Journal of Physiology (1997), 501.2, pp.355-362

pH-dependent interactions of Cd2" and a carboxylate blockerwith the rat C0C-1 chloride channel and its R304E mutant

in the Sf-9 insect cell line

G. Y. Rychkov *t, D. St J. Astill *, B. Bennetts *, B. P. Hughes t, A. H. Bretag *tand M. L. Roberts*t

*Department of Physiology, University of Adelaide, Adelaide 5005 and t Centrefor Advanced Biomedical Studies, University of South Australia, North Terrace, Adelaide,

South Australia 5000, Australia

1. Gating of the skeletal muscle chloride channel (ClC-1) is sensitive to extracellular pH. In thisstudy, whole-cell recording of currents from wild-type (WT) ClC-1 and a mutant, R304E,expressed in the Sf-9 insect cell line was used to investigate further the nature of the pH-sensitive residues.

2. Extracellular Cd2+ produced a concentration-dependent block of WT ClC-1 with an IC50 of1.0 + 0 1 mM and a Hill coefficient of 2-0 + 0 3. This block was sensitive to external pH,reducing at low pH, with an apparent pKa of 6-8 + 0 1 and a Hill coefficient for protonbinding of 3 0 + 03. Anthracene-9-carboxylate (A-9-C) block of WT ClC-1 was also pHsensitive, increasing at low pH, with an apparent pKa of 6-4 + 0 1 and a Hill coefficient forproton binding of 1P0 + 02.

3. Compared with WT ClC-1, R304E had a lower affinity for Cd2+ (IC50, 3 0 + 0'3 mM) but ithad a similar Hill coefficient for transition metal ion binding. The Hill coefficient for protonbinding to the Cd2+ binding site was reduced to 1-4 + 03. In contrast, the A-9-C bindingsite in R304E showed the same pH sensitivity and affinity for the blocker as that seen in WTClC-1.

4. ClC-1 has at least two binding sites for Cd2+, each of which has at least three residues whichcan be protonated. Binding of A-9-C is influenced by protonation of a single residue. Arg304 is not sufficiently close to the A-9-C binding site to affect its characteristics, but it doesalter Cd2+ binding, indicating that transition metal ions and aromatic carboxylates interactwith distinct sites.

5. The block of ClC-1 by transition metal ions and the apparent pKa of this block, together withthe apparent pKa for A-9-C block and gating are all compatible with the involvement of Hisresidues in the pore and gate of ClC- 1.

The skeletal muscle chloride channel, ClC-1, is a member ofa homologous family of voltage-dependent anion channelsexemplified by ClC-0, the chloride channel from the electricorgan of Torpedo (Jentsch, Gunther, Pusch & Schwappach,1995). Since the recent isolation of its cDNA, ClC-1 has beenexpressed in heterologous systems and shown to be a lowconductance, inwardly rectifying channel which showstime-dependent deactivation on hyperpolarization (Pusch,Steinmeyer & Jentsch, 1994; Astill, Rychkov, Clarke,Hughes, Roberts & Bretag, 1996).

From a knowledge of its primary structure and the presenceof a glycosylation site, Jentsch et al. (1995) have predicted atopology for the ClC-1 protein with multiple transmembrane

domains, and with both the N- and C-termini located on thecytoplasmic side of the membrane. Little is known, however,about the contribution of the various peptide domains tosuch properties of the channel as the structure of theconductance pathway, the determination of ionic selectivityor the control of gating between the open and closed states.Some information has come from the study of naturallyoccurring mutants, for example, where replacement of anaspartyl residue in the putative first transmembrane domainwith glycine (D136G) alters the channel kinetics, leading tothe conclusion that this site might be involved in the regionforming the voltage sensor (Fahlke, Riidel, Mitrovic, Zhou& George, 1995).

I To whom correspondence should be addressed.

