Download - Voltage-activated proton current in eosinophils from human blood

Journal of Physiology (1996), 496.2, pp.299-316

Voltage-activated proton current in eosinophils fromhuman blood

D. V. Gordienko, M. Tare, S. Parveen, C. J. Fenech, C. Robinsonand T. B. Bolton *

Department of Pharmacology and Clinical Pharmacology, St George8s Hospital MedicalSchool, Cranmer Terrace, London SWJ 7 ORE, UK

1. The resting membrane potential of freshly purified normodense human eosinophils bathedin and dialysed with quasi-physiological solutions was -63 + 2 mV (n = 100).

2. In voltage-clamp mode with quasi-physiological internal and external solutions, voltage stepsfrom the holding potential of -60 mV to levels positive to +20 mV resulted in development ofa quasi-instantaneous outward current and a slowly developing outward current. The instant-aneous current was absent when the cells were bathed in and dialysed with K+-free solution.

3. The slow outward current persisted following simultaneous replacement of K+, Na+ andmost of the Cl- with largely impermeant ions (tetraethylammonium, N-methyl-D-glucamineand methanesulphonate) and was augmented when the cell was dialysed with a solution ofincreased buffering capacity for protons. The observed reversal potential of the currentclosely followed the hydrogen equilibrium potential over a wide range of internal-externalpH combinations, indicating that the conductance underlying the slow outward current washighly selective for H+ ions.

4. Acidification of the pipette solution (increasing [H+]1) augmented the outward H+ currentand shifted its activation range negatively, whilst acidification of the external solution hadthe opposite effect. The voltage dependence of the current is modulated by thetransmembrane pH gradient so that only outward current could be activated. However,when the outward current was activated by a voltage step, rapid acidification of externalsolution produced an inward H+ current which rapidly deactivated.

5. The proton current was reversibly inhibited in a voltage-dependent manner by extracellularapplication of Zn2+. The apparent dissociation constants were 8 nm (at +40 mV), 36 nm (at+70 mV) and 200 nm (at +100 mV).

6. The proton current was augmented by exposure to 10 ,UM arachidonic acid. Thisaugmentation consisted of a shift of the voltage dependence of activation to more negativepotentials and enhancement of maximum conductance (H,max). The proton current recordedin eosinophils was significantly augmented under conditions of elevated cytosolic freecalcium concentration ([Ca2+]1). The threshold level of [Ca2P]i associated with this effect laybetween 0 1 and 1 uLM and was not measurably affected by cytosolic acidification.

7. Eosinophils from human blood possess a voltage-dependent H+ conductance (9H) whichnormally allows protons to move outwards only; raising [Ca2P]i was associated withaugmentation of 9H and intracellular acidification or arachidonate shifted its activationrange negatively towards physiological potentials.

Assessment of the role of the eosinophil in health and disease eosinophils are now incriminated as important effector cellshas proved difficult because these cells constitute only a in a number of diverse human diseases. These include thesmall percentage of the total leucocyte population. This host response to parasite infestation and tumours, thecontrasts with the relatively sophisticated understanding reaction to transplanted tissues, and both allergic and non-that exists for the more abundant neutrophil. Nevertheless, allergic inflammatory reactions of the major organ systems

(reviewed by Spry, 1988).

* To whom correspondence should be addressed.

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D. V Gordienko and others

Receptor-directed stimulation of eosinophils and neutrophilsleads to the release of a wide repertoire of chemicalmediators. Some of these exist preformed within secretorygranules, whilst others, such as eicosanoids and oxidants,are generated de novo when the cell is stimulated. Comparedwith neutrophils, there is a poor understanding of the eventsthat transduce receptor activation to the release of thesemediators from eosinophils. Furthermore, there is scantinformation concerning the contribution of plasma membraneelectrical conductances to the regulation of the function ofeosinophils, although the study of such events has proved tobe difficult even in the more numerous neutrophil.

In common with neutrophils, monocytes and macrophages,eosinophils possess a respiratory burst oxidase complex(NADPH: 02 oxidoreductase; Segal, Garcia, Goldstone,Cross & Jones, 1981; Bolscher, Koenderman, Tool, Stokman& Roos, 1990) which, upon cell activation, is fully assembledto undertake the single-electron reduction of oxygen toproduce superoxide anion (Cross & Jones, 1991). Thesuperoxide anion provides a basis for the subsequentproduction of other cytotoxic reactive oxidants includinghydrogen peroxide, -OH and hypohalous acids. Theproduction of the superoxide anion is electrogenic becauseelectrons proceed through an intracellular transport chainand reduce oxygen at, or proximal to, the extracellularsurface (Henderson, Chappell & Jones, 1987). During thisprocess NADPH is consumed by the oxidase andresynthesized by the hexose monophosphate shunt.Concomitant with the operation of these reactions is theintracellular accumulation of protons, which would lead tocytoplasmic acidification if there were an absence ofcompensatory mechanisms. In the neutrophil thesehomeostatic mechanisms include the Na+-H+ antiporter(Grinstein & Furuya, 1986; DeCoursey & Cherny, 1994a)and vacuolar H+-ATPase (Swallow, Grinstein & Rotstein,1988). Studies using potential- and pH-sensitive dyes, andwith the patch-clamp technique have also revealed inneutrophils and macrophages the existence of anelectrogenic compensatory mechanism involving a voltage-gated conductance for protons (Henderson et al. 1987;Henderson, Chappell & Jones, 1988; DeCoursey & Cherny,1993; Kapus, Romanek, Qu, Rotstein & Grinstein, 1993;Kapus, Romanek & Grinstein, 1994). However, theexistence of such pathways in the eosinophil has not beeninvestigated.

In this paper we provide what is, to our knowledge, the firststudy of the electrophysiological properties of freshlypurified human blood eosinophils. We provide evidence forthe existence of a highly selective proton conductance (9H)in eosinophils that has similar properties to the protonconductance described in other leucocytes, namely,neutrophils (DeCoursey & Cherny, 1993) and macrophages(Kapus et al. 1993, 1994). We postulate that the propertiesof the proton conductance suggest a possible role for it incellular pH homeostasis and phagolysosome formation wheneosinophils are activated. A preliminary account of some of

this work has previously been reported in abstract form(Gordienko, Tare, Parveen, Fenech, Robinson & Bolton,1995; Tare, Gordienko, Parveen, Fenech, Robinson &Bolton, 1996).

METHODSCell preparationBlood was taken from non-atopic healthy human donors (male andfemale, age range 23-53 years) by venipuncture, and anti-coagulated 9:1 (v/v) with 3-15% trisodium citrate. Followingcentrifugation at 400 g (20 min, 20 °C), the platelet-rich plasmawas discarded and the remaining fraction mixed 4: 1 (v/v) with6% (w/v) dextran in Dulbecco's phosphate-buffered saline (PBS).After allowing the erythrocytes to sediment at 1 g (45 min, 20 °C),the leucocyte-rich layer was removed and washed twice (389 g for10 min and then at 339 g for 7 min at 20 °C) in Ca!+- and Mg2+-freeHanks' balanced salt solution (HBSS) containing 2% (v/v) heat-inactivated fetal calf serum (FCS) and Phenol Red (10 jug ml-').Lysis of residual erythrocytes was achieved by resuspending thecell pellet in 0 2% (w/v) sodium chloride, shaking for 30 s and thenadding an equal volume of 1-6% (w/v) sodium chloride solution.After centrifugation (339 g, 7 min, 20 °C) the granulocyte pelletwas resuspended in HBSS (without Phenol Red and FCS) andlayered over discontinuous two-step gradients of Percoll(1'082 g ml-' and 1-094 g ml-'), with approximately 2-0 x 106 cellsper gradient. The density gradients were then centrifuged (389 g,30 min, 20 °C). Cells were recovered from the 1-082-1-094 g ml-'interface, washed in HBSS (339 g, 7 min, 20°C) and a samplestained with 2% (w/v) Crystal Violet for counting in a Neubauerhaemocytometer. The volume of the cell suspension was adjusted togive a maximum of 1 X 107 cells ml-' in ice-cooled HBSS, andmonoclonal antihuman CD16 was added (2 /sg per 107 cells). Themixed granulocytes were incubated at 4 °C for 15 min on a rollermixer (Denley Spiramix 5) and then washed 3 times with HBSS(339 g, 7 min, 4 °C). The granulocyte pellet was then resuspendedin ice-cold HBSS and incubated for 45 min at 4 °C with sheep anti-mouse IgG, labelled with superparamagnetic polymer particles,prior to magnetic removal of the CD16+ cells. The cell suspensioncontaining CD16- eosinophils was carefully aspirated and washed(339 g, 7 min, 20 °C) with HBSS containing 2% heat-inactivatedFCS. The cell pellet was finally resuspended in HBSS containing2% heat-inactivated FCS at a cell number of approximately1.0 x 106 cells ml-'. Viability of the preparations was determinedby Trypan Blue exclusion. Differential counts were performed afterHaematoxylin and Eosin staining of fixed cytocentrifuge smears.

The cell suspension was stored at 4 °C prior to use. When required,an aliquot of cell suspension was diluted in physiological saltsolution (BS I, Table 1) and allowed to attach to a glass coverslippre-coated with Sigmacote (Sigma) and mounted on the stage of aNikon Diaphot inverted phase-contrast microscope. The cells werethen thoroughly rinsed with appropriate external solution beforecommencement of the electrical recordings.

