply that these voltage-dependentCa2+ channels play a role in the regulation of secretion.However, some questions have to be answered to concludethat this might also be their function in RPE cells (Fig. 7).The first question is whether high voltage-activated Ca2+
channels can play a functional role in retinal epithelial cellswith a resting membrane potential of �40 to �45mV.Investigation of CaV1.3-mediated currents in other cellsystems showed that they have the most negative activationthreshold and potential for half maximal activation amongall L-type channel subtypes (Koschak et al., 2001; Michnaet al., 2003; Scholze et al., 2001). Furthermore, in a studyusing patch-clamp techniques in combination with mea-surements of intracellular free Ca2+ it could be shown thatL-type channels contribute to an increase in intracellularfree Ca2+at membrane potentials fixed to the restingpotential of RPE cells of about �40mV (Mergler andStrauss, 2002). This effect is achieved by activation of theCa2+/InsP3 second-messenger system. Intracellular appli-cation of InsP3 led to a phosphorylation-dependent shift inthe voltage-dependence of L-type channels to morenegative values, more close to the resting potential ofRPE cells (Mergler and Strauss, 2002). This increased thenumber of open channels mediating a larger membraneconductance for Ca2+ at the resting membrane potential.The resulting influx of extracellular Ca2+ into the cell leadsto an increase in cytosolic free Ca2+. A comparablemechanism was found for the bFGF-dependent stimula-tion of RPE cells (Rosenthal et al., 2001). Application ofbFGF leads to an increase in intracellular free Ca2+ by anifedipine-sensitive influx of Ca2+ into the cell throughL-type Ca2+ channels (Mergler et al., 1998). This effectwas not mediated by activation of the Ca2+/InsP3 second-messenger system. Instead, the bFGF-dependent rise in
on
ytosis
pithelial transport
aptation
mes Mitochondria
Glutamate
ATP
Ca2+
Ca2+
NMDA receptor
P2X receptor
Ca2+
Ca2+Na+ PMCA
NCX1
ls and their impact on Ca2+ homoeostasis in RPE cells. The ion channels
red. Connected second-messenger pathways are drawn in yellow (the box
roteins, which are not ion channels but contribute to changes in the Ca2+
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301284
intracellular free Ca2+ results from the physical interaction(Fig. 7) between the bFGF receptor FGFR2 and theCaV1.3 subunits (Rosenthal et al., 2001). This interactionleads to tyrosine phosphorylation of the Ca2+ channel anda subsequent shift in the voltage-dependent activationtowards more negative potentials, again closer to theresting potential of RPE cells. Also the resting activity ofL-type channels in the RPE is dependent on interactionwith a tyrosine kinase. L-type channels are constantlyactivated by the cytosolic tyrosine kinase pp60c�src (Strausset al., 1997, 2000). This regulatory effect is based on aphysical interaction of the CaV1.3 subunits and pp60c�src
(Strauss et al., 2000). Among the serine/threonine kinases,regulation of PKC seems to have the most importantimpact on L-type channel activity in RPE cells. Based onthe application of various blockers it was concluded that itis unlikely that protein kinase A or G have any effects onL-type channel activity (Strauss et al., 1997). PKC has twoeffects on L-type channel activity (Strauss et al., 1997). Oneis the direct activation of this channel. With basic PKCactivity, pp60c�src and PKC have additive effects on theL-type channel activity. However, a second function ofPKC emerges when PKC is further activated. Withstimulated PKC, not only the L-type channel activityincreases but also the effect of pp60c�src changes. Underthese conditions pp60c�src becomes an inhibitor of L-typechannel activity (Strauss et al., 1997). The underlyingmechanism is not clear. It is possible that different isoformsof PKC are responsible for these two different effects ofL-type Ca2+ channel regulation in RPE cells.