6196 355

G. Y Rychkov and others

Histidyl residues have frequently been implicated as beingresponsible for near-neutral pKa values associated withchannel function (De Biasi, Drewe, Kirsch & Brown, 1993;Tabcharani et at. 1993), and a number of studies in skeletalmuscle can be interpreted as suggesting that these residuesmight be involved in the function of its chloride channels.Hutter & Warner (1967a, b) showed that the restingmembrane conductance of frog skeletal muscle was pHdependent, with conductance changing sharply over the pHrange of 5-7, and that Zn2+ and Cu2+, which can complexwith the imidazole side-chain of histidine, blocked the effluxof 36CF- from frog skeletal muscle at pH 7-4 but had littleeffect at pH 5 0. Furthermore, the myotonia produced byZn2+ in isolated rat skeletal muscle is practically irreversibleat pH 7-4 but can be reversed by washing the muscle atpH 6-5 (Bretag, Fietz & Bennett, 1984; Bretag, 1987). Morerecent studies on ClC-1 expressed in Sf-9 insect cells haveshown that extracellular pH has direct effects on the gatingof this channel (Rychkov, Pusch, Astill, Roberts, Jentsch &Bretag, 1996).

In the present study we have analysed the effects ofextracellular pH on the pharmacology and gating of wild-type (WT) CIC-1 and its R304E mutant (Astill et al. 1996).This mutant, in which the arginine at position 304 isreplaced by glutamate, shows gating at pH 7-4 whichresembles that seen in WT C0C-1 at a lower extracellular pH(D. Astill and G. Rychkov, unpublished observations). Fromthe results of these experiments, important functional rolesare inferred for groups, possibly the imidazole side-chain ofhistidyl residues, which are titratable between pH 6 and 7.Comparison of the responses of R304E and WT CIC-1enables us to distinguish titratable residues which influenceblock by transition metals from those which modifyanthracene-9-carboxylate (A-9-C) block and channel gating.A preliminary account of some of these data has beenpublished in abstract form (Astill, Rychkov, Hughes,Bretag & Roberts, 1995).

METHODSExpression of C0C-1 in Sf-9 cellsMethods used for the expression of rat ClC-1 and for producing thesite-directed mutant R304E have been described in detailpreviously (Astill et al. 1996). Mutation of the arginine at position304 to glutamate was performed using the Altered Sites System(Promega, Madison, WI, USA) following the manufacturer'sprotocols. Cells were grown in Grace's insect cell culture medium(Gibco) supplemented with 0 333% (w/v) lactalbumin hydrolysate,0 333% (w/v) yeastolate (Difco, Detroit, MI, USA) and 10% (v/v)fetal bovine serum (CSL, Melbourne, Australia). Incubations wereat 28 °C in air. Cells were maintained in monolayer culture andpassaged at 80% confluence. Cells were then seeded at low densityin 35 mm Petri dishes and incubated until ca 50% confluence(24-48 h) before inoculation at a multiplicity of infection of 50 withthe baculovirus clones BVDA6.3 (WT) and BVDA6-R304E andfurther incubation for 28-30 h. Following incubation, infected cellswere seeded onto glass coverslips and maintained at room

temperature (21-23 °C) in cell culture medium until required. Priorto patch clamping, cells were rinsed in bath solution. Patch-clamp

experiments were performed between 28 and 34 h postinfection, atwhich time expression of the rat ClC-1 protein has been clearlydemonstrated (Astill et al. 1996).

ElectrophysiologyPatch-clamp experiments were performed in the whole-cellconfiguration at room temperature. The bath solution contained(mM): NaCl, 170; MgSO4, 2; and CaCl2, 2. For a pH higher than 6-6it was buffered with 10 mm Hepes adjusted with NaOH, and for apH less than 6-6 with 10 mm Mes titrated with NaOH. Pento-barbitone (0 5 mM) was added to block the native Cl- channels inSf-9 cells (Birnir, Tierney, Howitt, Cox & Gage, 1992). Cd2+ or Zn2+were added to the bath solution as CdCl2 or ZnSO4. Patch pipettesof 2-4 MQ2 were pulled from borosilicate glass and coated withSylgard (Dow Corning). The standard pipette solution contained(mM): KCl, 40; potassium glutamate, 120; EGTA-Na, 10; andHepes, 10; adjusted to pH 7'2 with NaOH. Currents were obtainedusing an EPC-7 amplifier (List, Darmstadt, Germany), filtered at3 kHz and stored on disk, using pCLAMP software (AxonInstruments). Potentials listed are pipette potentials expressed asintracellular potentials relative to outside zero. Corrections for theliquid junction potential between the bath and electrode solutions,estimated to be -14 mV by using JPCalc (Barry, 1994), wereapplied where specified in the figure legends.