Electrical recordingsWhole-cell membrane currents and membrane potentials weremeasured within 16 h of eosinophil isolation using conventionalwhole-cell patch-clamp technique (Hamill, Marty, Neher, Sakmann& Sigworth, 1981). Either an Axopatch-ID or an Axopatch 200A(Axon Instruments) was used as the input amplifier. All recordingswere made at room temperature (20-25 °C) in static bathing solution(-500 ,l volume). External solution changes were achieved in100-200 ms. Liquid junction potentials between pipette and bath

J Phy8iol. 496.2300



Proton current in human eosinophils

Table 1. Composition of bath solution (BS) and pipette solution (PS)

BS pH NaCl KCl CaCl2 MgCl2 Glucose HCl MSA NMDG Buffer Base(mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM)

I 7 0 140 5 2 1 6 - 10 Hepes 2A4mM NaOHII 7 0 10 5 2 1 6 - 130 1-0 Hepes 131P8 mM NaOHIII 7 0 10 2 1 6 135 5 10 Hepes 130-9 mMNaOHIV 7 0 - 2 1 10 70 100 Hepes TEA-OHV 6-5 2 1 - 10 70 1-00 Pipes TEA-OHVI 6-0 2 1 10 70 1-00Mes TEA-OHVII 5.5 2 1 10 70 1-00 Mes TEA-OHVIII 7 0 75 5 2 1 - 100 Hepes 24 mM NaOHIX 7 0 - 2 1 40 40 1-00Hepes TEA-OH

PS pH pCa NaCl KCI CaCl2 BAPTA HCl MSA NMDG Buffer Base(mM) (mM) (mM) (mM) (mM) (mM) (mM) (mM)

I 7 0 7 0 5 96-5 4.6 10 - 10 Hepes 48'8 mM KOHII 7 0 7 0 5 4-6 10 96-5 - 965 10 Hepes TEA-OHIII 7 0 7 0 - 2416 5 10 70 100 Hepes TEA-OHIV 6-5 7'0 1-74 5 10 70 100 Pipes TEA-OHV 6'0 7 0 0 97 5 10 70 100 Mes TEA-OHVI 5.5 7 0 0 3 5 10 70 100 Mes TEA-OHVII 6-0 7 0 5 75 0 97 5 1-00 Mes 64-3 mMKOHVIII 7 0 84i - 0 3 5 10 70 100 Hepes TEA-OHIX 7'0 - 0 03 10 70 - 100 Hepes TEA-OHX 7 0 8.0 - 0 7 10 10 70 100 Hepes TEA-OHXI 7 0 7 0 4-3 10 10 70 100 Hepes TEA-OHXII 7 0 6-0 8.9 10 10 70 - 100 Hepes TEA-OHXIII 6-0 8-0 0-2 10 10 70 100 Mes TEA-OHXIV 6-0 7 0 2-0 10 10 70 100 Mes TEA-OHXV 6-0 6-0 7 0 10 10 70 100 Mes TEA-OH

Selected biological buffers (Hepes, Mes or Pipes) were chosen according to their pKa to adjust pH to thevalues indicated in column 2. For solutions containing relatively impermeant ions (BS IV-VII and IX, andPS II-VI and VIII-XV), pH was set to the final value by neutralizing methanesulphonic acid (MSA) andappropriate buffer with TEA-OH. All pipette solutions contained 0-02 mm GTP. Pipette solutions I and IIcontained 1 mm MgATP and 5 mm creatine. Pipette solutions III-VI and VIII-XV contained 0 05 mMMgATP. In pipette solutions where pCa is indicated (column 3), the free calcium concentration ([Ca2+]) wasclamped by addition of CaCl2 at different concentrations (as indicated) for solutions of various pHcontaining 5 or 10 mM BAPTA and 0 05 or 1 mm MgATP. The concentrations of CaCl2 needed werecalculated using EQCAL software (Biosoft, Cambridge, UK). TEA-OH, tetraethylammonium hydroxide;MSA, methanesulphonic acid; NMDG, N-methyl-D-glucamine; BAPTA, 1,2,-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid.

solutions were measured as described (Neher, 1992) and nulled withan offset circuit before seal formation. None of the measured valuesexceeded 2 mV, so no further off-line corrections were introduced.Changes in electrode junction potential during substitution of thebath solution were minimized by connecting the ground electrodeto the bath solution via an agar-salt bridge. Fire-polished patchpipettes filled with pipette solution (PS I-XV, Table 1) hadresistances of 5-9 MQ. After tight seal formation between pipetteand cell membrane, fast capacitive transients were minimized usingelectrode capacitance compensation of the input amplifiers.Following formation of whole-cell configuration, voltage pulses of+ 10 mV were applied from the holding level of -60 mV in order toestimate input resistance, cell membrane capacitance and seriesresistance. The input resistance evaluated from the change insteady current produced by hyper- or depolarizing voltage stepswas 34 + 1-4 GQ2 (n = 218) when measurements were made with

K+-containing pipette and bath solutions and was 42-4 + 1-7 GQ(n = 200) in K+-free conditions. Whole-cell current records were

not corrected for leakage current. The cell membrane capacitancewas calculated as a ratio of total charge (estimated as integratedarea under the capacitive transients recorded with 10 kHz filtering)to the magnitude of the pulse (10 mV). In ninety-nine randomlyselected recordings, the cell membrane capacitance was 4 + 041 pF.Series resistance, taken as a ratio of the time constant ofexponentially decaying capacitive current to the cell membranecapacitance was 31 + 1 MQ2 (n = 99). Taking into account the slowkinetics and typically small amplitude of detected currents, no

compensation of cell capacitance and series resistance was used.The whole-cell currents were filtered at 2 kHz (-3 dB frequency) bya four-pole low-pass Bessel filter. Voltage protocols were generatedand data were collected either at 041 or 1-2 kHz using a CED 1401interfaced to a 386 PC running CED software (Cambridge

J Phy8iol.496.2 301




Electronic Design Ltd, Cambridge, UK). Data were analysed, fittedby functions (as described in the text) and plotted using MicroCalOrigin software (MicroCal Software, Inc., Northampton, MA,USA). Data are shown as representative traces obtained from atleast five different cells using similar experimental protocols, or asthe mean values + S.E.M. for the number of cells (n) examined.Comparative analysis of the data groups was performed usingStudent's t tests for paired or unpaired samples as appropriate. Tominimize the effects of possible spontaneous changes in theproperties of the conductance studied, a constant time was allowedfor cell dialysis before measurements were made (usually 2 min). Inthe series of experiments using zinc, the time between subsequentapplication of solutions of different concentrations was keptconstant. The sequence of solution changes was random whenexternal solutions of differing pH were applied. The properties ofeosinophils were observed to vary more between batches (or donors)than within batches. To take account of this, where the effects ofaltering intracellular pCa (pCaj) were investigated, cells weredialysed with solutions of different pCai sequentially andrepetitively, and a comparable number of cells in each batch wastested with each pCai.Reversal potential of the currentTo estimate the reversal potential (Erev) of the slow outwardcurrent, the current activated by a 2 s positive voltage step (up to+100 mV) was reversed by fast ramp repolarization. During theramp-like shift in membrane potential, a steady current (i,) flowsinto the membrane capacitance (Cm):

ic = Cm x d V/dt.When the time-dependent current is activated, the total currentthrough the membrane will be:

I= ic + ig+ il,where ig is a time-dependent current through the membraneconductance, gm, due to ion channel opening, and il is the currentthrough the leakage conductance (gl, about 25 pS). To measure thereversal potential of ig, it was necessary to subtract i0 and il. To dothis the same ramp was applied first after a positive pre-pulse of20 ms duration, which was sufficient to allow the membranecapacitance to be fully charged to the same voltage as during thelong voltage step. Assuming that negligible specific conductancesare activated during the short pre-pulse (gm 0), the currentduring the subsequent ramp is (ic + i,). Thus, the result of digitalsubtraction of the current recorded during the first ramp from thecurrent recorded during the second ramp represents:

(iC + ig + i1) - (ic + i1) = ig)and was used to construct the corrected current-voltagerelationship. Given that the cell membrane capacitance was onaverage 4 pF, a steady current of about 4 pA would flow into themembrane capacitance during ramp repolarization at a rate of1 mV ms-'. Thus, subtraction of the current recorded during thefirst ramp from the current recorded during the second ramp wouldalter Erev by about: AE= 4 pA/g8, assuming g8 >> gl, where 98 isequal to the slope conductance (gm) in the membrane potentialrange close to Erev-

Solutions and chemicalsThe compositions of the solutions used in this study are described inTable 1. In most cases they mainly contained the impermeant ionstetraethylammonium (TEA+) and methanesulphonate (CH3SO3-), asmall amount of Cl- (to prevent electrode polarization) and 100 mmof pH buffer selected according to pKa for pH values in the range5 5-7 0. Large concentrations of pH buffers were used to shunt the

intrinsic buffering capacity of the cell and to minimize depletion ofH+ after proton conductance activation (Byerly, Meech & Moody,1984; DeCoursey & Cherny, 1993, 1994a, b; Lukacs, Kapus,Nanda, Romanek & Grinstein, 1993). Previous microspectro-fluorimetric measurements of intracellular pH (pH1) (Demaurex,Grinstein, Jaconi, Schlegel, Lew & Krause, 1993) have shown thatcytosolic pH shifted to near that in the pipette within 1-2 min ofcell dialysis with a solution containing 100 mm pH buffer.Therefore, to ensure equilibration between the pipette solution andcytosol, data acquisition was commenced at least 2 min afterrupture of the cell membrane.