In summary, L-type channels in the RPE contribute tochanges in intracellular free Ca2+ not by changes in themembrane potential but by shifts of their voltage-dependence which increases the number of open channelsat the resting potential of RPE cells. The next question is,in which signalling pathways are L-type channels involved?A major regulation by tyrosine kinase implies that L-typechannels in the RPE are involved in growth factor-dependent Ca2+ signalling because many growth factorsact via stimulation of tyrosine kinase-dependent pathways.As mentioned above, this hypothesis has been proven forbFGF (Rosenthal et al., 2001). Other growth factors suchas insuline-like growth factor-1 (IGF-1) are also very likelycandidates as an IGF-1-dependent regulation of L-typechannel activity has been shown in smooth muscle cells andthe RPE is known to express IGF-1 receptors (Bence-Hanulec et al., 2000; Rosenthal et al., 2004; Slomiany andRosenzweig, 2004a, b).
Another question is, what changes in cell function mightbe achieved by increases in intracellular-free Ca2+-mediated by activated L-type channels? One effect ofmany growth factors is their influence on cell differentia-tion or cell function by changes in the gene expressionprofile. These changes are regulated by increased expres-sion of transcription factors (Fig. 7). In RPE cells the directopening of L-type channels by the dihydropyridinecompound BayK8644 resulted in an increased transcrip-
tion rate c-fos too (Rosenthal et al., 2005). Increased c-fos
expression is also induced by bFGF. However, the bFGF-dependent expression of c-fos was independent from L-typechannel activity. Thus stimulation of L-type channels in theRPE can change the expression of immediate early geneswhich is a mechanism playing a role in the growth factor-dependent modulation of gene expression by other growthfactors than bFGF (Rosenthal et al., 2005).As stated above, the subtype of L-type channels in the
RPE is the neuroendocrine subtype. This implies a role ofL-type channels in the regulation of secretion. The RPE isknown to secrete a variety of growth factors. Furthermore,the secretion of growth factors by the RPE is regulated byother growth factors (Campochiaro et al., 1994; Guillon-neau et al., 1997; Hackett et al., 1997; Nagineni et al., 2003;Ohno-Matsui et al., 2003; Rosenthal et al., 2004; Slomianyand Rosenzweig, 2004a, b) which in some cases have beenshown to stimulate L-type channels via tyrosine kinase-dependent phosphorylation. Thus the prominent role ofL-type channels in RPE cells is most likely the regulation ofgrowth factor secretion. Indeed, bFGF-induced secretionof VEGF was reduced to the basic secretion rate observedin the absence of bFGF after inhibition of L-type channels(Rosenthal et al., 2005).As stated above, L-type channels seem to participate in
the generation of the light-peak in the DC-ERG(Rosenthal et al., 2006). The light-peak is a signal in thehuman electro-oculogram (EOG, equivalent to the DC-ERG)which is thought to derive from activation of Cl� channelsin the basolateral membrane of the RPE (Gallemore et al.,1988; Gallemore and Steinberg, 1989a, 1993). In a recentstudy, it was shown that bestrophin-1, proposed to act as aCa2+-dependent Cl� channel (Qu et al., 2004; Qu andHartzell, 2004; Sun et al., 2002), additionally regulatesL-type Ca2+ channels in the RPE (Rosenthal et al., 2006).Furthermore, mutant bestrophins showed different effectson L-type channel properties. The possible interaction ofthe L-type channel with bestrophin-1, in other wordsinteraction of a Ca2+ channel and a Ca2+-dependent Cl�
channel, would put the activation of the Ca2+-dependentCl� channel into a close feedback mechanism therebyinfluencing transepithelial Cl� and fluid transport.In summary, L-type channels can contribute to changes
in intracellular free Ca2+ at the resting potential of RPEcells. The L-type channel-dependent changes in cytosolicfree Ca2+ participate in the regulation of growth factorsecretion and likely in the regulation of epithelial transportof Cl� and water. An influence on gene expression enablesan adaptation of transcriptional activity to the underlyingfunctional changes. To achieve this regulatory functionL-type channels mainly underlie the control by PKCand tyrosine kinase activity.