ChemicalsA-9-C (Aldrich) was prepared as the Na+ salt by neutralizing theacid with an equivalent amount of NaOH (1 M), allowing readydissolution.

Data analysisThe raw current data points were fitted with an equation of theform:

I = A1exp(t/ll) + A2exp(t/T2) + C,

where A1 and A2 represent the amplitude of the fast and slowexponential components of current deactivation, 7r and T2 representtheir time constants, t is time, and C represents the amplitude ofthe steady-state component. Peak (or instantaneous) current wasestimated by extrapolating this curve to the beginning of the pulse.

Titration curves and dose-response curves for Cd2+ and Zn2+ werefitted with sigmoidal functions of variable slope using a four-parameter logistic equation, to give estimates of pKa (the -log ofthe dissociation constant), IC50 and Hill coefficients. The data forapparent open probability (PO(V)) was fitted with Boltzmannfunctions of the form:

Po(V) = PO(OO) + (1 - PO0(a))1 + exp((V;- V)/k)

where PO(oo) is an offset, V is the transmembrane potential, VX isthe potential at which PO = (1 + P(oao))/2, and k is the slope factor.

Results are presented as means + S.E.M. The values for theparameters IC50, Hill coefficient, pK. and V; were obtained fromthe curves fitted to the pooled data from all cells tested, with threeor four cells represented at each data point.

RESULTSEffect of external pH on the block of C1C-1 bytransition metal ions and A-9-CLarge, rapidly deactivating inward currents are typicallyinduced in Sf-9 cells expressing ClC-1 when hyperpolarizingcurrent steps to -120 mV are applied following maximal

J Phy8iol.501.2356

J Physiol.501.2 pKa Of muscle chloride channels

3-2

1-6

0-8

0 50Time (ms)

WT

100

0-2 0-4 0-8 1-6

[Cd2+] (mM)

I I 13-2 6-4 1 2-8

Figure 1. The effect of extracellular Cd2" on currents through WT C0C-1 and the R304E mutantSf-9 cells expressing ClC-1 and exposed to a range of concentrations of Cd2+ in the bath solution were

stepped to a prepotential of +40 mV for 50 ms and then to a test potential of -120 mV for 50 ms. Thepeak current in the presence of Cd2+ is plotted as a proportion of the current measured in the absence ofCd2+. Results are means + S.E.M. (n = 3); WT, 0; R304E, 0. The inset shows typical WT currentsrecorded from one cell in the presence of a range of concentrations of Cd2 .

channel activation by preconditioning at +40 mV (Fig. 1).Addition of Cd2" (Fig. 1) to the control bath solution(pH 7-5) produced a concentration-dependent reduction ofcurrents through C0C-1 within 1 min. Application of Cd2+(2 mM), via the patch pipette to the interior of the cell, didnot affect peak current amplitude. The IC50 for the block by

external Cd2+ was 1P0 + 01 mm, while the Hill coefficientfor the concentration-response curve was 2-0 + 0-3. Asimilar effect was seen with extracellular Zn2+, which had an

IC50 of 0-35 + 0-04 mm and a Hill coefficient of 2-2 + 0 3(n = 4). This block by the transition metal ions from theextracellular side was found to be pH dependent. When the

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Figure 2. The effect of extracellular pH on the Cd2" block ofWT C0C-1 and the R304E mutantCurrents were recorded with 2-5 mm Cd2+ in the bath solution at a range of values of extracellular pH. Thevoltage protocol used was the same as in Fig. 1. The peak current at each pH in the presence of Cd2+ isexpressed as a proportion of the peak current measured at pH 6-0 in the absence of Cd2+. Results are

means + S.E.M. (n = 3); WT, 0; R304E, *. The inset shows typical WT currents recorded from a cellexposed to 2-5 mm Cd2+ at a range of values of extracellular pH.

357

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pH of the bath solution was varied at a constant externalCd2+ concentration of 2-5 mm, there was a maximal blockingeffect at pH 7 5, with little block at pH 6-0 (Fig. 2). Fromthe resultant titration curve, an apparent pKa of 6-8 + 01and a Hill coefficient of 3 0 + 03 could be extracted (Fig. 2).

