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Cell Calcium
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ypotonicity-induced TRPV4 function in renal collecting duct cells: modulationy progressive cross-talk with Ca2+-activated K+ channels
in Jin1, Jonathan Berrout1, Ling Chen, Roger G. O’Neil ∗
ept. of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston, TX 77030, USA
r t i c l e i n f o
rticle history:eceived 5 August 2011eceived in revised form1 November 2011ccepted 29 November 2011vailable online 26 December 2011
eywords:RPV4 channel
a b s t r a c t
The mouse cortical collecting duct (CCD) M-1 cells were grown to confluency on coverslips to assess theinteraction between TRPV4 and Ca2+-activated K+ channels. Immunocytochemistry demonstrated strongexpression of TRPV4, along with the CCD marker, aquaporin-2, and the Ca2+-activated K+ channels, thesmall conductance SK3 (KCa2.3) channel and large conductance BK� channel (KCa1.1). TRPV4 overexpres-sion studies demonstrated little physical dependency of the K+ channels on TRPV4. However, activation ofTRPV4 by hypotonic swelling (or GSK1016790A, a selective agonist) or inhibition by the selective antago-nist, HC-067047, demonstrated a strong dependency of SK3 and BK-� activation on TRPV4-mediated Ca2+
influx. Selective inhibition of BK-� channel (Iberiotoxin) or SK3 channel (apamin), thereby depolarizing2+
the cells, further revealed a significant dependency of TRPV4-mediated Ca influx on activation of bothK+ channels. It is concluded that a synergistic cross-talk exists between the TRPV4 channel and SK3 andBK-� channels to provide a tight functional regulation between the channel groups. This cross-talk maybe progressive in nature where the initial TRPV4-mediated Ca2+ influx would first activate the highlyCa2+-sensitive SK3 channel which, in turn, would lead to enhanced Ca2+ influx and activation of the less
2+ l.
Ca -sensitive BK channe
. Introduction
TRP channels are a ubiquitous superfamily of cationic channelshat, with a few exceptions, are calcium-permeable and gated by
diverse range of stimuli. TRPV4 is a particularly notable exam-le as it has been shown to be modulated not only by mechanicaltimuli, including shear stress and hypotonic cell swelling, but alsoy polyunsaturated fatty acids, some phorbol esters, and mod-rate heat [1–4]. In the renal collecting duct TRPV4 is stronglyxpressed and appears to play a role in mechanical control of Ca2+
ignaling dynamics [5,6]. However, once activated, many factorsan come into play in modulating the activity of the TRP channels,ncluding TRPV4, from phosphorylation status [7–9] to membranerafficking [13–15]. Indeed, alterations in membrane trafficking arenown to strongly influence TRPV4 activity [15] where we haveecently shown that in overexpression systems alterations in mem-
rane trafficking from the plasma membrane appears to underlie
major component of TRPV4 desensitization following activation13]. Since TRPV4 is known to associate with the actin cytoskeleton
∗ Corresponding author at: Department of Integrative Biology and Pharmacology,niversity of Texas Health Science Center, 6431 Fannin St., Rm MSB 4.132, USA.el.: +1 713 500 6316; fax: +1 713 500 7444.
E-mail address: [email protected] (R.G. O’Neil).1 These authors contributed equally to the study.
[16], it may be that insertion and retrieval cycles of TRPV4 fromthe plasma membrane may be a central component modulatingTRPV4-mediated intracellular Ca2+, [Ca2+]i, dynamics in collectingduct cells.
The TRPV4 channel is now known to be expressed in renal col-lecting duct cells where it appears to function as a flow sensor[6,9,17]. We have previously shown expression of TRPV4 in mouserenal collecting duct cells [6] and, most recently, that TRPV4 ismost strongly expressed in the aquaporin-2 positive cells (prin-cipal cells) of the cortical collecting duct (CCD) [5]. This segment ofthe collecting duct system is an important site of flow-sensitive K+
secretion where the Ca2+-dependent maxi-K channel, BK channel,appears to underlie the K+ secretion in a Ca2+-dependent manner[18–20]. Whether other Ca2+-dependent K+ channels participate inthis phenomenon is not known although our current study showsexpression of the highly Ca2+-sensitive SK3 channel in the collect-ing duct cell line, M-1 cells. Regardless, a dynamic interplay mayexist between the TRPV4 and Ca2+-dependent K+ channels in renalcollecting duct cells where Ca2+ influx leads to activation of the K+
channel which, in turn, would hyperpolarize the cell membrane andincrease the driving force for Ca2+ influx. Hence, the TRPV4 chan-nel may display a synergistic cross-talk with the calcium-activated
K+ channel to control calcium influx, membrane potential, and K+
secretion.Recent studies have now shown that some TRP channels may
associate with Ca2+-dependent K+ channels and tightly control the
+ channel activity [21,22]. Indeed, it has been shown for TRPC1hat it may associate with the Ca2+-dependent BK channel in vas-ular smooth muscle cells to control membrane potential [23] while
similar association of TRPA1 with small and intermediate Ca2+-ctivated K+ channels in vascular endothelial cells may serve aimilar function [24]. Other studies have shown that TRPV4 maylso play a key role in modulating Ca2+-activated K+ channels, par-icularly the BK channel, in a variety of cell types including vascularmooth muscle cells, endothelial cells and epithelial cells [25–30].n most studies this interaction between TRPV4 and BK (or other+ channels) appear to be an indirect, but functional, interplay.egardless, TRPV4 and other TRP channels may be important modu-
ators of Ca2+-dependent K+ channels which, in turn, may modulatea2+ influx via the TRP channels.