5.4.2. TRP channels
5.4.2.1. TRP channels in general. TRP channels representa large family of Ca2+-conducting cation channels (Clap-ham et al., 2003; Inoue, 2005; Ramsey et al., 2005). The
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301 285
name TRP derived from the Drosophila homologue TRP.TRP channels are represented by six families withindividual members who can contribute to a large varietyof signalling pathways or sensory function. The family ofTRPC (classic) and of the TRPM (metastatin like)channels are involved in intracellular signalling cascadesdue to their activation by G protein subunits, metabolitesof second-messenger cascades, or by depletion of cytosolicCa2+ stores. TRPV (vanilloid receptor) and TRPA (withankryn repeats) channels play a role in sensory mechanismsbecause they can be activated by a variety of physicalstimuli such as temperature or pH. One family representschannels in intracellular organelles (TRPML) and thefunction of the channels in other families is still unknown.
5.4.2.2. TRP channels in the RPE and their role in RPE
function. Early patch-clamp studies on rat RPE cellsindicated a possible presence of TRP channels (Poyer et al.,1996). Poyer et al. (1996) described G protein-activatedcation channels, which were selective for many differentmonovalent cations. Thus, it could be that the authorsdescribed currents mediated by TRPC channels. However,the cation currents described in the study by Poyer et al.seemed not to be carried by Ca2+ and it seemed that Gai-subunits or at least pertussis toxin-sensitive G-proteins andnot Gaq/11 subunits were responsible for their activation.These are not characteristic properties for TRPC channels.Nevertheless, in a recent publication, the expression ofTRPC1 channels was detected in the human RPE cell lineARPE-19 (Bollimuntha et al., 2005). As mentioned above,TRPC channels are integrated into the InsP3/Ca2+ secondmessenger pathway (Fig. 7) either by their function asstore-operated Ca2+ channels (SOCs) or by their activationby Gaq/11 subunits (Clapham et al., 2003; Inoue, 2005;Ramsey et al., 2005). In a study with combined measure-ments of membrane currents and changes in intracellularfree Ca2+ it could be shown that together with L-typeCa2+ channels another type of Ca2+-conducting ionchannel contributes to InsP3-induced increases in intracel-lular free Ca2+ (Mergler and Strauss, 2002). This studyused a standard protocol for induction of capacitativeCa2+ influx as a standard system to explore activation ofstore-operated Ca2+ influx. Thereby, a component ofCa2+ conductance insensitive to blockers for L-typechannels was detected. It might be that TRPC channelsunderlie this additional InsP3-induced membrane conduc-tance for Ca2+. In the RPE, TRPC channels could play arole in a couple of different agonist-controlled Ca2+-dependent signalling pathways. The RPE expresses P2Y(Peterson et al., 1997; Reigada et al., 2005; Reigada andMitchell, 2005; Ryan et al., 1999; Sullivan et al., 1997),muscarinic (Feldman et al., 1991; Friedman et al., 1988;Gonzalez et al., 2004) and adrenergic receptors (Edelmanand Miller, 1991; Feldman et al., 1991; Joseph and Miller,1992; Quinn and Miller, 1992; Quinn et al., 2001; Rymeret al., 2001). These receptors are known to act viastimulation of Gaq/11 proteins (Wettschureck and Offer-
manns, 2005) and to stimulate an increase in intracellularfree Ca2+ as second messenger.Since TRPC channels can be activated by Gaq/11
proteins these ion channels might play a role in theregulation of RPE function which are modulated by thesereceptors: adrenergic or purinergic regulation of trans-epithelial ion transport and muscarinergic or purinergicregulation of phagocytosis. Although the involvement ofTRPC channels in these regulatory pathways is not proven,the presence of these channels might further help toidentify so far unknown membrane conductances forCa2+ which contribute to the underlying Ca2+ signalling.Another feature of TRPC1 channels in RPE cells isthat they are interacting with beta-tubulin filaments ofthe cytoskeleton (Bollimuntha et al., 2005). This interac-tion enables a regulation of temporal and spatial distribu-tion of TRPC1 channels in the cell membrane. Thedifferentially regulated presence in the cell membranemodulates the effectiveness of an agonist to influenceRPE cell function.