Chemicals and drugs used. Adenosine 5'-triphosphate(magnesium salt), arachidonic acid (free acid), 1,2,-bis(2-amino-phenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), creatine,dextran from Leuconostoc mesenteroides (580 kDa), Hepes, Mes,Pipes and zinc chloride and were obtained from Sigma. Theremaining chemicals and reagents were obtained as follows:Percoll' from Pharmacia Biotech Ltd, Milton Keynes, UK; HBSSwith and without Phenol Red from Gibco BRL, Paisley, UK; heat-inactivated FCS from Labtech International Ltd, Uckfield, EastSussex, UK; murine monoclonal IgG, antihuman CD16 from TheBinding Site Ltd, Birmingham, UK; Dynabeads® M-450 sheepantimouse IgG from Dynal (UK) Ltd, New Ferry, Wirral, UK;Dulbecco's PBS from ICN Flow, Thame, Oxfordshire, UK.Tinctorial stains and all other reagents were obtained from BDH,Poole, UK, and were of the highest purity obtainable.

RESULTSNet outward currentWith quasi-physiological internal and external (PS I, BS I)solutions, the resting membrane potential of eosinophilsmeasured in current clamp mode was -63 + 2 mV(n = 100). Therefore, in voltage-clamp experiments, themembrane potential of eosinophils was held at -60 mV.Under these conditions, voltage steps to levels more positivethan +20 mV evoked outward currents that consisted oftwo kinetically distinct components: a rapid initial outwardcurrent was followed by a slowly activating component thatdid not reach steady state within 6 s (Fig. 1A). Whether CF-or K+ contributed to the outward current was determinedby ion substitution experiments. Reduction of externalchloride (by partial replacement of CF- with methane-sulphonate; BS II) was without effect on either of theoutward currents (Fig. IB). Average current-voltagerelationships constructed for the current at the beginningand at the end of the voltage steps, before and afterreduction of [Cl-]0, overlay for the voltage range tested(Fig. 1C). The slow component of the outward currentpersisted when cells were dialysed with, and bathed, inK+-free solution (K+ was replaced with NMDG+ in bothinternal and external solutions (BS III, PS II) and pH, wasadjusted using TEA-OH) whereas the fast initial currentwas substantially reduced (Fig. ID). In K+-free conditions,the current-voltage relationship for the current measured atthe beginning of the voltage steps was close to linear over thewhole voltage range tested. In contrast, the averagecurrent-voltage characteristics for the slow component ofthe outward current were similar in the presence or absence

302 J Phy8iol.496.2




of K+ (Fig. 1C and E). Thus, the voltage-gated slowoutward current was not carried by K+ or CF. To gainfurther insight into the ionic nature of the slow outwardcurrent, the reversal potential of the current was measuredin K+-free conditions. The voltage protocol used forestimating the reversal potential of the slow outwardcurrent is shown as the inset to Fig. IF (upper trace). The

A B

10 pA

2 s

10 pA

2 s

slow outward current activated by a voltage step to+100 mV was then reversed by fast (to minimize the effectof current deactivation) ramp repolarization to -80 mV. Toavoid a contribution of the capacitive current to themeasurements, two consecutive voltage steps were applied,the first being 20 ms and the second 2 s in duration.Assuming that negligible specific conductances are activated

C

I

-20

40 80V (mV)

EI (pA)

FI (pA)

10 pA|2 s

-40 0 40 80V (mV)

40 80V (mV)

*-5

-10

Figure 1. Whole-cell current evoked in human eosinophils by positive voltage stepsA, a representative family of currents activated in an eosinophil by 6 s long voltage steps applied in 20 mVincrements from a holding potential of -60 mV, in quasi-physiological external and pipette solutions (BS I,PS I). B, currents recorded in the same cell following reduction of [ClF]O from 151 to 21 mm (BS II, PS I).C, amplitudes of the currents measured at the beginning (0, BS I, PS I; v, BS II, PS I) and at the end(0, BS I, PS I; V, BS II, PS I) of the pulse are plotted versus the corresponding values of the pulsemembrane potential (means + S.E.M., n = 14). D, currents recorded in another cell using the same voltageprotocol as in A and B in the absence of K+ in internal and external solutions (BS III, PS II).E, corresponding current-voltage relationships for currents measured at the beginning (m) and at the end(O) of the pulses (BS III, PS II; means + S.E.M., n = 22). F, reversal potential of the slow outward currentin K+-free conditions (BS III, PS II). The value of the reversal potential was taken as the zero-currentvoltage measured during ramp repolarization. The inset shows the voltage protocol (upper) andcorresponding current trace (lower). Note that breaks in the traces represent a 30 s lapse in the recording.Two consecutive voltage steps (20 ms and 2 s in duration) were applied from -60 to +100 mV. Each voltagestep was terminated by ramp repolarization to -80 mV at a rate of 1 mV ms-1. The current-voltagerelationship shown arises from digital subtraction of the current obtained during the first ramp from thatduring the second (see Methods for detailed explanation). The calculated values for the equilibriumpotentials for Cl- (Ec1) and Nae (Ea) are indicated by the arrows.

D

10 pA

2 s

J Physiol. 496.2 303




during the first step, the result of digital subtraction of thecurrent recorded during the first ramp from the currentrecorded during the second ramp was used to construct thecorrected current-voltage relationship of the slow outwardcurrent as shown in Fig. iF The reversal potential (Erev)taken as the zero-current voltage during ramp repolarizationwas 0 1 + 0-6 mV (n = 24), thus differing from Ec,and ENaby 47 6 and 84 mV, respectively (Fig. 1F). The observedvalue of the reversal potential is consistent with theequilibrium potential for protons calculated according to theNernst equation (4 = 0, since the concentration of protonsin both pipette and external solutions was the same,pHi = pHo = 7 0). The result is also consistent with a non-selective conductance underlying the slow outward currentwhich fails to discriminate between K+, Cl-, Na+ and largeions such as TEA+, NMDG+ and CH3SO3-. To investigatewhether protons were the charge carrier, the effect ofaltering the transmembrane pH gradient on E wasexamined.

H+ selectivity: dependence of Erev on transmembranepH gradientMost of the experiments were performed using pipette andbath solutions containing the largely impermeant ions

A I (pA)

Erev (mV)

(11)4

1 I I I I 9 5le.~~~~~~~~~~~

-90 -60 -30(22) --,

(1 9)

(7) ,(10)*1 '

-60

30

-

-50

(6) ,'31 '1

I II

30 60EH (mV)

--30

--60

--90

TEA+ and CH3SO3-. The pH of both pipette and bathsolutions was clamped using 100 mm of the buffer Hepes,Mes or Pipes selected appropriately according to their pKa(PS III-VI, BS IV, VI and VII). The experimental protocolused for estimating the reversal potential of the currentunder different transmembrane pH gradients is shown inthe inset to Fig. 2A. The procedures for constructing thecurrent-voltage relationships have been described above.Examples of current-voltage relationships obtained at fivedifferent transmembrane pH combinations are shown inFig. 2A. The values of Er, taken as the zero-currentpotential were:

-86 mV (curve a, pH, 5*5, pHo 7 0, EH = -87-3 mV),-58 mV (curve b, pHTi 6-0, pHo 7 0, EH = -58&2 mV),-31 mV (curve c, pHi 6-5, pHo 7 0, EH = -29-1 mV),

3 mV (curve d,pHi 6-0, pHo 6-0, EH = 0 mV),and 27 mV (curve e,pHi 6-0, pHo 5 5, EH= +29-1 mV).

The current-voltage relationships labelled b, d and e wereobtained from an individual cell bathed successively in threeexternal solutions each differing only in pH.

Figure 2B summarizes the effect of altering the trans-membrane pH gradient on Erev of the current. Note that one

a

b

c Figure 2. Reversal potential of the voltage-gatedcurrent: dependence on internal-external pHA, changes in whole-cell current induced by ramp

d repolarization were used to determine the reversalpotential of the slow outward current in different

e internal-external pH combinations:curve a, pH, 5*5, pHo 7 0, Erev = -86 mV (PS VI, BS IV);curve b, pHi 6'0, pHo 7 0, Erev = -58 mV (PS V, BS IV);curve c, pHi 6-5, pHo 7 0, Erev = -31 mV (PS IV, BS IV);curve d, pHi 6-0, pHo 6-0, Erev = +3 mV (PS V, BS VI);curve e, pH, 6-0, pHo 5 5, Erev = +27 mV (PS V, BS VII).The inset shows the voltage protocol (upper section) andcorresponding current trace (lower section). The 30 s lapsein the recording is shown by the breaks in the traces. Thevoltage protocol was similar to that in Fig. I F, except thatcells were polarized to +60 mV and then repolarized byramps to -120 mV. The current-voltage relationshipsshown (curves a-e) were obtained by methods described inFig. 1. B, observed values of reversal potential are plottedagainst the theoretical Nernstian values for the hydrogenequilibrium potential (EH). *, internal-external solutionscontaining largely impermeant ions and minimal Cl- (BSIV and VI-VII; PS IV-VI); 0, internal-externalsolutions containing Nat, K+ and Cl- (PS VII, BS VIII);means + S.E.M., n given in parentheses. The dashed linerepresents the predicted values of reversal potential for apure proton conductance.

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series of experiments (denoted by the open circle in Fig. 2B)was performed with pipette and bath solutions containingNa+, K+ and Cl- at quasi-physiological concentrations(PS VII, BS VIII) with pHi = 6.0, pH. = 7 0 whereENa = +70 mV, 4 =-755 mV, Eci = -12 mV andEH =-58-2 mV. The dashed line on the graph shows thepredicted values of the reversal potential for a pure protonconductance.

