hyperpolarization-activated Cl- current, this current could be detected if the

Transcription

1 J. Phy8io. (1983), 342, pp With 17 text-figure8 Printed in Great Britain CHARACTERIZATION OF A CHLORIDE CONDUCTANCE ACTIVATED BY HYPERPOLARIZATION IN APL YSIA NEURONES BY DOMINIQUE CHESNOY-MARCHAIS From the Laboratoire de Neurobiologie, Ecole Normale Supe'rieure, 46, rue d'ulm Paris (Received 4 January 1983) SUMMARY 1. A voltage-clamp study was made of some properties of the non-synaptic hyperpolarization-activated C1- conductance recently described in Aplysia neurones loaded with Cl- ions (Chesnoy-Marchais, 1982). The experiments were performed on an identified family of neurones, which present cholinergic responses allowing an easy measurement of the equilibrium potentials of Cl- (EC1) and K+ ions (EK). 2. The Cl- selectivity of the hyperpolarization-activated conductance was deduced from four observations: (1) the extrapolated reversal potential of the hyperpolarization-activated current, Er, was close to the reversal potential ofthe cholinergic Cl- response, which is the equilibrium potential for Cl- ions, EC1. (2) Modifications of the intracellular or extracellular Cl- concentration induced changes of the reversal potential Er. (3) A prolonged and intense activation of the current lowered the intracellular Cl- concentration. (4) The current persisted after complete substitution of intracellular and extracellular cations by Cs+ ions, as well as after replacement of extracellular Na+ ions by Tris. 3. The steady-state Cl- conductance (ass) increases steeply with hyperpolarization. The kinetics of activation and deactivation are exponential and are characterized by the same voltage-dependent time constant (r), of the order of a few seconds or fractions of seconds. The curves g.8(v) and r( V) can both be fitted by a two-state model in which the rate constants are exponential functions of the membrane potential (e-fold change for mv). 4. The Cl- current is much more affected by changes of the intracellular Clconcentration than predicted simply from the change in Cl- driving force. Both the conductance and the time constant of activation are strongly modified. Modifications of the extracellular Cl- concentration do not always alter the amplitude of the hyperpolarization-activated Cl- current, but systematically affect its kinetics. 5. The hyperpolarization-activated current is abolished after prolonged exposure of the cell to an artificial sea water where N03- ions replace Cl- ions, as well as after intracellular injections of N03- ions. 6. Increasing the external ph shifts the g.(v) and r(v) curves to the left. Lowering the external ph has reverse but less pronounced effects. 7. In cells which were not loaded with Cl- ions and did not present the hyperpolarization-activated Cl- current, this current could be detected if the

2 278 D. CHESNO Y-MARCHAIS hyperpolarizing jump was preceded by short depolarizing pulses. In cells which were loaded with Cl- ions, the Cl- current became larger after a short depolarizing pulse. In the presence of extracellular C02+ ions, depolarizing pulses no longer increased the Cl- current. 8. The Cl- current is not affected by extracellularly applied DIDS (4,4'- diisothiocyano-2,2'-disulphonic acid stilbene), but is markedly reduced by intracellular injection of DIDS. 9. Extracellular Cs+ ions, which have been reported to block some cationic hyperpolarization-activated inward currents, do not reduce the hyperpolarizationactivated Cl- current. High concentrations of Cs+ produce complex effects which are probably due to an increased synaptic activity, but the hyperpolarization-activated Cl- current persists after complete substitution of the extracellular and intracellular monovalent cations by Cs+. INTRODUCTION It was recently reported (Chesnoy-Marchais, 1982) that, in Aply8ia neurones which have been loaded with Cl- ions, hyperpolarizing voltage jumps induce a slowly increasing inward current. Using neurones which allowed a precise measurement of the Cl- and K+ equilibrium potentials (as the reversal potentials of the rapid and slow cholinergic responses), it has been established that the hyperpolarization-activated inward current results from the activation of a voltage-gated Cl- conductance. More evidence for the Cl- selectivity of this conductance is presented here. Furthermore, some properties of the Cl- conductance are analysed. In particular, its voltage sensitivity and the effects ofchanging intracellular and extracellular Cl- concentrations on the kinetics of activation of the Cl- current are studied in detail. METHODS Preparation. All experiments were performed at room temperature on the A cells (Jahan-Parwar & Fredman, 1976) ofthe cerebral ganglion of the mollusc ApIy8ia californica. These neurones present the advantage of providing an easy measurement of the equilibrium potentials for Cl- ions (Ec1) and K+ ions (EX) as the reversal potentials of the fast and slow responses to cholinergic agonists (Gruol & Weinreich, 1979; Ascher & Chesnoy-Marchais, 1982). The ganglion was isolated and pinned onto the bottom of a small experimental chamber covered with a silicone resin. The connective tissue covering the cells was removed by dissection. We continuously perfused the dissected ganglion with control sea water (480 mm-nacl, 10 mm-kcl, 50 mm-mgcl2, 10mM-CaCl2, buffered to ph 7-6 with 5 mm-hepes (N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic acid NaOH). Electrical arrangement. Cells were voltage-clamped by using two separate micro-electrodes. The membrane current was read as a voltage drop across a 1 MCI resistor interposed in the feed-back loop. The gain of the loop was 500. The current was recorded on a paper recorder (Brush 280), and on magnetic tape (Racal). The voltage recording micro-electrode was filled either with K2SO4 (0 5 M) or with KCl (0'5-3M). The current micro-electrode was always filled with K2SO4 (0 5M). The bath was connected to ground via an agar-bridge (agar 3 %; sea water). It was absolutely necessary to avoid Ag-AgCl electrodes even when only very small modifications of the extracellular Clconcentration were done. The Cl- current is so voltage-sensitive that very small shifts of the 'ground' could induce pronounced modifications of the current. The Cl- independence of the agar ground was controlled by measuring in different solutions the bath potential with a low resistant 3M-KCI-filled micro-electrode. The small shift (of about 5 mv) which was observed in the case of