5.4.3. ATP receptors
5.4.3.1. ATP receptors in general. ATP can be released bydifferent cell types and can act as an autocrine or paracrineextracellular messenger (Burnstock, 2004; Burnstock andKnight, 2004). ATP can bind to two different receptortypes. These are the P2Y and P2X receptor families.Whereas the P2Y receptors are coupled to activation of Gproteins (mainly Gaq/11 or Gai/O subunits) (Burnstock,2004; Burnstock and Knight, 2004) and initiate anintracellular second messenger cascade, the P2X receptorsare ligand-activated ion channels (North, 2002). ATPbinding to P2X receptors results in the activation of acation channel, which can conduct Na+ as well as Ca2+.With this mechanism, ATP can directly contribute toincreases in intracellular free Ca2+ via stimulation of aninflux of extracellular Ca2+ into the cell. The P2Xreceptors represent a gene family of 7 members whichcan form homomeric or heteromeric ATP-activated ionchannels (North, 2002). Currents through P2X receptorchannels show fast inactivation and desensitization toATP. P2X receptors are expressed in a variety of tissuesand have been described to modulate synaptic currents,contractility, and secretion.
5.4.3.2. P2X receptors in the RPE and their role for RPE
function. So far the expression of P2X receptors in theRPE has not been shown by molecular biological orprotein biochemical methods. However, a study with ratRPE cells on ATP-dependent signalling revealed changes inthe membrane conductance which indicate the functionalpresence of P2X receptors (Ryan et al., 1999). Extracellularapplication of ATP resulted in the depolarization of RPEcells due to activation of cation channels (Fig. 7). Thiscation conductance shared some properties known of P2Xreceptors in heterologeous expression studies (North andSurprenant, 2000).
ARTICLE IN PRESSS. Wimmers et al. / Progress in Retinal and Eye Research 26 (2007) 263–301286
In the RPE, ATP-dependent signalling was found to playa role in the regulation of transepithelial ion and watertransport, and phagocytosis (Collison et al., 2005; Petersonet al., 1997; Quinn and Miller, 1992; Reigada et al., 2005;Reigada and Mitchell, 2005; Ryan et al., 1999; Sullivan etal., 1997). The main source for ATP seems to be the RPEitself which can release ATP in response to a variety ofstimuli such as bFGF or hypotonic challenge (Mitchell,2001; Reigada et al., 2005; Reigada and Mitchell, 2005).ATP that functions as an autocrine messenger can do so bybinding to different purinergic receptors such as P2Y, P2X,or adenosine receptors after degradation by ectonucleases(Mitchell, 2001; Reigada et al., 2005; Reigada andMitchell, 2005). P2Y receptor stimulation leads to anincreased transepithelial transport of Cl� and water(Peterson et al., 1997). The responsible receptor-mediatedsignalling promotes these effects in two ways. One is anincrease in intracellular free Ca2+ and the subsequentactivation of ion channels such as Ca2+-dependent Cl�
channels which directly provide a transportation pathwayfor ions (Peterson et al., 1997). So far, the ATP-inducedincreases in intracellular-free Ca2+ were found to arisemainly from release of Ca2+ from cytosolic Ca2+ storesindicating that these are mediated by activation of P2Yreceptors (Peterson et al., 1997; Ryan et al., 1999). Inaddition, it was found that ATP-dependent increases inintracellular free Ca2+ activate Ca2+-dependent K+
channels leading to hyperpolarization of the RPE cell(Ryan et al., 1999). This could be another mechanismby which ATP modulates transepithelial ion transport:modulation of the driving forces for ions to move acrossmembranes. In this case, the activation of K+ channelswould increase the driving force for Cl� to leave the cell.For the modulation of these driving forces the P2Y andP2X receptors can interact. This was found in a study inwhich the effects of ATP on the membrane conductancewere investigated (Ryan et al., 1999). Here, application ofATP first resulted in a cell depolarization, which wasfollowed by cell hyperpolarization. The first event wasfound to be dependent on the stimulation of P2X receptorswhereas the later event resulted from activation of Ca2+-dependent BK Ca2+ channels (see Section 3).