Dependence of Erev on the extent of current developmentAs in previous cases, Erev of the current was determinedusing the two-pulse/ramp protocol. However, the extent ofproton current development achieved before ramprepolarization was altered by successively increasing theduration of the second pulse (Fig. 3A). Current-voltagerelationships (recordings a-e, Fig. 3B) were constructed asdescribed previously. Erev was equal to the theoreticallypredicted value (pH, 5.5, pH. 7 0, EH = -87 mV) following0 5 s of current development. However, as the duration ofthe voltage pulse that activated the proton current wasincreased to 0 9 s (b), 1P3 s (c), 1-7 s (d) and 2 s (e), Erev wasshifted positively from 4 by 5, 11, 17 and 21 mV,respectively. The same experiments were performed on ninecells and the results are summarized in the inset to Fig. 3B,where the average values for ELev are expressed as a functionof the second pulse duration. These observations support the

contention that significant changes in transmembrane pHgradient occur during the flow of proton current, thusaccounting for the deviation of Erev from 4.In view of these results, the effect of changes in trans-membrane pH gradient on the proton current wasminimized in all further experiments by restricting pulseduration to 2 s and by using 100 mm buffer in pipette andbath solutions. Shorter test pulses were not used because theslow kinetics and small amplitude of the current precludedproper characterization of g9. However, it should be notedthat even 2 s pulses did not allow steady-state activation ofthe current.

Effects of intra- and extracellular acidification on theproton conductanceThe pH of the external solution (pHO) was fixed at 7 0(BS IV) whilst the pH of the pipette solution (pH1) wasclamped at 7 0, 6-5 or 6-0 (PS III-V). The families ofoutward currents shown in Fig. 4A-C were recorded in threedifferent eosinophils in response to gradually increasingvoltage steps.

It should be noted that increasing the buffering capacity forprotons in pipette solutions itself increases the magnitude ofthe slow outward current (compare Fig. ID and E withFig. 4A and D), implying that protons carrying the current

A

Figure 3. Reversal potential of thevoltage-gated current: dependence on theextent of slow outward currentdevelopmentA, the voltage protocol shown in the upperpanel was similar to that described in Figs IFand 2A, except that for each pair of pulses,the duration of the second pulse wassuccessively increased. The lower panel in Ashows a corresponding family of currentsobtained in an individual cell (internal pH 5-5and external pH 7 0, EH = -87-3 mV; PS VI,BS IV). For the second pulse, the duration ofthe voltage step preceding the ramp was:0 5 s (curve a), 0 9 s (curve b), 1-3 s (curve c),1I7 s (curve d) and 2 s (curve e). B, theinstantaneous current-voltage relationshipsare derived from current records shown inpanel A. The current-voltage characteristicsarise from digital subtraction of the currentobtained during the first ramp from thatduring the second ramp for each pair of pulses(curves a-e, correspondingly). Inset:dependence of Erev on the duration of thedepolarizing pulse (PS VI, BS IV;means + S.E.M., n = 9). Note that as theduration of the pulse activating the currentincreases, a greater positive shift of Erev awayfrom EH (-87-3 mV) occurs.

70 mV

-60 mV I4 J

360 r -120 mVN N N N

240 I

I (pA)

120

-60

B

E

2-0 2-5 3-0

e

dc

b

a

0 0-5 1-0 1-5Time (s)

J Physiol.496.2 305




originate from the protonated buffer (for details refer to theDiscussion).

When pipette solution contained 100 mm of the protonbuffer 2 s pulses were sufficient to evoke detectable time-dependent outward currents (Fig. 4A). Progressive loweringof pH, increased the magnitude of the outward current(Fig. 4B and C). This was accompanied by acceleration ofactivation kinetics (activation time constants at +100 mVderived from fitting of the data by a single-exponentialfunction were 6-5 + 1P6 s (pHi 7 0, n = 6), 4.4 + 0-6 s (pH16-5, n = 17) and 1-2 + 0-2 s (pH, 6-0, n = 33)).

The average current-voltage relationships for the currentmeasured at the end of the 2 s pulse for each pH

combination are shown in Fig. 4D. Apart from an increasein current magnitude, another feature is apparent. Thethreshold for activation of the current was shifted to the leftwith cytosolic acidification. This effect indicates thatchanges in pHi also had a direct effect on the voltagedependence of the H+ conductance. To correct for the effectsof pH on the driving force, chord conductance was plottedas a function of the test membrane voltage (Fig. 4E). Valuesof chord conductance (GH) were calculated according to theequation:

GH= IH/(Vm - Erev),where IH is the mean amplitude of the proton current (takenfrom Fig. 4D) at corresponding test membrane potentials

ApH; = 7.0, pHo = 7.0

.AM ~~~~~r50 pA |

600 ms

DI (pA)

-80

BpH, = 6-5, pHo = 7-0

CpH, = 6-0, pHo = 7-0

50 pA I r 50 pA[

600 ms 600 ms

E

- 240

0 40 80 -80 -40 0 40 80V (mV) V (mV)

Figure 4. Influence of internal acidification on the slow outward currentA-C, families of whole-cell currents recorded in three different cells in response to 2 s long pulses, steppingfrom a holding potential of -60 mV to levels ranging from -70 to +100 mV, with 10 mV increments. Bothinternal and external solutions contained relatively impermeant ions. The pH of the external solution wasfixed at 7 0 (BS IV). The pH of the internal solutions was clamped at 7 0 (A, *, PS III), 6-5 (B, *, PS IV)and 6-0 (C, V, PS V). D, current-voltage relationships for the current measured at the end of the 2 s pulse(as in A-C) with internal pH of 7 0 (0, n = 5), 6-5 (U, n = 13), 6-0 (V, n = 17) (means + S.E.M.). E, effect ofinternal acidification on the voltage dependence of the proton conductance. Chord conductance (OH) wascalculated from the average amplitudes of the proton currents (taken from D, *, *, V, correspondingly) asdescribed in the text.

J Phy8iol. 496.2



Proton current in hi

(Vm) and Erev is assumed to be equal to EH. However, itshould be noted that this assumption leads to anunderestimation of H+ conductance at very positivemembrane potentials, where the effect of changing thetransmembrane pH gradient during large currentdevelopment is more pronounced. Cytosolic acidificationshifts the threshold for activation of the conductance in thenegative direction. The threshold, defined as the mostnegative voltage at which time-dependent outward currentcould be detected, was: +20 mV (pHi 7 0), -10 mV (pH, 6 5),-30 mV (pHi 6 0) (Fig. 4E). As expected, acidification ofthe external solution had the opposite effects to thosedescribed above. Three families of whole-cell currentsshown in Fig. 5A-C were recorded in an individualeosinophil consecutively bathed in three external solutionsof differing pH: 7 0 (Fig. 5A, BS IV), 6f5 (Fig. 5B, BS V) and

uman eosinophils 307

6-0 (Fig. 5C, BS VI). The pH of the pipette solution wasfixed at 6-0 (PS V). The voltage protocol was identical tothat used in the preceding series of experiments.Acidification of the bathing solution diminished themagnitude of outward currents but enhanced the magnitudeof the tail currents, consistent with the applied alterations inprotonmotive force (Fig. 5A-C). The average current-voltagecharacteristics for the currents measured at the end of the2 s pulse under different external pH conditions and thecorresponding chord conductance-voltage relationships areshown in Fig. 5D and E, respectively. Comparison ofconductance-voltage relationships reveals that protonconductance (9H) is also sensitive to pH of the extracellularmedium. Progressive acidification of external solutionshifted the threshold of activation of proton conductancefrom -30 mV (pHo 7 0) to +10 mV (pHo 6 5) and to

ApHi = 6-0, pH, = 7.0

BpH1 = 6-0, pHo = 6-5

CpH, = 6-0, pHo = 6-0

50 pA | 50 pA |600 ms 600 ms

-240

0 40 80V (mV)

-80 -40 0 40 80V (mV)

Figure 5. Effects of external acidification on the slow outward currentA-C, families of whole-cell currents recorded in a single eosinophil dialysed with a pipette solution in whichpH was fixed at 6f0 (PS V). The pH of the external solutions was clamped at 7'0 (A, 0, BS IV), 6-5 (B, *,BS V) and 6-0 (C, V, BS VI). The voltage protocol used was the same as that described in Fig. 4. D, current-voltage relationships for the current measured at the end of the 2 s pulse at external pH of 70 (0, n = 17),6-5 (i, n = 6) and 6-0 (V, n = 7) (means + S.E.M.). E, effect of external acidification on the voltagedependence of the proton conductance. Mean values of the current shown in D were used to calculate thechord conductance (GH; *, m, V correspondingly).

D

I (pA)

50 pA|

600 ms

E

J Phy8iol.496.2




+30 mV (pHo 6 0). Thus, the voltage dependence of channelgating is modulated by pH on both sides of the cellmembrane such that only outward proton currents could beactivated by positive stepping of the membrane potential.

When the outward current was activated by a voltage stepat a certain pH1-pHO combination, rapid acidification of theexternal solution reversed the current, an example of whichis shown in Fig. 6. When the cell was dialysed with asolution of pH, 6-5 (PS IV) and bathed in a solution of pHo7 0 (BS IV), a prolonged voltage step (for 50 s) to +30 mVresulted in the development of an outward proton current(Fig. 6A). This outward current reached a peak within 10 sand then gradually declined in amplitude to a quasi-steady-state level. The observed decay of the current during thelong voltage step does not reflect time-dependentinactivation of the H+ conductance (because no voltage-dependent inactivation of 9H was observed; data notshown), but could be explained in terms of changing of thetransmembrane pH gradient during H+ currentdevelopment as discussed above (see Fig. 3).