3 H YPERPOLARIZATION-ACTI VATED Cl- CONDUCTANCE 279 the replacement of control sea water by the mannitol sea water was carefully corrected for during the experiments where such a substitution was done. Compo8ition of the extracellular 8soluton&. The composition of the various extracellular solutions which were used is given in Table 1. The substitutions were nearly iso-osmotic. The so-called 65 % mannitol (or 65 % isethionate) solution was prepared by mixing 35 % (v/v) control and 65 % (v/v) mannitol (or isethionate) solution. Thus, in these low Cl- solutions, the Cl- concentration was onehalf that in the control. When replacing Cl- ions by other anions (such as NO3- or isethionate), it was necessary to control the concentration of free Ca2+, since Cal+ ions are known to complex readily with various anions TABLE 1. Composition of various extracellular solutions. The control Na salt was NaCl. All solutions contained 5 mm-hepes-naoh, ph 7-6. The concentrations are indicated in mm Extracellular Na salt solution or substitute KCl MgCl2 CaCl2 Control Isethionate NO Mannitol (see for example Kenyon & Gibbons, 1977). Therefore, during the preparation of the various solutions, the Cal+ activity of the solutions was measured with a Ca2+-sensitive electrode (Radiometer F 2002); the reference electrode was a KCl-calomel electrode (Radiometer K 401). The Cal+ activities of the control and test solutions were adjusted to the same value. In the case of the substitution of Cl- by isethionate ions, it was necessary to put 15 mm-cacl2 in the isethionate solution in order to obtain the same Ca2+ activity as in control sea water. In the case of the NO3- solution, the complexation of Ca2+ ions was so marked that it was not possible to obtain the same Ca2+ activity as in control sea water. Therefore, 20 mm-cacl2 was included in the NO3- solution and the CaCl. concentration was lowered to 5 mm, instead of 10 mm, for the control in experiments where NO,- substitutions were done (Figs. 7 and 8). (See note added in proof.) The ph 6-0 and ph 9-0 solutions were identical to the control sea water apart from the buffer: they did not contain HEPES but contained either 10 mm-potassium bipthalate NaOH (ph 6-0 and no KCl) or 5 mm-sodium tetraborate HCl (ph 9 0). [1]i is used below to represent the intracellular concentration of an ion I, and [I]e to represent its extracellular concentration. Intracellular injections. In order to inject ions (such as Cl-, H+, EGTA, etc.) by ionophoresis inside the cell, I used double-barrelled pipettes. One pipette barrel was filled with the drug and the second one was filled with KSO4 (0-5 M). This double-barrelled pipette was inserted into the cell just after the insertion of the two recording electrodes. Ionophoretic currents were generated by WPI microionophoresis programmer units (WPI 160). A continuous braking current of about 10 na was usually applied to avoid leakage during control measurements performed before the intracellular injections. Nystatin experiments. Treatment with nystatin (experiment of Fig. 16) was performed according to Russell, Eaton & Brodwick (1977). The dose of nystatin was very critical: doses higher than 20 mg 1-l always damaged the neurones. Before treatment with nystatin, the ganglion was perfused with a Cs+-loading solution (300 mm-cscl, 394 mm-sucrose, 10 mm-mgso4, 5 mm-hepes-csoh, ph 7-6). Nystatin was then applied in this solution for 30 min followed by 30 min washing with the nystatin-free Cs+-loading (517 mm-cscl, 10 mm-cacl., solution before perfusion with the Na+-free, K+-free, Cs+ solution 50 mm-mgcl3, 5 mm-hepes-cs OH, ph 7-6). Before recording, the cell bodies were isolated by cutting the axons, in order to avoid Cs+-induced synaptic activity. For these experiments, the intracellular micro-electrodes were filled with CsCl (0-5 M) or CsSO4 (0 5 M).