5.4.4. Glutamate receptors
5.4.4.1. Glutamate receptors in general. Like ATP, theneurotransmitter glutamate can bind to either ligand-gatedion channels, the so called ionotropic glutamate receptors(Kew and Kemp, 2005; Mayer, 2005), or to receptors whichactivate G-protein-coupled intracellular second-messengercascades, the metabotropic glutamate receptors (Conn,2003; Kew and Kemp, 2005).
5.4.4.2. Glutamate receptors in the RPE. In independentstudies, the functional expression of glutamate receptorshas been described in the RPE (Fig. 7). The RPE expressesboth metabotropic as well ionotropic glutamate receptors(Feldman et al., 1991; Fragoso and Lopez-Colome, 1999;
Lopez-Colome et al., 1993, 1994; Uchida et al., 1998).Stimulation of either metabotropic or ionotropic glutamatereceptors in the RPE result in an increase in the cytosolic-free Ca2+ (Feldman et al., 1991; Fragoso and Lopez-Colome, 1999). The stimulation of metabotropic receptorsleads to an increase in intracellular free Ca2+ viastimulation of a phospholipase C-dependent intracellularsecond messenger cascade. Interestingly, this increase wasreduced after application of the L-type channel blockernifedipine (Fragoso and Lopez-Colome, 1999). This mightbe enabled by the InsP3-dependent stimulation of L-typeCa2+ channels, which has been described above. However,glutamate-dependent increases in intracellular-free Ca2+
were also reduced in the presence of the ionotropic NMDAreceptor (N-methyl-D-aspartate receptor) antagonists. Thusglutamate also activates NMDA receptors in RPE cells.Because of the expression of two different receptorsubtypes of glutamate receptors, glutamate initiates acomplex pattern of intracellular Ca2+ signalling in RPEcells. However, the effects of this Ca2+ signalling for RPEfunction are not entirely clear. Recently, it could be shownthat stimulation of NMDA receptors in the RPE activatesthe release of ATP from RPE cells (Reigada et al., 2006). Inthe dark, photoreceptors release glutamate at their synapticterminals. It is likely that part of this glutamate reachesRPE cells and leads to an adaptation of the RPE functionto the requirements of photoreceptor function in the dark.One of these functions might be the regulation of thephagocytic activity of RPE cells which is controlled bychanges in the illumination of the retina (Greenberger andBesharse, 1985; Lopez-Colome et al., 1994).
6. Na+
In addition to its role as charge carrier in excitable cellswhere a Na+ influx is responsible for the depolarization ofthe plasma membrane, Na+ plays a central role in manytransport processes of diverse organic molecules like aminoacids, sugars, and neurotransmitters where the steep Na+
gradient is used for secondary active transport of thesemolecules. Therefore, the Na+ homeostasis of a cell is ofgreat importance for its normal function.Two main families of Na+ channels exist: the voltage-
gated Na+ channels (Nav1.1–Nav1.9) and the epithelialNa+ channels (ENaCa, b, g and d). These two familieshave very different functions. While voltage-gated channelsare found almost exclusively in excitable cells where theyare responsible for cell depolarization, ENaC are involvedin Na+ homeostasis in epithelial cells.
6.1. Na+ ions in RPE cell function
Over the apical membrane of the RPE cells a Na+
gradient is established by the Na+/K+-ATPase. Besidesthe contribution to the negative membrane potential, thisgradient is used for the transport of various molecules fromthe subretinal space into the cells. These molecules include
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K+ and Cl�, HCO3�, H+, lactate, GABA and taurine.
These transport mechanisms are involved in volumeregulation of the subretinal space, pH regulation, and theremoval of metabolic products and neurotransmitters. Forthese tasks, Na+ is permanently secreted by the RPE intothe subretinal space. While it is well known that Na+
leaves the cells through the apical membrane by the Na+/K+ ATPase activity, it is still a subject of speculation howthe Na+ enters the cells through the basolateral membrane.The steep chemical gradient between the extracellularspace and the cytosol provides ideal conditions forNa+-conducting ion channels to perform Na+ influxthrough the basolateral membrane of the RPE.