The concentration-response curve for block of C0C-1 byA-9-C was shifted to the left by changing the extracellularpH from 7-5 to 5-5 (Fig. 3A), indicating that the affinity forA-9-C was also pH dependent, but in the opposite directionto transition metal ion binding. The IC,O for A-9-C changedfrom 7-2 + 0-2 ,gM at pH 7-5 to 0'29 + 0 04 /SM at pH 5.5.There was no appreciable decrease in affinity for A-9-Cwhen pH was raised from 7-5 to 8-0, and it did not increasefurther when pH was lowered from 5-5 to 5 0. Titration ofthe block of A-9-C (1 ,CM) gave a value for the apparent pK.of 6-4 + 0 1, and a Hill coefficient of 1 0 + 0-2 (Fig. 3B).

Effect of extracellular pH on gating andpharmacology of R304EUnder control conditions (bath pH 7 5), only subtledifferences were observed in the general characteristics ofgating of the R304E mutant relative to WT (Fig. 4).Differences that could be significant in understanding thesequence-function relation for gating were the shift of theapparent open probability curve of R304E by 17 mV tomore negative potentials (Fig. 4C) and the decreased relativeamplitude of the slow component (A2) of the inward currentat large hyperpolarizing voltages (compare tail currents at-100 mV in Fig. 4A and B). As the pH of the bath solutionwas lowered, fast gating of the R304E mutant showed thesame behaviour as fast gating of the WT channel in that

A1*0-

0-8

0-6

0-4 -

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deactivation of the current became less marked, and atpH 5.5 the steady-state current was over 90% of theinstantaneous current (Fig. 5, inset). The titration curve forthe R304E mutant gave the same pKa and Hill coefficientfor proton binding as that obtained for the WT (pKa,6X1 + 0-1 and 6-0 + 0 1; Hill coefficient, 1.1 + 041 and1-2 + 0 1 for WT and R304E, respectively) and showed thatthe increased steady-state component of the inward currentseen in the mutant at normal pH could not be diminishedby raising bath pH (Fig. 5).

In comparison with WT, the R304E mutant had a loweraffinity for Cd2+, with an IC50 of 3 0 + 0 3 mm (Fig. 1),although the Hill coefficient for Cd2+ binding (2-2 + 0-2 forR304E) was similar to that of the WT channel. A plot of thetitration curve for current amplitude in the presence of2-5 mm extracellular Cd2+ (Fig. 2) showed a shift to theright compared with that for the WT channel and was muchflatter. The apparent pKa for the block was 7-2 + 0 1 with aHill coefficient of 1-4 + 0 3 (n = 3).

By contrast with its diminished ability to bind Cd2+, R304Ehad the same affinity for A-9-C as the WT, and the titrationcurve for the effect of pH on A-9-C block did not deviatesignificantly from that for WT (Fig. 3).

DISCUSSIONFrom work on whole muscle, it is well known that thetransition metals Zn2+ and Cd2+ block Cl- permeability, asestimated by 36Cl- efflux, and also block Cl- conductance, asdetermined by cable analysis (Hutter & Warner, 1967a;

B1*0-

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5 6 7pH

8 I8 9

Figure 3. The effect of extracellular pH on the block of WT C0C-1 and the R304E mutant byA-9-CThe voltage protocol used was the same as in Fig. 1. A, concentration-response curves for the blockingeffect of A-9-C on peak current at pH 7 5 (0, n = 5) and pH 5-5 (O, n = 4). B, the peak currents at variouspH values in the presence of 10-6 M A-9-C are expressed as a proportion of the peak current at pH 7-5 inthe absence of A-9-C. Results are means + S.E.M. (n = 3); WT, 0; R304E, 0. The inset shows WT currentsrecorded from one cell in the presence of 10-6 M A-9-C at different external pH values.

358 J Physiol.501.2

pKa Of muscle chloride channels

Bretag et al. 1984). Our observations on whole-cell Cl-currents in Sf-9 insect cells expressing ClC-1 are in goodagreement with this previous work and provide moreinformation about the mechanism of transition metal block.The Hill coefficient of approximately 2 derived from thedose-response curve for block of inward currents by Cd2+indicates that ClC-1 has multiple transition metal bindingsites. It has been proposed that another member of thisfamily of ion channels, ClC-0, functions as a dimer,appearing to have two parallel conductance pathways(Hanke & Miller, 1983). While it has not been possible todemonstrate this in CIC-1, because the conductance is toosmall to allow direct recording of single-channel events,there is indirect evidence for a multimeric structure(Steinmeyer, Lorenz, Pusch, Koch & Jentsch, 1995) and adouble-barrelled arrangement could account for two Cd2+binding sites.