After a 1 min interval, the same voltage step was applied.When the proton current reached peak amplitude, rapidswitching of external solution to one of pH 5'5 (thusshifting 4 from -29-1 mV to +87'3 mV) evoked aninward current. However, this current exhibited rapid

30 mV

-60 mV

A 75

I (pA)

0

-50

B 75

I (pA)

C

pHi 6-5

deactivation (not unlike the tail currents previouslydescribed; Fig. 6B). A subsequent voltage step failed to elicitan outward current whilst the external pH was maintainedat 5-5 (Fig. 6C).

Voltage-dependent block of the proton current by zincThe proton conductance observed in a variety of cell typesis known to be sensitive to the blocking actions ofpolyvalent metal cations (Thomas & Meech, 1982; Byerly etal. 1984; Mahaut-Smith, 1989b; DeCoursey & Cherny, 1993;Demaurex et al. 1993). The effect of one of the most potentand widely used blockers, Zn2+, was studied in humaneosinophils. Addition of 25 uM ZnCl2 to the extracellularmedium reversibly inhibited the proton current (Fig. 7A-C).The current-voltage relationships and corresponding chordconductance characteristics (Fig. 7D and E) show that theblocking action of Zn2+ was voltage dependent.

Concentration dependency of blockade by Zn2+ was studiedwhen the proton current was activated by stepping themembrane voltage to three different levels: +40, +70 and+100 mV (Fig. 8). Figure 8A shows an example of originalcurrent traces recorded in control and after addition of 10-8,10-7 10-6 and 10-3 M Zn2+ to the external solution andgrouped according to the three test voltage levels. At eachtest voltage, addition of 1 mm Zn2+ resulted in completeblockade of the time-dependent outward current. Therefore,

LpHo 7 0

pHo 7-0

75 r

I (pA)

0

pHo 5.5

Figure 6. Reversal of the slow outward current byrapid switching of external pHThe pH of pipette solution was clamped at 6-5 (PS IV)and the voltage was stepped from -60 mV to +30 mV(upper panel, voltage trace). A, the slow outward currentrecorded when the cell was bathed in an external solutionof pH 7 0 (BS IV; EH = -29 mV). B, once the outwardcurrent was activated, rapid switching of the externalpH to 5-5 (BS VII; EH = +87 mV) reversed the current.Note that the inward H+ current exhibited rapiddeactivation. C, when the cell was bathed in a solution ofpHo 5.5, the voltage step failed to activate the H+current (EH = +87 mV).

pHo 5-5

10 s

J Phy8iol.496.2308

-50 L) by guest on July 12, 2011jp.physoc.orgDownloaded from J Physiol (



to avoid contribution of leakage current to furthercalculations, the currents recorded in the presence of 1 mMZn2+ were subtracted from current traces at each testvoltage. The varying potency of Zn2+ at three differentmembrane potentials can be appreciated in Fig. 8B. Forseventeen cells tested, the leakage-corrected currents wereexpressed as a fraction of the control currents. These datawere averaged and then plotted against Zn2+ concentration([Zn2+]0) for the three different voltage levels tested. Themean experimental points (n = 10-17) for each voltage levelwere well approximated by the Hill equation (see legend toFig. 8), with a Hill coefficient of 0 5. From this plot and thederived dissociation constants it can be seen that thepotency of Zn2+ was greater at less positive voltage levels.

A BControl

0

1 s

Modulation of proton conductance by arachidonic acidPrevious studies in neutrophils have suggested thatarachidonic acid is capable of activating electrogenictransmembrane proton effiux (Henderson & Chappell, 1992)and augments the voltage-activated hydrogen current(DeCoursey & Cherny, 1993). Consequently, we investigatedits effects on the voltage-dependent proton current ineosinophils. A typical experiment is shown in Fig. 9. Aftercontrol currents had been evoked in response toprogressively increasing positive voltage steps (Fig. 9A), thecell was exposed to 10 uM arachidonic acid for 1 min andthe same voltage protocol was repeated. Whilst theamplitude of the current at its onset was virtuallyunaffected, arachidonic acid accelerated the activation

25 /M Zn2+

IV

60 pA

1 s

CWashout

60 pA

1 s

DI (pA)

I Iy w %v v'

-60 -30

E- 400 GH(nS) r 25

0

-300 /

- 200

- 100

..f%' V V v V V V

0 30 60 90 120V (mV)

2-0 "!O8-20~~~~0- 1*5 00o

0//

1-05

20-5

.v 'v 'V W V V V

-60 -30 0 30 60 90 120V (mV)

Figure 7. Inhibition of H+ current by external Zn2+ applicationA, families of whole-cell currents obtained in an individual cell in response to 2 s pulses stepping from aholding potential of -60 mV to levels ranging from -50 to +110 mV, with 10 mV increments (PS V,BS IV). B, inhibition of the current by 6 min exposure to 25 /M Zn2'. C, the current was restored followingwashout of Zn2+ for 6 min. D and E, corresponding current-voltage, and chord conductance-voltagerelationships. For both graphs, current amplitude was measured at the end of the 2 s pulse in control (0),25 uM Zn2+ (v) and after washout of Zn2+ (0). Chord conductance (GH) was calculated assumingH -58 mV.

J Physiol.496.2 309




kinetics (activation time constants at +100 mV derived fromsingle-exponential fitting were 0 7 + 0-06 s for control,0 5 + 0 04 s for arachidonic acid (n = 12)) and enhanced theamplitude of the slow outward current (Fig. 9B). As can bejudged by the tail currents, the rate of channel deactivationwas also increased in the presence of arachidonic acid. Theseobservations (made with largely impermeant ions in pipetteand bath solutions, BS IV, PS V) and the fact thatarachidonic acid fails to affect the outward current reversalfollowing rapid switching of pH in external solution (datanot shown) suggest that arachidonic acid modulates thevoltage-gated H+ conductance rather than invokes anotherconductive pathway. The effect of arachidonic acid was notdue to its conversion to prostaglandins or leukotrienesbecause neither the cyclo-oxygenase inhibitor indomethacinnor the 5-lipoxygenase inhibitor BW A4C (N-(3-phenoxy-cinnamyl)-acetohydroxamic acid) inhibited the effect (datanot shown).

In all of twelve cells tested, arachidonic acid significantlyaugmented the proton current at all voltages at which the

A

current had been measured in the control period. This isillustrated by the current-voltage relationships in Fig. 9C.Chord conductance was calculated from the average currentamplitude at each test voltage (from Fig. 9C) and expressedas a function of test membrane potential (Fig. 9D). As canbe seen, arachidonic acid apparently increases themaximum H+ conductance (9Hmax) and modulates thevoltage dependence of 9H such that the half-activationvoltage is shifted by at least 20 mV in the negativedirection.

Augmentation of proton current under conditions ofelevated cytosolic free calcium concentrationA rise in cytosolic calcium is an obligatory trigger for thechain of events leading to chemotaxis and/or mediatorrelease by eosinophils (Brundage, Fogarty, Tuft & Fay,1991; Kernen et al. 1991; Elsner et al. 1994). To investigatewhether the conductive properties of eosinophil plasmamembrane could be affected by [Ca2P], (and intracellularcalcium buffering capacity), whole-cell currents recordedfrom eosinophils dialysed with solutions in which [Ca2P]

+100 mv

+70 mV

U

+40 mV

B

B

1.0-..

izn /Ic

Control

10-8 M Zn2+

10-7

10-6

0*5 s

Figure 8. Blockade of H+ current by Zn2+,dependence on membrane potentialA, three families of whole-cell currents shownwere recorded from an individual eosinophil(PS V, BS IV). The membrane potential washeld at -60 mV and was then stepped for 1 s to+40, +70 and +100 mV as indicated. Thisvoltage protocol was first applied in controlsolution and then repeated in solutionscontaining increasing concentrations of Zn2+(10-8, 10-7, 10-6 and 10-3 M as indicated on theright of the panel). B, modulation by themembrane potential of the blockade by Zn2+.Currents expressed as a fraction of the controlcurrent ('Zn/Ic) are plotted versuscorresponding Zn2+ concentrations ([Zn2+]O).Data are shown as mean values + S.E.M.(n = 10-17). Current amplitudes weremeasured in control and in different Zn2+concentrations at the end of the 1 s pulse tothree different levels: +40 mV (V) +70 mV (-)and +100 mV (0), as shown in A. Curves weredrawn according to the Hill equation:'zn/Ic = 1/[1 + ([Zn2+]0/K)n] where n, the Hillcoefficient, was equal to 0 5, and K, theapparent dissociation constant, was equal to8 x 10-9 M (V, +40 mV), 3'6 x 10 M (H,+70 mV) and 2 x 10-7 M (0, +100 mV). Thesevalues were chosen for the best fit usingLevenberg-Marquardt non-linear least-squaresminimization algorithm.