The purpose of the present study was to elucidate the underlyingotential pathways controlling TRPV4-mediated [Ca2+]i dynam-
cs following hypotonic-induced stimulation of TRPV4 in mouseenal collecting duct cells, the M-1 cells. We demonstrated thathese cells express TRPV4 and the aquaporin-2 water channel (CCD
arker) along with two Ca2+-dependent K+ channels, the SK3 andK�. Somewhat surprisingly, hypotonic stimulation was not asso-iated with alterations in plasma membrane expression of TRPV4espite the biphasic nature of [Ca2+]i changes. Further, TRPV4 didot appear to be closely associated with BK or SK3 expression. How-ver, a dynamic functional cross-talk between TRPV4 and the K+
hannels was demonstrated with the K+ channel activation gen-rating a maximal stimulation of TRPV4-mediated Ca2+ influx. Asuch, these studies may have direct implications relating to theechanisms of modulation of Ca2+ signaling and flow-dependent
+ secretion in the CCD.
. Materials and methods
.1. Cell culture and transfection
Mouse kidney M-1 (cortical collecting duct) cells from Ameri-an Type Culture Collection were grown in standard DMEM/Ham’s12 media with growth supplements (Sigma, D8437) and 10%BS at 37 ◦C, pH 7.4 [9]. Cells were seeded onto coverslips andrown to confluency at 37 ◦C [6]. In some studies a recom-inant mouse TRPV4 construct, tagged with the fluorophoreVenus at the C-terminus, was used to transiently transfect-1 cells (TRPV4-mVenus) using the Effectene Kit (Qiagene)
s before [13]. Transfected cells were used within 24–48 h ofransfection.
.2. Immunoblotting and immunocytochemistry
M-1 cells were grown on 10 cm tissue culture plates, rinsed withce-cold PBS, harvested (scraped), lysed and subjected to separa-ion by sodium dodecyl sulfate-polyacrylamide gel electrophoresisSDS-PAGE) as previously described [6,13]. Whereupon, the sepa-ated protein were transferred from the gels to PVDF membranesnd immunoblotted. Primary antibodies used for Western blottingncluded antibodies against TRPV4 (anti-TRPV4 at 1:500 dilution,lomone Labs), BK-� (anti-BK-� at 1:200 dilution, Alomone Labs),nd SK3 (anti-SK3 at 1:200 dilution, Alomone Labs). Binding of pri-ary antibodies was detected with secondary antibody conjugated
o HRP using an ECL detection reagent (Amersham).In initial studies of TRPV4 expression and trafficking to/from
he plasma membrane, a highly enriched plasma membrane frac-
ion from M-1 cells was used for immunoblotting. The enrichedlasma membrane fraction of M-1 cells grown on 10 cm tissue cul-ure plates was achieved with a sucrose gradient-based membranerotein extraction kit (Biovision, Inc.). An intermediate preparation
51 (2012) 131– 139
of a whole cell lysate was applied to the sucrose gradient, subjectedto centrifugation, and the resultant pellet (plasma membrane frac-tion) solubilized in a modified RIPA buffer (0.1% Na deoxycholate,0.01% SDS, 1% NP-40, and 20 mM Mg acetate in PBS) containingprotease inhibitors (Sigma–Aldrich). The BCA assay (Thermo Sci-entific) was used to measure protein concentrations. Membranealiquots were subjected to SDS-PAGE separation and detected onPVDF membranes. Lack of contamination by cytosolic components(anti-�-tubulin, Sigma–Aldrich) was verified for each preparation.TRPV4 band intensities were quantified with Image J (version1.42q, NIH) for differing treatments. TRPV4 channel intensitieswere normalized to the plasma membrane marker, pan cadherin(anti-pan cadherin, AbCam), as a loading control. All experimentswere repeated 3–4 times.
A Nikon A1 confocal microscope was used for immunoflu-orescent imaging to assess cellular localization of the channelproteins as described before [6,13,31]. Cells on coverslips werewashed, fixed in 4% paraformaldehyde (RT, 20 min), and stained,as above, with primary antibodies against TRPV4, BK-�, SK3,along with aquaporin-2 (anti-AQP-2 tagged with ATTO 550,1:200 dilution, Alomone Labs) and plasma membrane markerpan cadherin (anti-pan cadherin, 1:125 dilution, Sigma–Aldrich).Cells were subsequently stained with secondary antibody labeledwith FITC (anti-AQP-2-ATTO-550 directly labeled), mounted withVectraShield mounting media (Vector Laboratories) and imagedwith a Nikon A1 confocal microscope. In some studies phal-loidin (Alex Fluor 647 phalloidin) was used for staining ofF-actin.