6.2. Na+ channels of the RPE
In cultured RPE cells from rat, newt, and humans,voltage-gated Na+ channels have been observe in patch-clamp investigations (Botchkin and Matthews, 1994; Sakaiand Saito, 1997; Wen et al., 1994). Nevertheless, it has alsobeen shown that these Na+ channels appear only in RPEcells when they are cultured for at least 24 h. Thus, thereshould be other Na+-conducting proteins in the basola-teral membrane. By immunohistochemistry and molecularbiological methods, different members of the other familyof Na+ channels (ENaC) have been detected in the RPE(Dyka et al., 2005; Golestaneh et al., 2002; Mirshahi et al.,1999). Though, the physiological data providing evidencefor their expression in RPE cells are still missing, theircontribution to the transepithelial transport seems verylikely since such a coordinated transport activity betweenthe Na+/K+ ATPase and ENaC localized in oppositemembranes in Na+ transport is well known from otherepithelial cell types e.g. in the collecting duct in the kidney.Because of the missing electrophysiological data we willnot go further into detail regarding the Na+ channels inthe RPE.
7. Ion channels and RPE disease
Ion channel research improved the understanding ofmany diseases in two ways.
On the one hand, analysis of ion channel functionresulted in a better understanding of the physiology of cellfunction in general (Jurkat-Rott and Lehmann-Horn,2004). This led also to new insights into the patho-physiology of diseases and, thus, into the development ofnew treatments.
Together with genetic analyses of genes coding for ionchannels the term of channelopathies emerged (Celesia,2001; Hubner and Jentsch, 2002; Jentsch et al., 2004;Lehmann-Horn and Jurkat-Rott, 1999; Striessnig et al.,2004). Channelopathies are disorders, which result frommalfunction of ion channels. These can be due to gain offunction, loss of function, or by dominant-negative effectsleading, for example, to a decreased number of channels inthe cell membrane.
7.1. Change or loss of ion channel function leading to RPE
diseases
Mainly changes in Cl� channel function were found tolead to degeneration of the retina.Although not a channelopathy, the inactivation of the
ClC-2 channel gene in mouse results in a phenotype verysimilar to the clinical picture of retinitis pigmentosa in man(Bosl et al., 2001). A comparable cause for retinitispigmentosa was not found in the human disease, importantaspects of RPE function can be discussed from this mousemodel. Analysis of transepithelial transport properties ofthe ClC-2 knock-out mouse revealed that the RPE in thesemice show no transepithelial potential (Bosl et al., 2001)indicating the absence of transepithelial transport of Cl�
and water. The first conclusion, which can be drawn fromthis is that ClC-2 channels might provide the mostimportant efflux pathway for Cl� across the basolateralmembrane according to the model of transepithelialtransport which has been described in the chapters above.The second conclusion, which can be drawn from theobservations in the ClC-2 �/� mouse is the importance ofthe transepithelial transport of water and Cl� from thesubretinal space to the blood side for the maintenance ofphotoreceptor function and survival. The absence of thetransport leads to the degeneration of photoreceptors.More important for a human disease are the effects of
mutations in the gene coding for bestrophin-1, the VMD2
gene (Marquardt et al., 1998; Petrukhin et al., 1998).Mutations in the VMD2 gene cause an inherited form ofmacular degeneration with juvenile onset, Best’s vitelliformmacular dystrophy (Best, 1905; Cross and Bard, 1974;Godel et al., 1986; Weingeist et al., 1982). It is discussedthat the mutations in the VMD2 gene lead to a changedbasolateral membrane conductance for Cl� to explain animportant symptom for the diagnosis of Best’s disease.Thus Best’s macular dystrophy might be understood as achannelopathy. This diagnostic symptom is the reductionof the light-rise or light-peak in the patient’s electro-oculogram of the Best patients (Cross and Bard, 1974). Ananalysis of