10-12 10-1o 10-8 10-6 10-4 10-2

[Zn2+]o (M)

splommw

310 J Physiol.496.2




was weakly buffered (003 mm BAPTA, PS IX; n = 10)were compared with those from the cells in which [Ca2+]iwas clamped to 8-5 x 10-9M (5 mm BAPTA-0-3 mM Ca2+,PS VIII; n = 13). Experiments were performed oneosinophils derived from the same batch and bathed in thesame external solution (BS IX). Outward current at the endof a 2 s step was significantly augmented at test membranevoltages in the range 0 to +120 mV when [Ca2+], wasweakly buffered (Fig. IOA). These observations were madewith internal and external solutions containing largelyimpermeant ions, suggesting that the conductance for H+,rather than any other ionic species, was being modulated.However, to investigate the possible involvement of a C2+-activated chloride conductance in the whole-cell current

A

changes observed (because both pipette and bath solutionscontained Cl- in our experimental conditions), the reversalpotential was measured using the two-pulse/ramp protocolas described earlier. The expected reversal potential for theproton current was 0 mV when pH, = pHo = 7 0. If anyCa2+-activated ClF conductance was invoked during dialysisof the cell with weakly buffered Ca!+ solution, the reversalpotential of the current would be shifted towards Eci(Eci = -38-4 mV). Figure lOB reveals that there was noshift of Erev towards Ec,. Instead, in ten cells Erev of thecurrent was +4-9 + 1P9 mV. The dependence of Erev on thetransmembrane pH gradient was further confirmed in aseparate series of experiments where the cells were dialysedwith Ca2+-unclamped (30 #M EGTA) pipette solution of

BControl 10 FSM arachidonic acid

0

0

100 pA

800 ms

100 pA

800 ms

DI (pA) 800

600

-40 0 40 80V (mV)

GH (nS)

-80 -40 0 40 80V (mV)

Figure 9. Augmentation of H+ current by 10 FM arachidonic acidA, a family of whole-cell currents recorded in an eosinophil (internal pH 6-0, external pH 7 0; PS V, BS IV).The membrane potential was held at -60 mV and was then stepped for 2 s in 20 mV increments topotentials from -80 to +100 mV. B, augmentation of the currents after 1 min exposure to 10/tMarachidonic acid. The currents were recorded in the same cell as in A, using the same experimental protocol.C, current amplitude measured in control (0) and in arachidonic acid (0) at the end of each pulse is plottedversus test membrane potential. D, corresponding chord conductance-voltage relationships in control (0)and arachidonic acid (0). In C and D the values are shown as means + S.E.M. (n = 12) and the asterisksindicate significant differences between control and results in the presence of 10sM arachidonic acid(P <0-05).

C

-80

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pH 6-0 (pH. = 7 0, EH = -58 mV). In this case, Erev wasfound to be -52 + 2 mV (n = 4) whilst EC, = -118 mV([Cl-]1 = 10 mm, [CF-]0 = 16 mM). Furthermore, underthese conditions increasing [Cl-]. to 106 mm failed toaugment the outward current (in contrast to that which

AI (pA)

0

50 pA

1 s

-120 -80

*

40 80

would be expected in the case of a chloride conductance) andErev remained unaltered (data not shown). On the otherhand, it should be noted that close correspondence of Erev toEH, calculated on the basis of nominal pH, and pHo values,indicates that the enhancement of the proton current, in

B

V (mV)

C D

120 -120 -80 -40 0 40 80 120V (mV) V (mV)

Figure 10. Potentiation of the H+ current under conditions of elevated cytosolic free calciumconcentrationA, recordings were commenced 2 min after formation of whole-cell configuration with eosinophils obtainedfrom the same batch. The cells were dialysed with a solution either weakly (PS IX, 0, n = 10) or strongly(PS VIII, *, n = 13) buffered for Ca2+ and bathed in the same external solution (BS IX). The membranepotential was held at -60 mV and stepped for 2 s to levels ranging from -80 to +120 mV, in 20 mVincrements. Current amplitude measured at the end of the 2 s pulse was plotted against corresponding testmembrane potential. Data are shown as mean values + S.E.M. Inset shows two current records elicited byvoltage steps to 120 mV in two different eosinophils: [Ca2+], clamped to 8 5 nM (Ca2+-5 mm BAPTA, 0)and unclamped (0 03 mm BAPTA, 0). B, the reversal potential of the slow outward current observed in a

weakly buffered cell. The value of the reversal potential was taken as the zero-current voltage measuredduring ramp repolarization. The experimental approach (voltage command and corresponding current traceare shown in the inset) was the same as in Fig. 1F C and D, average current-voltage relationships obtainedin eosinophils from four batches. The experimental protocol was the same as in A, but [Ca2+]i was clampedto three different levels: 10 nm (PS X, n= 11; PS XIII, n= 14, v), 100 nm (PS XI, n= 10; PS XIV,n = 14, 0) and 1 #SM (PS XII, n = 9; PS XV, n = 18, *) using Ca2+-10 mm BAPTA buffer, whilst pHi was

either 7 0 (C, PS X-XII) or 6-0 (D, PS XIII-PS XV). In each of the four batches tested, equal numbers ofcells within each batch (donor) were dialysed with solutions of different pCai sequentially and repetitively.Insets show the traces of currents elicited by voltage steps to +120 mV. * Significant difference (P < 0-05)between: * and * or V (C); and * and * (D). tSignificant difference (P < 0 05) between * and V (D).

-120

J Physiol.496.2




cells where [Ca2+]i was weakly buffered, does not result fromcytosolic acidification which could be elicited by thedisplacement of H+ by Ca2' from intracellular binding sites(Thomas & Meech, 1982).

To determine the level of [Ca2+]i which is associated withdetectable augmentation of proton current and whether thislevel would be affected by intracellular acidification,eosinophils were dialysed with solutions of pHi equal toeither 7 0 or 6'0 and [Ca2P], was strongly buffered to threelevels: 10 nm (PS X or XIII), 100 nm (PS XI or XIV) and1 /M (PS XII or XV) in each of four different batchestested. Data were then grouped according to pCai and pH1and averaged. The results of these experiments aresummarized in Fig. 10C (pHi = 7 0) and D (pHi = 6 0),where average current-voltage relationships for the currentsmeasured at the end of 2 s pulses are shown. It can be seenthat regardless of the pHi, the augmentation of the currentwas apparent when [Ca2+]i was 1 /ZM, whilst there was nosignificant difference between the current-voltagerelationships obtained when [Ca2+]i was 10 and 100 nM.

DISCUSSIONThis paper describes the first study of the passive and activeelectrophysiological properties of human peripheral bloodeosinophils characterized using the patch-clamp technique.With pipette and bath solutions of quasi-physiologicalcomposition, an outward current of low amplitude wasobserved when cell membrane potential was stepped from-60 mV to levels positive to +20 mV. This outward currentconsisted of an instantaneously activated component and adistinct, slowly developing current. The instantaneouscurrent was eliminated when K+ was replaced with NMDG+and/or TEA+ in pipette and bath solutions. Therefore in allexperiments in which the slow outward current wascharacterized, K+ was replaced with TEA+ in both externaland pipette solutions. It should be noted that TEA+ isknown to partially inhibit proton currents in somepreparations (e.g. Byerly et al. 1984; Meech & Thomas,1987). However, it is clear that TEA+ is not considered aneffective blocker of gH (DeCoursey & Cherny, 1994b). Sincerobust proton currents could still be recorded in eosinophilsin symmetrical TEA+ solutions, we have used TEA+ toseparate the slow outward current from the instantaneouslyactivated component.

The slowly developing current was not carried by potassiumor sodium. Its reversal potential differed significantly fromthose of Na+ or K+ and the current persisted when solutionsconsisting of relatively impermeant ions were substituted. Itwas also unaffected by changes in the transmembranegradient of Cl-. The amplitude of the slowly developingcurrent was augmented when the cells were dialysed withsolutions of increasing buffering capacity for protons(compare Fig. ID and E with Fig. 4A and D), and itsreversal potential was governed by the transmembrane pHgradient and not dependent upon the presence or absence of

K+, Na+ or Cl- in the solutions. The relative H+/Cl-permeability calculated using the Goldman-Hodgkin-Katzvoltage equation (Hodgkin & Katz, 1949) was more than1-8 x 106. Furthermore, the average slope for thedependence of Erev on the transmembrane pH gradient was58 mV (pH unit)-1, which is virtually the same as the valueof 58 2 mV (pH unit)-' predicted by the Nernst equation forpure proton conductance. The practicalities ofdistinguishing between H+ and OH- as charge carriers areextremely difficult (for reviews see Lukacs et al. 1993;DeCoursey & Cherny, 1994b), particularly if the conductivemechanism is subject to direct allosteric regulation byprotons (see below) or if the transmembrane pH gradientchanges when the current is activated (e.g. see Fig. 3).However, on the balance of evidence presently available, weshall refer to the slowly developing current in eosinophils asa hydrogen ion or proton current. For present purposes wealso assume that the current is conducted by means of ionchannels, but in the absence of evidence from single channelrecordings the possible involvement of an electrogenic pumpor some other electrogenic means of proton translocationcannot be formally excluded.