As to the nature of a possible binding site for transitionmetals, Zn2+ and Cd2+ bind readily to histidine, cysteineand acidic residues of protein molecules. An argumentfavouring the involvement of His residues is sustained bythe sensitivity to pH of ClC-1 block by Cd2+, where the

A

Q0.

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apparent pKa is 6-8. Values between 6-5 and 7 are typicalfor the imidazole side-chain of His when incorporated in aprotein. In contrast, the pKa of the sulfhydryl group ofcysteine is about 10-7 and that of carboxyl groups is around4, although there are examples where the acid dissociationconstants of particular groups have been shifted by severalunits by close proximity to neighbouring charged residues(reviewed by Hanke & Miller, 1983). A Hill coefficient of 3for the titration curve of the effect of Cd21 on ClC-1 currentamplitude suggests that the binding site consists of at leastthree residues, each of which can be protonated. Positiveco-operativity of proton binding can be explained byassuming that protonation of one of the residues involved inCd2' binding reduces affinity for the blocking cation andthus facilitates protonation of the next residue. It is notpossible to say, at this stage, whether these residues comefrom separate subunits of a multimer.

The effectiveness of another blocker of ClC-1, A-9-C, alsodepends on the external pH. In contrast to Cd2' binding,the potency of negatively charged A-9-C is increased whenexternal pH is lowered. A pKa of approximately 6-4 for theinfluence of pH on block by A-9-C is again consistent with

WT

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CU0. 0-4-0.

R304E

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-180 -120 -60Voltage (mV)

0 60

0 50 100Time (ms)

150

Figure 4. The effect of point mutation of Arg 304 to Glu on apparent open probability (PO) atnormal pHCurrent traces in Sf-9 cells expressing WT (A) and R304E (B) ClC- l were elicited by test pulses from -140to +80 mV in 20 mV increments, with a constant 'tail' pulse to -100 mV. Apparent PO was determinedfrom the tail currents by normalizing to the maximal tail current flowing after the most positive test pulses.C, apparent P. values are plotted as means + S.E.M. (n = 8; WT, 0; R304E, 0) with the continuous linesrepresenting fits of a Boltzmann distribution. Liquid junction potentials have been corrected.

II.-

J Physiol.501.2 359

3. Y Rychkov and others

the possible involvement of His residues. Previous studieshave shown that A-9-C competes with Cl- for the bindingsite (Astill et al. 1996) and that A-9-C is effective only fromthe external solution (G. Rychkov, unpublished observation).An existing model for A-9-C binding suggests that itconsists of a positively charged residue with a hydrophobicpocket in the vicinity (Bryant & Morales-Aguilera, 1971;Palade & Barchi, 1977). Greater affinity for the negativelycharged A-9-C at low pH might then be explained byprotonation of the binding site to increase its positivity, or itmight be that protonation of a relevant His residue altersthe characteristics of the site which binds A-9-C. A Hillcoefficient of 1 for the interaction between pH and block byA-9-C indicates that protonation of a single site is involvedin increasing the potency of the aromatic acid.

In recent studies, we have shown that the fast gating ofClC-1 is controlled by external Cl- in the same way as thefast gating of ClC-0 (Pusch, Ludewig, Rehfeld & Jentsch,1995; Rychkov et al. 1996). Cl- binding in the pore seems tobe essential for channel opening, and dissociation of Cl-from its binding site during hyperpolarization has beenproposed as a possible explanation for inward currentdeactivation (Rychkov et al. 1996). Reduced inward currentdeactivation as seen when the external pH was lowered inthese experiments suggests that protonation of a residue inthe channel increases the affinity of the regulatory site forCl-. The titration curve of channel gating, shown in thiswork, gives a pKa of about 6 1 and suggests that protonationof a single residue may be responsible for the change ingating behaviour. Again, the residue most compatible withthis pKa is His.