2.3. Measurement of intracellular calcium
The fura 2 fluorescence ratiometric method was used to mea-sure intracellular calcium levels, [Ca2+]i, in M-1 cells grown toconfluency on coverslips as done extensively before [6,9]. Cells oncoverslips were loaded with fura-2/AM (2–10 �M) for 45–60 min atroom temperature, washed, attached to the bottom of a perfusionchamber and imaged with InCa Imaging Workstation (IntracellularImaging, Inc.) at 37 ◦C. Typically 10–30 cells (ROIs) were simulta-neously monitored on a coverslip and the results averaged for eachexperiment. Intracellular calcium was estimated from the fura-2fluorescence by excitation at 340 nm and 380 nm and calculatingthe ratio of the emission intensities at 511 nm in the usual man-ner every 1–3 s. Results are presented as the emission ratios anddenoted F340/F380. In some studies, cells were subjected to intra-cellular fura 2 calibration and the ratios converted to intracellularcalcium activity, [Ca2+]i, as described by Grynkiewicz et al. [32]using methods previously outlined [6,9].
2.4. Chemicals and solutions
M-1 cells were typically studied in a control isotonic (Ctl) mediacontaining (in mM): 145 NaCl, 5.4 KCl, 0.5 MgCl2, 0.4 MgSO4, 3.3NaHCO3, 2.0 CaCl2, 10 HEPES, 5.5 glucose, pH 7.4, having an osmo-larity of 310 mOsm/L (Ctl or ISO media). The hypotonic media(HYPO) was identical to the Ctl media except 45 mM NaCl wasremoved to provide an osmolality of 220 mOsm/L. In some stud-ies, the Ca2+ was removed from the standard solutions and 0.5 mMEGTA added, pH 7.4, to chelate any residual Ca2+ (0 Ca2+) as typicallydone [6].
The following drugs and chemicals were used in the study:GSK1016790A (GSK101, Santa Cruz Biotechnology; stock solution,10 �M in DMSO), HC-067047 (Tocris Bioscience; stock solution
100 �M in DMSO), iberiotoxin (IbTX, Alomone Labs: 50 mM stockin PBS), apamin (Alomone Labs; 100 mM stock in PBS), andEGTA, ethyleneglycol-bis (�-aminoethyl ether)-N,N,N′,N′-tetra-acetic acid (Sigma Chemical).
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Data are presented as mean values ± SEM. Student’s t-test orNOVA were used to test for statistical significance, as appropriate,ith P < 0.05 considered significant.
. Results
.1. Expression of TRPV4 in aquaporin-2-positive M-1 cells
We have recently shown that TRPV4 is expressed in theouse cortical collecting duct [5,6] with strong expression in the
quaporin-2 (AQP2)-expressing cells, indicative of the principalell (PC). We now show similar staining patterns in M-1 cellsFig. 1A). Immunostaining of confluent M-1 monolayers showstrong expression of both AQP-2 and TRPV4 in M-1 cells with signif-cant co-localization at, or near, the plasma membrane (Fig. 1A, topanel, merged). These results demonstrate CCD PC-like propertiesf the M-1 cells.
TRPV4 can also associate with the actin cytoskeleton whichay promote trafficking between the cytosol and plasma mem-
rane [16]. Staining of M-1 cells for �-tubulin (microtubules) and-actin shows a highly organized actin cortical band around theeriphery of the cells, as evidenced by the phalloidin staining pat-erns (Fig. 1B), typical of epithelial cells [33,34]. In contrast, the
icrotubule staining was not highly organized in these cells. TRPV4xpression and the actin cortical band demonstrate significant co-ocalization indicative for a membrane protein with associations toctin and membrane trafficking.
.2. Activation of TRPV4 by hypotonic swelling (HYPO) withinimal TRPV4 trafficking
It is well documented that TRPV4 is a mechanically sensi-
ive channel that can be activated by hypotonic cell swelling and
echanical stresses [see 35]. We have previously demonstratedhat such mechanical stress can rapidly lead to activation of TRPV4n TRPV4-transfected HeLa or HEK cells and in M-1 cells [6,9]. In
ig. 1. Immunofluorescent imaging and immunostaining of M-1 cells. (A) Immunofluores, red). Merged images (right panel) show significant co-localization of TRPV4 and AQar = 20 �m. (B) Immunofluorescent staining of M-1 cells for TRPV4 (anti-TRPV4, green),hows well-organized actin cortical bands near the periphery of the cell while �-tubuleear the cell membrane as apparent in the merged image. Scale bar = 20 �m.