For a proton conductance, the free concentration of chargecarrier is several orders of magnitude smaller than that forthe current of any other species. At pHi 6-0, an eosinophilof diameter 10 sm would contain 5 x 10O19 mol of protons.At a membrane potential of -20 mV this amount of protonscould be extruded from the cell in 1 s by a current of0 04 pA if the proton conductance was a hundredth of thatactually measured. The currents actually recorded inexperiments were often 104 times larger, implying that theprotons carrying the current were being releasedcontinuously from the protonated buffer during currentdevelopment. Using 100 mm buffer to fix pH1 at 6-0 (Meswith a pKa of 6-15 will be protonated by 58-5% under theseconditions), the total buffered charge in a cell of 10 #smdiameter would be 3 nC, whereas the charge efflux during avoltage step to +30 mV of 50 s duration was up to 6-5 nC.The apparent inactivation of the current during voltagesteps of long duration (e.g. see Fig. 6) and the systematicminor deviation of Erev from EH across the range of pHgradients tested (e.g. Fig. 3) probably reflect depletion ofintracellular protons due to a rapid local depletion of H+ atthe inner surface of the membrane and a slower depletion ofH+ in the bulk cytosol. These changes would be expected todistort the kinetic- and voltage-dependent characteristics ofthe proton conductance and we therefore did not examinethe steady-state characteristics or kinetics of the H+ current.Proton depletion and its consequences in other cell typeshave been reported and discussed in detail elsewhere(DeCoursey, 1991; DeCoursey & Cherny, 1994 b).

Proton currents have previously been identified in a numberof cell types including neurones (Thomas & Meech, 1982;Byerly et al. 1984; Meech & Thomas, 1987), oocytes (Barish& Baud, 1984), alveolar epithelial cells (DeCoursey, 1991;DeCoursey & Cherny, 1995; Cherny, Markin & DeCoursey,

J Phy8iol. 496.2 313




1995), neutrophils (DeCoursey & Cherny, 1993) andmacrophages (Kapus et al. 1993). The current we have nowidentified in eosinophils has a qualitative similarity to thosepreviously reported, particularly in neutrophils where thethreshold for current activation by a positive shift in themembrane potential is dependent upon the transmembranepH gradient (DeCoursey & Cherny, 1993). Although manyvoltage-gated channels are sensitive to a reduction in pHothrough the neutralization of negative charges on theexterior aspect of the cytoplasmic membrane, the effect ofexternal acidification on the current-voltage relationship foreosinophils was greater (ca 60 mV (pH unit)-') than themaximum shift of 37 mV (pH unit)-' predicted by theGouy-Chapman-Stern theory (Gilbert & Ehrenstein,1970). This discrepancy may reflect direct allostericmodulation of the gating mechanism by H+. Cytosolicacidification shifted the voltage dependency of 9H in thenegative direction, consistent with the features of 9Hobserved in HL-60 cells (Demaurex et al. 1993),macrophages (Kapus et al. 1993), neurones (Byerly et al.1984; Mahaut-Smith, 1989a) and alveolar epithelial cells(Cherny et al. 1995; DeCoursey & Cherny, 1995). Changingthe transmembrane pH gradient generally shifts thethreshold for H+ current activation to levels more positivethan EH, thus establishing a role for 9H in the facilitation ofH+ efflux but not uptake.

Sensitivity to Zn2+ and Cd2+ is an established characteristicof H+ currents in other cell types (reviewed by Lukacs et al.1993; DeCoursey & Cherny, 1994b). A positive shift in thegH-voltage relationship and a reduction in the rate ofproton current activation indicated that the protonconductance of eosinophils underwent an effective voltage-dependent block following external application ofmicromolar concentrations of Zn2+. The concentrationdependence of the blockade was well approximated by theHill equation with a coefficient <1, suggesting negativeco-operativity in the binding of Zn2+ to the presumptive H+channel. The potentiation of blockade by externalacidification (at +40 mV the apparent dissociation constantwas shifted from 8 nm at pH. 7 0 to 60 nM at pH. 7 5) isfurther evidence supporting the view that Zn2+ and H+ mayinteract with the same site on the presumptive protonchannel.

The question arises of the possible functions of a protoncurrent that, in resting eosinophils, only becomes activatedat positive membrane potentials. The extreme selectivityand large capacity of the pathway suggest that it might beone mechanism contributing to the regulation of cellular pH.Eosinophils have a high capacity to generate the superoxideanion in response to activation of their respiratory burstoxidase by a diverse range of soluble and particulate stimuli.Superoxide anions, and other reactive oxidant speciesderived from them, have a cytotoxic potential and havebeen detected in increased amounts in diseases associated

Trush, Rembish & Liu, 1995). The NADPH: 02oxidoreductase required for superoxide anion formation isexpressed in 2- to 3-fold greater amounts in eosinophilsthan in neutrophils (Segal et al. 1981; Bolscher et al. 1990)highlighting the importance of this pathway to the functionof these cells. Activation of the respiratory burst results inthe consumption of NADPH and the production of protonswhich, in the absence of compensating mechanisms, wouldproduce cytoplasmic acidification. Cytoplasmic acidificationwould also be favoured during the subsequent regenerationof NADPH by the hexose monophosphate shunt. Little isknown about pH homeostasis in eosinophils, but inneutrophils and macrophages at least three mechanismscontribute: an electroneutral Na+-H+ antiporter (Grinstein& Furuya, 1986; DeCoursey & Cherny, 1994a), anelectrogenic vacuolar H+-ATPase (Swallow et al. 1988) and aproton conductance similar to that now reported ineosinophils (Henderson et al. 1987, 1988; DeCoursey &Cherny, 1993; Kapus et al. 1993, 1994). Thus, the existencein eosinophils of an electrogenic pathway facilitating protonextrusion would provide homeostasis of intracellular pH,compensate for the electrogenic production of superoxideanion, and provide a mechanism for the acidification ofphagolysosomes in microbial or parasitic killing. Our dataclearly establish the possibility that the proton conductancecould become activated when the cytoplasm is acidified orwhen the overall transmembrane pH gradient is altered tofacilitate proton efflux. Furthermore, we have also shownthat the amplitude and/or the voltage dependence of thecurrent is modulated by other factors that are relevant tothe function-directed activation of eosinophils. An elevationin intracellular Ca2+ is a key event in granule exocytosis andnewly generated mediator release in eosinophils (Kernen etal. 1991; Aizawa et al. 1992). In our experiments we foundthat the proton conductance was augmented underconditions of elevated cytosolic free calcium concentrations.The threshold calcium concentration necessary to elicitdetectable enhancement of voltage-gated proton conductancewas found to lie between 041 and 1 UM, that is, within therange which has been detected during eosinophil activation(Kernen et al. 1991). It should be noted that theseobservations differ from those made in snail neurones whereelevation of intracellular calcium up to 10 /M was withouteffect on the H+ current (Byerly et al. 1984). However, weare presently unable to comment definitively on whether theproton conductance is calcium dependent per se, or whetherany other Ca2+-dependent mechanisms potentiate thishydrogen conductive pathway. It is clear, however, that theaugmentation of the proton current in [Ca2+],unclamped/elevated conditions could not be attributed toacidification caused by the displacement of H+ by Ca2+ fromshared intracellular binding sites (as was judged by thereversal potential of the proton current).

Activation of eosinophils by many receptor-directedagonists also results in the liberation of arachidonic acidwith eosinophilic inflammation (Sanders, Zweier, Harrison,from its storage sites in membrane lipids and its subsequent

J Physiol.496.2314



J Phy8iol. 496.2 Proton current in human eosinophils 315

oxidative metabolism by cyclo-oxygenase and lipoxygenasepathways. Mobilization of arachidonic acid within theeosinophil is an important component of the activation ofthe respiratory burst oxidase and degranulation (Rossi,1986; Henderson & Chappell, 1992; Aebischer, Pashe &Jorg, 1993). Eosinophils may also come into contact withhigh concentrations of arachidonic acid released by other celltypes at sites of severe inflammation (Hammarstrom,Hamberg, Samulesson, Duell, Stawiski & Voorhees, 1975).In the presence of arachidonic acid, the eosinophil protonconductance exhibited an accelerated activation, a negativeshift in the voltage dependence of activation and an increasein 9H,max. The augmentation of the whole-cell current inthe presence of arachidonic acid is unlikely to be due to theintroduction of non-proton conductances. Furthermore, thechange in voltage dependency indicates that it cannotsimply be accounted for by the recruitment of additional H+channels. It is thus possible that arachidonic acid directlyaffects the rate of gating of the presumptive proton channel.A role for arachidonate in gating the neutrophil protonchannel has previously been suggested on the basis ofexperiments using cytoplasts (Henderson & Chappell, 1992).In contradiction to an earlier report (Nanda, Romanek,Curnette & Grinstein, 1994), a more recent study hasprovided persuasive evidence that the arachidonic acid-activatable H+ channel of human neutrophils comprises thelarge subunit (gp9l -phox) of the respiratory burst oxidasecomplex (Henderson, Banting & Chappell, 1995). Thissuggests that the conductance that we have identified mayinvolve the eosinophil equivalent of this protein.

In conclusion, this study presents a description of some ofthe passive and active electrophysiological properties ofhuman eosinophils, with particular characterization of ahighly selective voltage-gated proton conductance. Furtherwork will be necessary to establish the precise physiologicalrelevance of this conductance in relation to eosinophilfunction in health and disease.

AEBISCHER, C. P., PASHE, I. & JORG, A. (1993). Nanomolar arachidonicacid influences the respiratory burst in eosinophils and neutrophilsinduced by GTP-binding protein. A comparative study of therespiratory burst in bovine eosinophils and neutrophils. EuropeanJournal of Biochemistry 218, 669-677.

AIZAWA, T., KAKUTA, Y., YAMAUCHI, K., OHKAWARA, Y., NITTA, Y.,TAMURA, G., SASAKI, H. & TAKASHIMA, T. (1992). Induction ofgranule release by intracellular application of calcium andguanosine-5'-O-(3-thiotriphosphate) in human eosinophils. Journalof Allergy and Clinical Immunology 90, 789-795.