6

Fahlke et al. (1995) have used the alternative argument,involving an aspartate with an anticipated pKa of 4 1shifted to about 6, in explaining the effects of the D136Gmutation in human ClC-1. Inward currents, in this mutant,show slow activation on hyperpolarization at pH 7 4, unlikethe WT channel in which currents deactivate rapidly underthese conditions. When WT ClC-1 is exposed to low pH andheld at positive potentials between pulses, it too shows anactivating inward current on hyperpolarization (Rychkov etal. 1996). Protonation, at low extracellular pH, of theaspartate in position 136, would remove the negative chargeand might be expected to produce an effect on gatingpractically identical to that of the D136G mutation whereneutral Gly replaces the negative Asp. Comparison of theactivating inward currents in WT ClC-1 at low pH andDI36G does not support such a simple relationship, however,as those in D136G activate at least 10 times more slowlyand deactivate at more negative potentials (Fahlke et al.1995; Rychkov et al. 1996). It is also clear that Asp 136cannot be involved in A-9-C or Cd2+ binding because thesensitivity of the D136G mutant to either A-9-C (Fahlke etal. 1995) or Cd2+ (Kiirz, Wagner, George & Riidel, 1996) issimilar to that in WT ClC-1, whereas removing negativecharge on the WT channel by exposing it to low pH lowersthe IC50 for block by A-9-C 20-fold and completelyeliminates the block by Cd2+ (2-5 mM).

We have studied the pH-sensitive features of the R304Emutant because it has an increased steady-state openprobability at hyperpolarized potentials compared with WTClC-1. This increased open probability resembles the effectof low external pH on WT ClC-1, suggesting that the

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Figure 5. The effect of extracellular pH on deactivation ofWT C0C-1 and the R304E mutant

Sf-9 cells expressing ClC-I were stepped to a prepulse of +40 mV for 100 ms and then to a test potential of-120 mV for a further 100 ms. The quasi-steady-state current at the end of the -120 mV step is plotted asa proportion of the peak current at the start of the test pulse. Results are the means + S.E.M. (n = 5);WT, 0; R304E, 0. The inset shows typical R304E current traces recorded at a series of values ofextracellular pH.

360 J Physiol.501.2

pKa Of muscle chloride channels

R304E mutation may have altered the pKa of the residueinvolved in gating so that it was protonated at pH 7-4 andthat this mutant might be useful in locating the gatingregion of the channel. Titration of R304E gating indicated,however, that it has a pKa of 6 1, that the high open

probability remained even at high external pH and that,consequentially, the altered open probability of the mutantwas not a result of protonation. Further comparison of theresponses of R304E and WT ClC-1 to alterations ofextracellular pH and to the application of transition metalions does, however, provide further insight into the natureof ClC-1. In R304E the affinity of the Cd2+ binding site isreduced and greater competition from protons is suggestedby an increase in the apparent pKa. Reduction of the Hillcoefficient for protonation of the Cd2+ binding site in themutant indicates that a steric change has occurred so that itis no longer possible for Cd2+ to co-ordinate with all of theresidues that formed the site in the WT channel. Increasedaffinity for protons by each residue in the binding site,indicated by the increase in the apparent pKa, might bereflecting an increased nucleophilicity, induced, in turn, bythe local electrostatic effect of replacing a positive Arg witha negative Glu. It could also be a reflection of a reduced Cd2+binding affinity of all groups within the binding site. WhileArg 304 might be sufficiently close to the transition metalion binding site to alter its characteristics, it does not appear

to be in the vicinity of the gating region or the A-9-Cbinding site since the pKa for gating as well as for the blockof conductance by A-9-C are identical in the WT and mutant.Together, these results support the contention that thosetitratable sites involved in inward current deactivation andA-9-C block are likely to be distinct, and distant, from thosewhich influence transition metal binding, but that in eachcase they could be His residues.

Recently, it has been shown that Zn2+ blocks human ClC-1with an IC50 and Hill coefficient similar to those shown herefor rat ClC-1 (Kiirz, Wagner, George & Riidel, 1997). On thebasis of studies with protein-modifying reagents, theseauthors suggest that a mixed site involving cysteine andhistidine residues may be involved in binding Zn2+, althoughthey have not shown modification of the effects of transitionmetal ions by these reagents. Studies using site-directedmutagensis of cysteine and histidine residues potentiallyinvolved in transition metal binding, channel gating andA-9-C binding should resolve the nature of the residuesresponsible for the characteristics of ClC-1.