51 (2012) 131– 139 133
the current study we took advantage of this mode of TRPV4 activa-tion to evaluate the affect of channel activation on [Ca2+]i dynamicsand membrane trafficking. As shown in Fig. 2A, exposure of M-1 cells to hypotonic media (reducing the osmolality from 310 to220 mOsm/kg) lead to an influx of Ca2+ with biphasic changes in[Ca2+]i [6]. Typically [Ca2+]i reached a peak increase within 1–3 minof stimulation followed by relaxation to a pseudo-plateau levelover 5–10 min. This response is primarily related to hypotonicity-induced activation of TRPV4 as application of the selective TRPV4antagonist, HC-067047 [36], reduced the HYPO-induced changes in[Ca2+]i in a dose-dependent fashion (Fig. 2A and C, pre-incubatedwith HC-067047 for 5 min). Addition of 100 nM HC-067047, adose sufficient to completely abolish GSK101-induced activationof TRPV4 (see Fig. 4D), reduced the peak HYPO-induced [Ca2+]ichange to 17 ± 4% of control HYPO responses observed in theabsence of the blocker (Fig. 2C). Similarly, upon removal of extra-cellular Ca2+ (0 Ca2+), the HYPO-induced increase in [Ca2+]i wasreduced to 20 ± 5% of control values, demonstrating that most ofthe increase in [Ca2+]i in the presence of extracellular Ca2+ wasdue to Ca2+ influx. In addition, the small increase in [Ca2+]i under0 Ca2+ conditions, as for the case with 100 nM HC-067047, likelyreflects release of Ca2+ from internal stores upon HYPO stimulation(also see [6]).
The reason for this transient nature of the TRPV4-mediatedresponse to HYPO is not known. We have shown that TRPV4 activa-tion in TRPV4-transfected HeLa cells leads to a rapid desensitizationof the TRPV4 currents that appears to largely reflect retrieval ofTRPV4 from the plasma membrane [13]. However, in the M-1 cellswhich endogenously express TRPV4 this does not appear to bethe case. As shown in Fig. 3, expression of TRPV4 in the post-nuclear supernatant (PNS, cell lysate) or in a highly purified plasmamembrane fraction (PM) did not significantly change followingHYPO exposure (normalized to pan-cadherin, a plasma membrane
marker). A summary of the TRPV4 band intensities (normalized torelative intensity of 1 at 0′) did not significantly change for up to30 min of HYPO exposure (Fig. 3B) demonstrating a fundamentaldifference from that observed in heterologous expression systems
cent staining of M-1 cells for TRPV4 (anti-TRPV4, green) and AQP-2 (anti-aquaporinP-2 proteins within the cytoplasm and at, or near, the plasma membrane. Scale
�-tubulin (anti-alpha tubulin, red), and F-actin (phalloidin, blue). F-actin staining shows little defined organization. Co-localization of TRPV4 with actin is apparent
134 M. Jin et al. / Cell Calcium 51 (2012) 131– 139
Fig. 2. Effect of HYPO and the TRPV4 antagonist, HC-067047, on M-1 [Ca2+]i levels. (A) [Ca2+]i response to HYPO (220 mOsm) in the presence and absence of HC-067047(100 nM) and extracellular Ca2+ (0 Ca2+). In the absence of blocker (A), HYPO induces a biphasic time course with a peak increase in [Ca2+]i within 1–3 min followed byrelaxation to pseudoplateau levels. Under 0 Ca2+ conditions, the increase in [Ca2+]i is markedly reduced except for a small transient increase in [Ca2+]i , indicative of Ca2+
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elease from internal stores. (B) Effect of HYPO on peak [Ca2+]i levels (n = 10). (C) Suiven as a % of the control HYPO response. Data are presented for the HYPO respC-067047 and for 0 Ca2+ (n = 2). *P < 0.05.
13; unpublished data]. Hence, it appears that the biphasic naturef the TRPV4-mediated [Ca2+]i changes are not related to changesn TRPV4 trafficking to, or from, the plasma membrane in M-1 cells.hese results, however, cannot rule out parallel changes in TRPV4nsertion and retrieval rates, leaving the abundance of TRPV4 at thelasma membrane relatively constant.
.3. Selective activation of TRPV4 by the agonist GSK1016790Aith minimal TRPV4 trafficking
The GSK1016790A (GSK101) compound was recently identifieds a selective agonist of TRPV4 [37,38]. We have demonstratedelective activation of TRPV4 by GSK101 in TRPV4-expressing HeLaells [13] as also shown by others [39,40]. In the current stud-es GSK101 was used to selectively activate TRPV4 in confluent
onolayers of M-1 cells as an alternative approach to activateRPV4. GSK101 was shown to potently activate Ca2+ influx in-1 cells, as evident from the rapid increase in the F340/F380
atio (Fig. 4A), corresponding to an increase in [Ca2+]i levels from5 ± 28 to 455 ± 55 nM (n = 7, Fig. 4B). A modest biphasic responseas still apparent, but with an elevated plateau phase compared
o HYPO stimulation. This is similar to that shown before inRPV4-transfected HeLa cells [13]. However, GSK101 had no affectn [Ca2+]i levels when extracellular Ca2+ was removed (Fig. 4C).ndeed, on average GSK101 did not significantly increase [Ca2+]ievels in the absence of extracellular Ca2+ (Fig. 4D) demonstratingts affect only on Ca2+ influx pathways. To further assess the speci-city of GSK101 on TRPV4, the effect of prior incubation (5 min)ith HC-067047 (100 nM) was evaluated. As shown in Fig. 4C, HC-
67047 completely abolished activation of TRPV4 by GSK101 asvidence by the abolition of an increase in [Ca2+]i. Hence, GSK101ppears to solely activate Ca2+ influx pathways, i.e., TRPV4, withittle or no influence on Ca2+ stores.