BARISH, M. E. & BAUD, C. (1984). A voltage-gated hydrogen ioncurrent in the oocyte membrane of the axolotl, Ambystoma. Journalof Physiology 352, 243-263.

BOLSCHER, B. G. J. M., KOENDERMAN, L., TooL, A. T. J., STOKMAN,P. J. & Roos, D. (1990). NADPH:02 oxidoreductase of humaneosinophils in the cell-free system. FEBS Letters 268, 269-273.

BRUNDAGE, R. A., FOGARTY, K. E., TuFT, R. A. & FAY F. S. (1991).Calcium gradients underlying polarization and chemotaxis ofeosinophils. Science 254, 703-706.

BYERLY, L., MEECH, R. & MOODY, W. (1984). Rapidly activatinghydrogen ion currents in perfused neurones of the snail, Lymnaeastagnalis. Journal of Physiology 351, 199-216.

CHERNY, V. V., MARKIN, V. V. & DECOURSEY, T. E. (1995). Thevoltage-activated hydrogen ion conductance in rat alveolar epithelialcells is determined by the pH gradient. Journal of GeneralPhysiology 105, 861-896.

CROSS, A. R. & JONES, 0. T. G. (1991). Enzymic mechanisms ofsuperoxide production. Biochimica et Biophysica Acta 1057,281-298.

DECOURSEY, T. E. (1991). Hydrogen ion currents in rat alveolarepithelial cells. Biophysical Journal 60, 1243-1253.

DECOURSEY, T. E. & CHERNY, V. V. (1993). Potential, pH, andarachidonate gate hydrogen ion currents in human neutrophils.Biophysical Journal 65, 1590-1598.

DECOURSEY, T. E. & CHERNY, V. V. (1994a). Na-H' antiport detectedthrough hydrogen ion currents in rat alveolar epithelial cells andhuman neutrophils. Journal of General Physiology 103, 755-785.

DECOURSEY, T. E. & CHERNY, V. V. (1994b). Voltage-activatedhydrogen ion currents. Journal of Membrane Biology 141,203-223.

DECOURSEY, T. E. & CHERNY, V. V. (1995). Voltage-activated protoncurrents in membrane patches of rat alveolar epithelial cells. Journalof Physiology 489, 299-307.

DEMAUREX, N., GRINSTEIN, S., JACONI, M., SCHLEGEL, W., LEW, D. P.& KRAUSE, K.-H. (1993). Proton currents in human granulocytes:regulation by membrane potential and intracellular pH. Journal ofPhysiology 466, 329-344.

ELSNER, J., OPPERMANN, M., CZECH, W., DOBOS, G., SCHOPF, E.,NORGAUER, J. & KAPP., A. (1994). C3a activates reactive oxygenradical species production and intracellular calcium transient inhuman eosinophils. European Journal of Immunology 24, 518-522.

GILBERT, D. L. & EHRENSTEIN, G. (1970). Use of a fixed charge modelto determine the pK of the negative sites on the external membrane.Journal of General Physiology 55, 822-825.

GORDIENKO, D. V., TARE, M., PARVEEN, S., FENECH, C., ROBINSON, C.& BOLTON, T. B. (1995). Identification of voltage-activated protoncurrents in human eosinophils. British Journal of Pharmacology116, 31P.

GRINSTEIN, S. & FURUYA, W. (1986). Cytoplasmic pH regulation inphorbol ester-activated human neutrophils. American Journal ofPhysiology 251, C55-65.

HAMILL, 0. P., MARTY, A., NEHER., E., SAKMANN, B. & SIGWORTH,F. J. (1981). Improved patch-clamp techniques for high-resolutioncurrent recording from cells and cell-free membrane patches.Pfluigers Archiv 391, 85-100.

HAMMARSTRO5M, S., HAMBERG, M., SAMULESSON, B., DUELL, E. A.,STAWISKI, M. & VOORHEES, J. J. (1975). Increased concentrations ofnonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetra-enoic acid, prostaglandin E2, and prostaglandin F2M in epidermis ofpsoriasis. Proceedings of the National Academy of Sciences of theUSA 72, 5130-5134.

HENDERSON, L. M., BANTING, G. & CHAPPELL, J. B. (1995). Thearachidonate-activatable NADPH-oxidase-associated H+ channel.Evidence that gp9l -phox functions as an essential part of thechannel. Journal of Biological Chemistry 270, 5909-5916.

HENDERSON, L. M. & CHAPPELL, J. B. (1992). The NADPH-oxidase-associated H+ channel is opened by arachidonate. BiochemicalJournal 283, 171-175.



316 D. V Gordienko and others J Physiol. 496.2

HENDERSON, L. M., CHAPPELL, J. B. & JONES, 0. T. G. (1987). Thesuperoxide-generating NADPH oxidase of human neutrophils iselectrogenic and associated with an H+ channel. BiochemicalJournal 246, 325-329.

HENDERSON, L. M., CHAPPELL, J. B. & JONES, 0. T. G. (1988).Internal pH changes associated with the activity of the NADPHoxidase: further evidence for the presence of a H+ conductingchannel. Biochemical Journal 251, 563-567.

HODGKIN, A. L. & KATZ, B. (1949). The effect of sodium ions on theelectrical activity of the giant axon of the squid. Journal ofPhysiology 108, 37-77.

KAPUS, A., ROMANEK, R. & GRINSTEIN, S. (1994). Arachidonic acidstimulates the plasma membrane H+ conductance of macrophages.Journal of Biological Chemistry 269, 4736-4745.

KAPUS, A., ROMANEK, R., Qu, A. Y., ROTSTEIN, 0. D. & GRINSTEIN, S.(1993). A pH-sensitive and voltage-dependent proton conductancein the plasma membrane of macrophages. Journal of GeneralPhysiology 102, 729-760.

KERNEN, P., WYMANN, M. P., VON TSCHARNER, V., DERANLEAU,D. A., TAI, P. C., SPRY, C. J, DAHINDEN, C. A. & BAGGIOLINI, M.(1991). Shape changes, exocytosis, and cytosolic free calciumchanges in stimulated human eosinophils. Journal of ClinicalInvestigation 87, 2012-2017.

LUKACS, G. L., KAPUS, A., NANDA, A., ROMANEK, R. & GRINSTEIN, S.(1993). Proton conductance of the plasma membrane: properties,regulation, and functional role. American Journal of Physiology265, C3-14.

MAHAUT-SMITH, M. P. (1989a). Separation of hydrogen ion currents inintact molluscan neurones. Journal of Experimental Biology 145,439-454.

MAHAUT-SMITH, M. P. (1989b). The effect of zinc on calcium andhydrogen ion currents in intact molluscan neurones. Journal ofExperimental Biology 145, 455-464.

MEECH, R. W. & THOMAS, R. C. (1987). Voltage-dependent intra-cellular pH in Helix aspersa neurones. Journal of Physiology 390,433-452.

NANDA, A., ROMANEK, R., CURNETTE, J. T. & GRINSTEIN, S. (1994).Assessment of the contribution of the cytochrome b moiety of theNADPH oxidase to the transmembrane H+ conductance ofleukocytes. Journal of Biological Chemistry 269, 27280-27285.

NEHER, E. (1992). Correction for liquid junction potentials in patchclamp experiments. Methods in Enzymology 207, 123-131.

Rossi, F. (1986). The 02- forming NADPH oxidase of thephagocytes: nature, mechanisms of activation and function.Biochimica et Biophysica Acta 853, 65-89.

SANDERS, S. P., ZWEIER, J. L., HARRISON, S. J., TRUSH, M. A.,REMBISH, S. J. & Liu, M. C. (1995). Spontaneous oxygen radicalproduction at sites of antigen challenge in allergic subjects.American Journal of Respiratory and Critical Care Medicine 151,1725-1733.

SEGAL, A. W., GARCIA, R., GOLDSTONE, A. H., CROSS, A. R. & JONES,0. T. G. (1981). Cytochrome b-245 of neutrophils is also present inhuman monocytes, macrophages and eosinophils. BiochemicalJournal 196, 363-367.

SPRY, C. J. F. (1988). Eosinophils. A Comprehensive Review and Guideto the Scientific and Medical Literature. Oxford University Press,Oxford.

SWALLOW, C. J., GRINSTEIN, S. & ROTSTEIN, 0. D. (1988). CytoplasmicpH regulation in macrophages by an ATP-dependent and dicyclo-hexylcarbodimide-sensitive mechanism. Possible involvement of aplasma membrane proton pump. Journal of Biological Chemistry263, 19558-19563.

TARE, M., GORDIENKO, D. V., PARVEEN, S., FENECH, C., ROBINSON, C.& BOLTON, T. B. (1996). Electrophysiological properties of humaneosinophils. Journal of Physiology 491.P, 90-91P

THOMAS, R. C. & MEECH, R. W. (1982). Hydrogen ion currents andintracellular pH in depolarised voltage-clamped snail neurones.Nature 299, 826-828.

AcknowledgementsThis work was supported by the Medical Research Council (M.T.and S.P.) and by Pfizer Central Research (D.V.G. and C.J.F.). Wethank Dr A. V. Zholos for helpful discussions.

Author's permanent addressD. V. Gordienko: Department of Nerve-Muscle Physiology, A. A.Bogomoletz Institute of Physiology, National Academy of Sciences,4 Bogomoletz Street, Kiev 252024, Ukraine.

Author's email addressT. B. Bolton: [email protected]

Received 2 February 1996; accepted 10 July 1996.