ASTILL, D. ST J., RYCHKOV, G. Y., CLARKE, J. D., HUGHES, B. P.,ROBERTS, M. L. & BRETAG, A. H. (1996). Characteristics of skeletalmuscle chloride channel ClC-1 and point mutant R304E expressedin Sf-9 insect cells. Biochimica et Biophysica Acta 1280, 178-186.

ASTILL, D. ST J., RYCHKOV, G. Y., HUGHES, B. P., BRETAG, A. H. &ROBERTS, M. L. (1995). The involvement of histidine in the functionof ClC-1, the skeletal muscle chloride channel. Proceedings of theAustralian Physiological and Pharmacological Society 26, 268P.

BARRY, P. H. (1994). JPCalc, a software package for calculating liquidjunction potential corrections in patch-clamp, intracellular,epithelial and bilayer measurements and for correcting junctionpotential measurements. Journal of Neuroscience Methods 51,107-116.

BIRNIR, B., TIERNEY, M. L., HOWITT, S. M., Cox, G. B. & GAGE, P. W.(1992). A combination of human acl and ,B1 subunits is required forformation of detectable GABA-activated chloride channels in Sf9cells. Proceedings of the Royal Society B 250, 307-312.

BRETAG, A. H. (1987). Muscle chloride channels. Physiological Reviews67, 618-724.

BRETAG, A. H., FIETZ, M. J. & BENNETT, R. R. J. (1984). The effectsof zinc and other transition metal ions on rat skeletal muscle.Proceedings of the Australian Physiological and PharmacologicalSociety 15, 146P.

BRYANT, S. H. & MORALES-AGUILERA, A. (1971). Chloride conductancein normal and myotonic muscle fibres and the action of mono-carboxylic acids. Journal of Physiology 219, 367-383.

DE BIASI, M., DREWE, J. A., KIRSCH, G. E. & BROWN, A. M. (1993).Histidine substitution identifies a surface position and confers Cs+selectivity on a K+ pore. Biophysical Journal 65, 1235-1242.

FAHLKE, C., RUDEL, R., MITROVIC, N., ZHOU, M. & GEORGE, A. L. JR(1995). An aspartic acid residue important for voltage-dependentgating of human muscle chloride channels. Neuron 15, 463-472.

HANKE, W. & MILLER, C. (1983). Single chloride channels fromTorpedo electroplax: activation by protons. Journal of GeneralPhysiology 82, 25-45.

HUTTER, 0. F. & WARNER, A. E. (1967a). Action of some foreigncations and anions on the chloride permeability of frog muscle.Journal of Physiology 189, 445-460.

HUTTER, 0. F. & WARNER, A. E. (1967 b). The pH sensitivity of thechloride conductance of frog skeletal muscle. Journal of Physiology189, 403-425.

JENTSCH, T. J., GUNTHER, W., PUSCH, M. & SCHWAPPACH, B. (1995).Properties of voltage-gated chloride channels of the ClC gene family.Journal of Physiology 482.P, 19-25S.

KtRZ, L. L., WAGNER, S., GEORGE, A. L. & RtDEL, R. (1996).Inhibition of human muscle chloride channels by histidine andcysteine-reactive compounds. Pflilgers Archiv 431, R86.

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AcknowledgementsWe are grateful to Professor T. J. Jentsch and Dr K. Steinmeyer ofthe Centre for Molecular Neurobiology, Hamburg, for providing therat ClC-1 clone and to Dr B. Birnir of the John Curtin School ofMedical Research, Australian National University, Canberra, forthe Sf-9 cell line. This work was supported by the NeuromuscularResearch Foundation of the Muscular Dystrophy Association ofSouth Australia, the Australian Research Council and the ResearchCommittee of the University of South Australia. D.StJ.A. held aRoss Stuart Postgraduate Scholarship of the Muscular DystrophyAssociation of South Australia.

Author's email addressM. L. Roberts: mroberts@physiol.adelaide.edu.au

Received 1 October 1996; accepted 28 February 1997.