y effect of TRPV4 inhibition by HC-067047 and 0 Ca2+ on the peak change in [Ca2+]i
in control (HYPO, n = 5, 100% response), 1 nM (n = 4), 10 nM (n = 3), 100 nM (n = 3)
TRPV4 expression at the plasma membrane was likewise ana-lyzed via immunoblots in a similar manner as done for HYPOexposure, described above. When TRPV4 intensities were nor-malized to the plasma membrane marker, pan cadherin, it wasapparent that plasma membrane levels of TRPV4 likewise werenot significantly altered for up to 30 min of GSK101 exposure(see Fig. 3C). Again, this differs from that observed with GSK101stimulation in the heterologous expression systems where TRPV4activation was followed by rapid net retrieval from the plasmamembrane [13].
3.4. Expression of BK and SK3 channels and their dependency onTRPV4 expression
A preliminary screen of M-1 cells identified expression of twoCa2+-dependent K+ channels in confluent monolayers of M-1 cells,the BK� and SK3 channels. As shown in the immunofluorescentimages of Fig. 5A, expression of both BK� and SK3 were appar-ent in M-1 cells. Modest colocalization with the plasma membranemarker, pan cadherin, demonstrates both cytoplasmic and plasmamembrane, or near plasma membrane, expression for both K+ chan-nels.
Western blot analysis of confluent M-1 cells also demonstratedstrong expression of both BK� and SK3 (Fig. 5B). Both K+ channelsalso appeared to functionally interact with TRPV4 (see below). We,therefore, assessed whether expression of the K+ channels may bedependent upon TRPV4 expression since TRPV4 appears to be aninitiating component. To begin to assess for a potential dependenceof BK� and SK3 on TRPV4, M-1 cells were transiently transfected (2
days) with a mouse TRPV4 construct (mVenus-tagged TRPV4 cDNA)to yield TRPV4-overexpressing M-1 cells. Immunoblots of wild typeM-1 cells (wt) and TRPV4-transfected M-1 cells (tr) were evaluated.As shown in Fig. 5B, TRPV4 was strongly expressed in wt M-1 cells
M. Jin et al. / Cell Calcium
Fig. 3. Effect of HYPO and GSK101 on plasma membrane (PM) expression of TRPV4in M-1 cells. (A) Western blot of TRPV4 expression in the post-nuclear fraction(PNS, whole cell) and plasma membrane fraction (PM) following HYPO exposureover a 30-min time period. (B) Time course of the relative TRPV4 expression abun-dance (TRPV4/pan cadherin) following HYPO exposure. Values are normalized tothe isotonic, control (ISO), condition (0 min) (n = 3). HYPO stimulation of TRPV4 didnot lead to alterations in net TRPV4 expression levels at the plasma membrane.(C) Time course of the relative TRPV4 expression levels (TRPV4/pan cadherin) fol-l(e
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owing GSK101 (10 nM) exposure. Values are normalized to the control condition0 min) (n = 3). GSK101 stimulation of TRPV4 did not lead to alterations in net TRPV4xpression levels at the plasma membrane.
also see Fig. 1B, TRPV4) displaying the expected double band near00 kDa. In TRPV4-mVenus transfected M-1 cells (tr), the additionalands of the TRPV4-mVenus construct were also apparent at higherolecular weights as expected (reflecting the mVenus tag). TRPV4
ransfection enhanced TRPV4 expression levels as expected. How-ver, BK� expression levels appeared to decline modestly (BK�, wtersus trs) while SK3 expression levels tended to increase mod-stly (SK3, wt versus trs). A strong dependence of BK� and SK3xpression on TRPV4 was not readily apparent from this analysis.mmunoprecipitation analysis was not performed since our pri-
ary anti-TRPV4 antibody did not immunoprecipitate well. Hence, dependence of BK or SK3 expression on TRPV4 expression did notppear to be present in the M-1 cells.