4 280 D. CHESNO Y-MARCHAIS A na 4 s < -70 I~~~~~nA ~70 4 s -90 5nA s Time, t (s) 3 C D LO2 0 I* *VH) VH III I Vt(mV) Er Fig. 1. Hyperpolarization-induced slow relaxations. A, relaxations recorded after hyperpolarizing jumps from -50 to -60, -70 and -90 my ('on/' relaxations) and after returning to -50 mv (' off' relaxations). B, semi-logarithmic plot of the 'on ' relaxations. The slow increases in inward current occurring during the hyperpolarizing jumps from -50 my to V, as well as the slow decreases in inward current occurring after the return to -50 mv were exponential. This allowed the precise measurement, by extrapolation of the exponential relaxations, of the magnitude of these current changes (AI( V) and AI( V -* 50); see Fig. 1 D for notations; the zero current level is arbitrary). C, measurement of the reversal potential of these hyperpolarization-activated slow inward currents, by extrapolation of the ratio I(-50 V)/AI(V -50) (see eqn. (3) p. 282). This reversal potential, Er, was found very close to the Cl- equilibrium potential, Eci, which was measured as the reversal potential of the cholinergic Cl- response. The value of Eslwas measured both before and after the hyperpolarizing pulses, and was not affected by these pulses. EC1 and Er are indicated by the arows. The voltage dependence of the steady-state conductance is given by Fig. 2A (a). The time constants tr(v) of the exponential relaxations are plotted in Fig. 2B (t). RESULTS Hyperpolarization-activated Cl- current This section presents the three parameters which have been used to characterize the hyperpolarization-activated current: the time constant of the relaxations, the reversal potential and the steady-state value of the hyperpolarization-activated C1 conductance underlying this current.

5 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE Exponential slow relaxation. r( V) curve. Fig. 1 A illustrates the hyperpolarizationactivated C1- current in a neurone which was loaded with C1- ions, in order to increase the current. The membrane potential was held at -50 mv and was raised for a few seconds to more hyperpolarized levels V (between -60 and -100 mv). After the instantaneous current change which was induced by such a hyperpolarizing jump, there was a slow increase in inward current ('on' relaxation), which reached its new steady-state value (IS( V)) after a few seconds. When the potential was brought back from V to -50 mv, the current slowly returned to IS(-50) (off relaxation). It is clear in Fig. 1 A that, as V was made more hyperpolarized, the 'on' relaxation became larger and faster (note the two different scales of Fig. 1 A). This is illustrated by the semi-logarithmic plot of Fig. 1 B which shows that the 'on' relaxations were nearly exponential and had a much shorter time constant at -90 mv (r(-90) = 0 7 s) than at -70 mv (r(-70) = 2-5 s) and -60 mv (r(-60) = 35-5 s) (see also Fig. 2B (0)). The 'off' relaxations, recorded at -50 mv on return from the various potentials V, were also exponential and their time constant r(- 50) was not dependent on V. Furthermore, for any potential V between -50 and -90 mv, allowing both the measurement at V of an 'on' relaxation, after a hyperpolarizing jump from the holding potential VH (-40 or -50 mv) to -100 mv, and the measurement of an 'off' relaxation, on repolarization to V after a pre-pulse from VH to -100 mv, it was found that the time constants ron( V) and Toff(V) of these relaxations were identical (Chesnoy-Marchais, 1982). These results indicate that the activation and the deactivation of the Cl- current have the same kinetics and may be characterized by the same T( V) curve. This is a rather simple situation, which is in agreement with a two-state model (see Discussion) and which in practice allowed the use of either 'on' or 'off' relaxation in building the r( V) relation. In most experiments, the value of T corresponding to the holding potential VH was obtained from 'off' relaxations while the other values r(v) were obtained from the 'on' relaxations, recorded during hyperpolarizations from VH to V. However, when the 'on' relaxation was too small, 7r(V) was measured from the 'off' relaxation recorded at V on repolarization from -90 or -100 mv. Estimation of the reversal potential, Er. The method which had been used previously to estimate the reversal potential of the hyperpolarization-activated current, Er, required two series of voltage jumps: hyperpolarizing jumps from the holding potential VH to various potentials V, and repolarizing jumps to the potentials V following a jump from VH to a hyperpolarized level (e.g. -95 mv) (see Chesnoy- Marchais, 1982, fig. 2a). Fig. 1 C reports the measurement of Er by another method which requires only one series of rectangular hyperpolarizing jumps (from the holding potential VH to various potentials V) but requires the measurement of both AI( VH-. V) (the slow increase in inward current occurring during the 'on' relaxation at V) and AI( V -_ VH) (the slow decrease in inward current occurring during the 'off' relaxation at VH (see Fig. 1 D for notations). Assuming that AI(VH-. V) results from the activation at V of a conductance, corresponding to an increase in the number of 'open channels' (or 'active units') from n,,(vh), steady-state value at VH, to nss(v), steady-state value at V, and that AI(V -. VH) results from the reverse deactivation of this conductance, it appears 281