the chick DC-ERG revealed that an increase inthe basolateral membrane conductance for Cl� withsubsequent depolarization of the basolateral membranerepresents the underlying mechanism contributing to thelight-rise (Gallemore et al., 1988, 2004; Gallemore andSteinberg, 1989b). It is believed that a light-peak substanceis released from the retina after illumination (Gallemoreet al., 1988, 2004; Gallemore and Steinberg, 1989b). Thissubstance diffuses to the RPE, binds to a receptor atthe apical membrane, and initiates an intracellular secondmessenger cascade, which in turn most likely results inthe activation of a Ca2+-dependent Cl� channel in thebasolateral membrane of the RPE. Thus, mutations in theVMD2 gene lead to a decrease in the basolateral membraneconductance for Cl� in the RPE. Following this lineof reasoning it became a very attractive hypothesisthat the VMD2 gene product itself might function as
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a Ca2+-dependent Cl� channel and that the mutations leadto a loss in the Cl� channel function. Indeed, the VMD2
gene product bestrophin-1 appears to function asCa2+-dependent Cl� channel (see Section 4) (Sun et al.,2002). Furthermore, 15 different mutant bestrophins wereinvestigated so far and it seems that all mutations lead to aloss in Cl� channel function with a dominant negativeeffect on the wild-type allele. However, the hypothesis thata loss of the Cl� channel function of bestrophin causes theleading symptom and the retinal degeneration in Best’sdisease is not supported by recent studies of the clinicalpicture of patients with VMD2 mutations and by a studyinvestigating light-peak increases in bestrophin-1 �/�mice. Patients carrying mutations in the VMD2 gene canhave a macular degeneration but normal electro-oculo-grams (Kramer et al., 2000; Pollack et al., 2005; Renneret al., 2005; Wabbels et al., 2004, 2006). Or in other casesthe changes in the patient’s light-peak appeared secondaryto the onset of the macular degeneration (Wabbels et al.,2004). Thus not all mutations, which lead to maculardegeneration seem to result in a loss of Cl� channelfunction. This impression is supported by an investigationof a bestrophin-1 knockout mouse model. In these mice,the absence of bestrophin-1 did not reduce the light-peakamplitude in the DC-ERG (Marmorstein et al., 2006).Furthermore, at low light-intensities, the bestrophin-1 �/�mice showed increased light-peak amplitudes compared tothe wild-type mice. Thus, either the lack of the Cl� channelfunction in the RPE could be compensated for by anotherCl� channel, which could also be the case in the humanRPE and would have comparable changes in the humanelectro-oculogram. Or bestrophin-1 might have additionalfunctions to the Cl� channel function involved in genera-tion of the light peak and in the aetiology of the humanmacular degeneration, if mutations have changed thisfunction. This additional function might be an influence ofbestrophin-1 on the Ca2+ homeostasis of the RPE as it hasbeen discussed in the chapter about Ca2+ channels of theRPE (Rosenthal et al., 2006).
Another disease which might have an impact on RPEfunction is cystic fibrosis (Blaug et al., 2003). The geneproduct of the disease-causing gene, CFTR, is alsoexpressed in the RPE (Blaug et al., 2003; Reigada andMitchell, 2005; Weng et al., 2002; Wills et al., 2000). CFTRis known to function as Cl� channel (Jentsch et al., 2002).Although cystic fibrosis patients show a decreased signal inthe electro-oculogram which is also discussed to beassociated with an activation of Cl� channels in the RPE(Blaug et al., 2003), the loss of CFTR Cl� channel functionin patients with cystic fibrosis seems not to have anydegenerative effects on the macula (Blaug et al., 2003).
In summary, loss of ion channel function in the RPE canhave severe effects on the retina and lead to retinaldegeneration. Such a mechanism is discussed to be thecause for an inherited form of macular degeneration, Bestvitelliform macular dystrophy. However, such a mechan-ism has not to been proven to be the case in Best disease.