.5. Functional coupling between TRPV4 and both BK and SK3 in-1 cells
Does hypotonicity-induced activation of TRPV4 and the asso-iated Ca2+ influx depend upon the Ca2+-dependent K+ channels?
e were particularly interested in assessing whether activationf BK or SK3 would, in and of itself, also modulate intracellular
alcium levels since this would, in part, control K+ secretion by col-ecting duct cells. As shown in Fig. 6A and B, HYPO leads to thexpected biphasic elevation in [Ca2+]i due to TRPV4-mediated Ca2+
nflux (and Ca2+ release). Prior to HYPO treatment, addition of the
51 (2012) 131– 139 135
selective BK antagonist, iberiotoxin (IbTX, 100 nM), in the isotonicstate (Fig. 6B, arrow) had no effect on [Ca2+]i, results consistentwith an inactive BK channel (Fig. 6D). In contrast, in the presence ofIbTX the HYPO-induced changes in [Ca2+]i were markedly bluntedas shown in representative double test protocols (Fig. 6A versusB). In these studies, cells were first exposed to HYPO conditions,returned to control isotonic media (Ctl) for 5–10 min, then fol-lowed by a second HYPO exposure in the presence of IbTX (Fig. 6A).IbTX led to a significant inhibition of the Ca2+ response (Fig. 6A).Reversing the order with IbTX added during the first exposure tohypotonic media produced a similar level of inhibition (Fig. 6B).On average IbTX treatment reduced the peak change in the HYPO-induced [Ca2+]i by 32 ± 6% (n = 6, Fig. 6D). Hence, the HYPO-induced[Ca2+]i is enhanced with BK activation (no IbTX), but depressed inthe presence of BK inhibition (IbTX).
In a similar manner as observed for IbTX, addition of the selectiveSK antagonist, apamin (300 nM), likewise had no effect on [Ca2+]i inisotonic mediate (Fig. 6C, arrow and D), but markedly depressed thehypotonicity-induced peak change in [Ca2+]i. On average, apaminlead to an inhibition of the peak response by 28 ± 4% (n = 4, Fig. 6E).This is slightly less than that observed with IbTX, but the over-all affect is similar. However, when IbTX and apamin are addedtogether, the inhibition of the peak response is even greater, aver-aging near 70% inhibition (Fig. 6E). Hence, while neither BK� orSK3 appeared to be active prior to TRPV4 stimulation (Fig. 6D),TRPV4 activation by HYPO media induced a K+ channel-sensitiveCa2+ influx where blockade of the either the BK or SK3 channels, orboth, markedly reduces [Ca2+]i. This apparent cross-talk betweenTRPV4 and the Ca2+-dependent K+ channels displays a synergy thatmay modulate both cell signaling events and potentially alter netfluxes in polarized epithelial cells such as K+ secretion in the renalcollecting duct (see below).
Finally, the effect of IbTX on GSK101 Ca2+ influx was also testedin a few studies. As for the HYPO situation, activation of TRPV4by addition of GSK101 (10 nM) likewise revealed an IbTX-sensitivecomponent of Ca2+ influx. On average IbTX lead to a 24 ± 4% reduc-tion in the TRPV4-mediated [Ca2+]i response to GSK101 (data notshown). Further testing with GSK101 was not performed sincethe high Ca2+ influx associated with GSK101 activation of TRPV4appears to “push” the cells into a high-Ca2+ resistance state as wehave previously described (see [13]).
4. Discussion
Stimulation of renal collecting duct M-1 cells by mechanicalstress leads to rapid activation of TRPV4 and Ca2+ influx [6]. Thepresent studies demonstrate that exposure of M-1 cells to hypo-tonic swelling conditions leads to a biphasic elevations in [Ca2+]iwhich is characterized by a transient peak elevation followedby relaxation to a more prolonged pseudo-plateau phase withmodestly elevated [Ca2+]i level. We have previously shown for tran-siently transfected TRPV4-HeLa cells that following activation ofTRPV4 the channel would rapidly desensitize, largely as a resultof retrieval of TRPV4 from the plasma membrane into the cytosol[13]. We had anticipated a similar trafficking outcome for the M-1cells which endogenously express TRPV4. However, we show in thisstudy that hypotonic activation of TRPV4 in M-1 cells does not leadto net retrieval of TRPV4 from the plasma membrane (Fig. 3) despitethe biphasic nature of the [Ca2+]i response (Fig. 2). TRPV4 levelsat the plasma membrane remain relatively constant over manyminutes of hypotonic stress. It may be that hypotonic stimulation
may simultaneously activate both TRPV4 insertion and retrievalfrom the plasma membrane so that the net abundance remainsunchanged, although this was not the case for TRPV4 in HeLa over-expression system [13]. In this regard it is interesting that Heller
136 M. Jin et al. / Cell Calcium 51 (2012) 131– 139
Fig. 4. Effect of the selective TRPV4 agonist, GSK101, on M-1 cell [Ca2+]i levels. (A) Typical [Ca2+]i trace from one coverslip (average of 10 cells) showing effect of TRPV4activation following addition of 10 nM GSK101. The [Ca2+]i response is slightly biphasic reaching a peak within 1–6 min. (B) Summary of GSK101 affects on [Ca2+]i (n = 7). (C)Representative time course showing the effect of GSK101 on [Ca2+]i in the presence and absence of extracellular Ca2+ (0 Ca2+). Note the abrupt increase in [Ca2+]i followingre-introduction of Ca2+ with prior treatment by GSK101. (D) Summary of the affect of GSK101 on the peak change in the F340/F380 ratio in the presence and absence of 0C
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a2+ (–Ca2+, n = 2) and HC-067047 (100 nM, n = 4). *P < 0.05.