6 282 D. CHESNO Y-MARCHAIS that the ratio of these values, AI( VH-. V)/AI(V -+ VH), is a normalized value of the elementary current, iei(v), carried by each single active unit at the potential V (eqn. (3)): AI(VH-. V) = (nss(v)-nss(vh)) X iei(v), (1) AI(V-. VH) = (nss(v)-nss(vh)) X iei(vh), (2) AI( VH -+ V) _ie( V) (3) AI( V _+ VH) ie ( VH) Therefore, the potential at which this ratio changes its sign is the reversal potential of the hyperpolarization-activated current. Fig. 1 C shows that at least between -50 and -90 mv, the elementary current could be considered as a linear function of V. Assuming that the elementary current did not rectify above -50 mv, the reversal potential, E, was measured by linear extrapolation. The value of Er which was obtained by this method was very close to the value of EC1 which was measured as the reversal potential of the cholinergic Cl- response (Fig. 1 C). A few experiments were done to try to measure directly the reversal potential of the 'off' relaxations obtained by repolarizing from -100 mv to various potentials V close to EC1. These experiments were not very successful. When the membrane was repolarized from -100 mv to a potential V more depolarized than EC1 (EC1 was usually above -40 mv since the neurones were loaded with C1-), the slowly decreasing outward current, which was expected from the slow deactivation ofthe Cl- conductance, was not always observed. On the contrary, a slowly decreasing inward current (or increasing outward current) was often observed on return to V. This observation probably results from additional voltage-gated currents such as Ic (Adams, Smith & Thompson, 1980) which contaminated the Cl- relaxation. Calculation of the Cl- conductance. In order to obtain simple exponential Clrelaxations during hyperpolarizing pulses, and to avoid the complex relaxations which may result from the simultaneous change ofseveral voltage-gated conductances, it was usually sufficient to set the holding potential VH below -40 mv. This allowed the measurement of the time constant of activation and the estimation of the reversal potential. However, if one attempted to evaluate the steady-state Cl- conductance, g8s( V), from the slow increase in inward current that occurs after the hyperpolarizing jump (AI(VH-. V); see Fig. 1D), the choice of the holding potential was more difficult. AI( VH-. V) being given by eqn. (1 a) (see below), it was necessary to choose a holding potential at which the Cl- conductance was not activated: AI(VH-* V) = ( 9SS(V)-9SS(VH)) X (V-Er) (la) It was estimated that the Cl- current was not activated at the holding potential when hyperpolarizing or depolarizing jumps from VH, of at least 10 mv amplitude, did not induce any slow relaxation similar to the Cl- ones. When this was the case, the steady-state conductance, g.( V), was calculated according to eqn. (4): gs()=ai( VH -* V) 4 = (V-Er) (4) The value of Er which was used to calculate g,8( V) was usually the value of EC1 (obtained from the reversal potential of the cholinergic Cl- response) rather than the extrapolated reversal potential of the hyperpolarization-activated current.