7.2. Involvement of ion channels in signalling cascades
leading to degenerative processes in the retina
Normal ion channel function in the RPE can play a rolein the aetiology of degenerative diseases of the retina. If anion channel has an important contribution to a signalpathway leading to the initiation of processes which markimportant steps in a chain of events leading to pathologicchanges in cell function then this ion channel may play akey role in the aetiology of a disease. This may be the casefor L-type Ca2+ channels. As mentioned above, the RPE isable to secrete a variety of growth factors in health and indisease. Several lines of evidence indicate that growthfactors secreted by the RPE promote the development ofchoroidal neovascularization in age-related macular degen-eration (Amin et al., 1994; Frank, 1997; Holz et al., 2004;Lambooij et al., 2003; Oh et al., 1999; Rosenthal et al.,2004), the most common cause for blindness in industrial-ized countries. In this disease the complication of choroidalneovascularization accounts for the major component ofvision loss (Ambati et al., 2003). In the chain of eventswhich lead to choroidal neovascularization the RPE seemto play a central role by its secretion of the majorangiogenic factor, VEGF (Amin et al., 1994; Frank,1997; Krzystolik et al., 2002; Lambooij et al., 2003; Lopezet al., 1996; Rosenthal et al., 2004). The secretion of VEGFby the RPE is under control of other growth factors whichmight be secreted by photoreceptors or by the RPE itself(Frank, 1997; Kondo et al., 2003; Mousa et al., 1999;Nagineni et al., 2003; Rosenthal et al., 2004, 2005;Slomiany and Rosenzweig, 2004a, b; Witmer et al., 2003).Thus, the secretion of growth factors by the RPE hasseveral effects in the initiation of choroidal neovasculariza-tion. These are autocrine and paracrine stimulation. Asmentioned above, the L-type Ca2+ channels in the RPEregulate secretion of VEGF. Due to regulation of L-typechannels by other growth factors or cytosolic tyrosinekinase, which are stimulated by other growth factors,L-type Ca2+ channels are also able to fulfil the autocrinestimulation pathway of VEGF secretion. Thus, with thisimportant function, L-type channels might play a key rolein the initiation of choroidal neovascularization in age-related macular degeneration and could be a target for thedevelopment of strategies to prevent this severe complica-tion (Strauss et al., 2003). This hypothesis is supported bythe observation that freshly isolated RPE cells frompatients with age-related macular degeneration and chor-oidal neovascularization still show Ba2+ currents withproperties of L-type Ca2+ channels (Strauss et al., 2003). Asensitive reaction of L-type channels to degenerativeprocesses in the retina was detected in studies of L-typechannel function in RPE cells of an animal model forhuman retinitis pigmentosa, the Royal College of Surgeons(RCS) rat. Here, the L-type Ca2+ channels showed anincreased activity due to a changed regulation (Mergleret al., 1998). Although the consequences of these effectshave not been further explored these observations show
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that L-type channel dependent Ca2+-signalling is sensi-tively adapted to the changed patho-physiologic situationin the degenerative process of the retina.
8. Summary
The patch-clamp technique provides a strong tool in theidentification and characterization of ion fluxes acrossmembranes. In the RPE it has been used to support thedata previously obtained by other techniques like molecularbiological, protein biochemical, and other electrophysio-logical studies with Ussing chambers. The patch-clampstudies not only confirmed these data but also extended ourknowledge on ionic currents across the RPE membranes.This in turn led us to the development of new ideas of howion channels may be involved in RPE cell functions.
So, it could be confirmed that a K+ conductancethrough inwardly rectifying K+ channels contributes tothe K+ homoeostasis in the subretinal space. Consideringthe additional expression of voltage-gated and Ca2+-activated K+ channels, the picture of K+ transport bythe RPE could be extended.
Comparably, the proposed Cl� conductance in thebasolateral membrane of RPE cells was confirmed bypatch-clamp studies. This technique proves to be importantfor the identification of the function of a macular diseaseassociated protein, bestrophin. Based on patch-clamp data,bestrophin is now thought to be a Cl� channel and/or amodulator of voltage-gated Ca2+ channels. These Ca2+
channels have been shown by the patch-clamp technique tobe involved in RPE cell functions like the secretion ofgrowth factors and in transcellular transport. However, theinvestigation of ion channels in the RPE is not finished anda closer investigation of bestrophin function, glutamatereceptors, P2X receptors, and TRPC channels will furtherunderstanding and open new aspects to the role of the RPEin visual function.
Acknowledgements
The authors thank Tyson Kinnick for the help, forscientific input and stimulating fruitful discussions. Thework was supported by the Deutsche Forschnugsge-meinschaft grant DFG STR480/8-2 and DFG STR480/9-1.
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