nd coworkers [14] have recently demonstrated that PACSIN 3, annhibitor of the endocytotic machinery [41], is highly expressed athe luminal border of TRPV4-expressing renal cells. It was showno bind to TRPV4 and lead to TRPV4 retention in the luminal cell
embrane, supposedly due to inhibition of TRPV4 endocytosis. Fur-hermore, it was subsequently shown that expression of PACSIN 3an lead to inhibition of TRPV4 activation by hypotonic swelling inRPV4-transfected HEK cells [42]. Hence, it may be that PACSIN 3n M-1 CCD cells serves to retain TRPV4 in the plasma membraneollowing hypotonic swelling with subsequent inhibition, or par-ial inhibition, of TRPV4 activity leading to the biphasic nature ofhe hypotonic response. This concepts remains to be fully testedn future studies as it may reflect a critical pathway in control ofRPV4 activity in the CCD. Other pathways and modulators couldlso play a role in this process.
An alternative pathway that may modulate TRPV4-mediateda2+ influx in M-1 cells may be the Ca2+-dependent K+ channelsnd control of the membrane potential. It is well known that in thearly collecting duct system, including the CCD, Ca2+ plays a majorole in modulating K+ secretion and, hence, K+ balance [18–20].+ secretion in this segment has been shown to be flow sensitiverising from Ca2+-dependent K+ secretion largely through activa-ion of BK. We have recently identified TRPV4 as a flow-sensor inhe M-1 collecting duct cells [6] and shown its expression at theuminal membrane of the mouse CCD [5] while Suzuki and cowork-rs have demonstrated a marked reduction in flow-induced K+
ecretion in CCD from mice lacking the TRPV4 gene [17]. While BKould appear to be the dominant effector pathway in this response,
ur present studies open up the possibility that SK3 could be aewly identified effector in this process. Both of these channelsppear to be activated upon exposure of M-1 cells to hypotonicwelling conditions (or by agonist activation of TRPV4). To this
end we demonstrate in these studies that there appears to be asynergistic cross-talk between TRPV4 and the K+ channels sinceTRPV4 activation is needed to activate BK and SK3 while at thesame time activation of BK and SK3 appears to enhanced Ca2+ influx(via TRPV4) as a result of hyperpolarization of the plasma mem-brane (see discussion, below). While such cross-talk has not beenexplicitly described before for the renal collecting duct system, it isimplicit from studies in cells expressing similar types of Ca2+ and K+
channels as outlined below. Hence, the TRPV4-mediated cross talkwith the downstream K+ channels may underlie flow-dependent K+
secretion where the apparent synergism between channels wouldserve to drive this entire process and modulation of K+ balance.
Ca2+-dependent K+ channels are widely expressed in bothexcitable and non-excitable cells where they are thought to playa major role in modulating membrane potential [43,44]. Some celltypes, such as vascular endothelial cells, express a wide range ofCa2+-dependent K+ channels, including SK, IK, and BK channels,where the channels function to control membrane hyperpolar-ization as an important component of the endothelial-derivedhyperpolarizing factors underlying vascular dilation/blood pres-sure modulation [45–47]. Similarly, Ca2+-dependent K+ channelshave been widely described in a diverse range of both excitableand non-excitable cells [43,48–50] where they again play a rolein modulating membrane potential. Important for the control ofthese channels is alterations in [Ca2+]i that can arise from Ca2+
release from internal stores or, the more likely scenarios, fromstimulation of Ca2+ influx into the cells from both TRP channelsand various voltage activated Ca2+ channels [25,51]. Furthermore,
functional associations of channels within a common microdomainor tethered complex can greatly alter any cross-talk although suchassociations are largely unknown for this channel grouping [see 44].Hence, the interplay between Ca2+ influx channels and the range of
M. Jin et al. / Cell Calcium 51 (2012) 131– 139 137
Fig. 5. Expression of BK� and SK3K+ channels in M-1 cells. (A) Immunofluorescent staining of M-1 cells for BK� (anti-BK�, green) and pan cadherin (anti-pan cadherin,red) (upper panel) and for SK3 (anti-SK3, green) and pan cadherin (anti-pan cadherin, red) (lower panel). Merged images show modest collocation of BK� and SK3 with pancadherin at the plasma membrane. Scale bar = 20 �m. (B) Western blot analysis of TRPV4, BK�, and SK3 expression levels in wild type (wt) M-1 cells and in TRPV4-transfected( h mVei V4 ovo
Cct
tawt(mTliaicrt
tBcbsa
tr) M-1 cells. Note, the TRPV4 construct used for transfection (TRPV4 tagged witmmunoblot. TRPV4 expression levels were, as expected, greatly elevated with TRPverexpression levels.
a2+-dependent K+ channels can vary greatly from one setting, orell type, to the next. In the renal collecting duct cells this appearso be dominated by TRPV4 and its cross-talk with BK and SK3.
We also hypothesize that within the collecting duct cells thathe cross-talk between TRPV4 and the K+ channels likely reflects
variable synergism or progression in K+ channel activation. Thisould arise because of the differences in the Ca2+-sensitivity of
he K+ channels being expressed: SK3 is highly Ca2+-sensitivemid-nanomolar range) while BK has a low Ca2+-sensitivity (low
icromolar range) [see 43,44]. Hence, it may be that the initialRPV4-mediated Ca2+ influx would first activate the SK3 channel,eading to modest membrane hyperpolarization and increased Ca2+
nflux which, if [Ca2+]i were high enough, would lead to subsequentctivation of BK with a further hyperpolarization and more Ca2+
nflux. Other signaling pathways controlling the channel activitiesould, in turn, modulate the response. Hence, some diversity in theesponse is anticipated because of the variable Ca2+-sensitivities ofhe channels being expressed.
Finally, from an analysis of the driving forces for Ca2+ influx inhe M-1 cells, it is apparent that cross-talk between TRPV4 andK/SK3 can be very significant, and lead to important functional
oupling. To what extent can there be a reciprocal modulationetween channel types leading to increased Ca2+ influx as a con-equence of membrane hyperpolarization following K+ channelctivation? If it is assumed that upon TRPV4 activation the [Ca2+]i
nus) runs higher on immunoblots and, therefore, leads to multiple bands on theerexpression (tr) whereas BK� and SK3 showed little or no dependence on TRPV4
can approach at least 200 nM (likely more, see Figs. 2B and 3B),the chemical driving force for Ca2+ entry with 1 mM extracellularwould be near −110 mV [ECa = 30 mV × log (1 × 10−3/200 × 10−9)].With a resting membrane potential of near −40 mV for the M-1 cells [52], the total driving force for Ca2+ entry would be near−150 mV (−110 + −40 mV). Assuming the Vm were to hyperpolar-ize to −80 mV with maximal K+ channel activation, but reducedto near 0 mV with K+ channel inhibition, the driving force for Ca2+
entry would change from near −190 mV to near −110 mV for thetwo states, respectively. This equates to a 30–50% change in theCa2+ driving force depending upon the state of K+ channel activa-tion. Hence, the potential range of BK/SK3-induced modulation ofthe Ca2+ driving force can be very substantial when considering Vmchanges only. This scenario is also consistent with the observedchanges in [Ca2+]i upon inhibition of the BK and SK3 channelsreported in the current study (see Fig. 6). Hence, the cross talk in M-1 cells and CCD cells may be a major factor in setting both the rateof calcium influx and the extent of membrane hyperpolarization.
In summary, the current study has demonstrated thathypotonicity-induced activation of TRPV4 in renal collectingduct cells leads to activation of Ca2+ entry without alterations in
membrane trafficking to the plasma membrane. Further, this Ca2+
influx leads to activation of two Ca2+-dependent K+ channels, BKand SK3, both of which display a functional synergistic cross-talkleading to modulation of channel functions. It is likely that such
138 M. Jin et al. / Cell Calcium 51 (2012) 131– 139
Fig. 6. Effect of BK antagonist, IbTX, and SK3 antagonist, apamin, on [Ca2+]i response to HYPO in M-1 cells. (A) Representative trace showing the effect of HYPO on [Ca2+]i
followed by a repeat HYPO stimulation in the presence of IbTX (100 nM). (B) Representative tracing showing that IbTX under isotonic control conditions had no affect on[Ca2+]i (arrow), and reversing the order of IbTX on the HYPO response showed the same inhibition by IbTX on HYPO-induced [Ca2+]i changes. (C) Typical [Ca2+]i responseto HYPO followed by HYPO response in the presence of apamin (300 nM). (D) Summary response of resting [Ca2+]i ratio upon addition of IbTX (n = 3), apamin (n = 3), andIbTX + apamin (n = 3) under control, isotonic (ISO), conditions. The inhibitors had no affect on the resting [Ca2+]i levels. (E) Summary showing the HYPO-induced changesin [Ca2+]i ratio (�), relative to the control HYPO response (100%), in the presence of IbTX (68 ± 6%, n = 6), apamin (72 ± 4%, n = 4), and IbTX + apamin (28 ± 10%, n = 3). Bothi 2+ bine 2+
rKc
A
a
R[
[
nhibitors modestly reduced the HYPO-induced increase in [Ca ]i ratio, but the com
egulation may underlie Ca2+ signaling events and flow-dependent+ secretion in the TRPV4-expressing segments of the mammalianollecting duct.
cknowledgements
This work was supported by NIH grants to RGO, R01 DK070950nd R21 DE018522.
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