7 HYPERPOLARIZATION-ACTI VATED Cl- CONDUCTANCE In some cases (particularly when the intracellular Cl- concentration was high), in order not to activate the Cl- conductance at the holding potential, it was necessary to set VH at a depolarized level where other voltage-gated conductances were activated. The resulting currents sometimes contaminated the Cl- relaxations so much that they prevented the evaluation of g,8( V). Effects of the intracellular Cl- concentration The hyperpolarization-induced Cl- relaxations shown in Fig. 1 were usually nearly undetectable if the neurone had not been loaded with C1- ions, but they became very large as the intracellular Cl- concentration was increased (Chesnoy-Marchais, 1982). This effect was much larger than expected from a simple change in driving force for Cl- ions. For example, changing EC1 from -50 to -40 mv (by intracellular injection of Cl- ions) increases the driving force for Cl- ions at -100 mv only by a factor 1P2; however, this may enhance the slow increase in Cl- current observed during a jump from -50 to -100 mv (AI( )) by a factor higher than 10. The effects of intracellular Cl- ions were therefore analysed in more detail. Three methods were used to modify the intracellular Cl- concentration. The easier one consisted of first loading the neurone with Cl- ions (by the leak of the KCl- filled micro-electrode) in order to obtain a first set of data, and then to hyperpolarize the neurone for several minutes through a K2S04-filled micro-electrode. This, as shown below, induced a slowly reversible lowering of the intracellular Cl- concentration, and thus allowed the recording ofa second set of data. The results obtained by this method are presented first and were compared to those obtained by using successive intracellular ionophoretic injection of Cl- ions (second section). The effects of altering the intracellular Cl- concentration by cholinergic stimulation are reported in a third section. Lowering the intracellular Cl- concentration by prolonged hyperpolarization8. In the experiments previously described, the duration of the hyperpolarizing jumps used to study the hyperpolarization-activated Cl- conductance was usually no longer than necessary to allow measurement of the 'steady-state' Cl- current ( < 40 s). 283 It was determined that such short duration hyperpolarizing jumps did not modify EC1. However, if hyperpolarizing jumps of several minutes duration were applied, the situation was more complex and differed depending upon whether the jump did, or did not, activate the Cl- conductance. If it did not (for example because the intracellular Cl- concentration was too low), the current trace was stable as long as the potential was maintained hyperpolarized, and EC1 was not modified by this prolonged hyperpolarization (this observation was already made by Ascher, Kunze & Neild (1976)). However, if the hyperpolarization did activate the Cl- conductance, the 'on' relaxation observed during the first seconds was followed by a much slower decrease of the inward current which lasted for minutes. This very slow decrease in inward current, occurring at hyperpolarized membrane potentials at which the Clconductance is activated, was simultaneous with a progressive change in EC1, which, initially between -20 and -40 mv, got progressively nearer to -60 mv during the prolonged hyperpolarization. The changes in EC1 which were induced by prolonged hyperpolarizations most probably resulted from the outward net flux of Cl- ions which occurred during the hyperpolarization. In the experiment reported in Fig. 2, the neurone was loaded with Cl- ions by the

8 284 D. CHESNO Y-MARCHAIS leak of the voltage-recording micro-electrode until EC, reached a stable value of -24 mv. A first set of records was then obtained. Then, the neurone was hyperpolarized for 5 min at -100 mv. This brought ECi from -24 mv to -38 mv corresponding to a decrease of the intracellular Cl- concentration by a factor 1-7. Fig. 2 shows the voltage dependence of the hyperpolarization-activated Clconductance (A), as well as the voltage dependence of its kinetics of activation (B) for the two values of EC,. 150 *B A *5 0-2 I V (mv) V (mv) Fig. 2. Effects ofa prolonged hyperpolarization lowering the intracellular Cl- concentration on the steady-state conductance and the kinetics of activation of the Cl- current. Before the prolonged hyperpolarization (@), ECi was stable at -24 mv. (The neurone had been loaded with Cl-.) After the hyperpolarization to -100 mv during 5 min (0), EC1 was around -38 mv. This indicates that the prolonged hyperpolarization had lowered the intracellular C1- concentration from 235 to 135 mm. A, the steady-state C1- conductance 98( V) was calculated from 'on' relaxations from -40 mv according to eqn. (4). This was possible because the Cl- conductance was not activated at -40 mv. B, the prolonged hyperpolarization, which lowered the intracellular Cl- concentration, markedly reduced the steady-state conductance of the hyperpolarization-activated Cl- current, and affected its kinetics of activation. Same neurone as in Fig. 1. The decrease of the intracellular Cl- concentration strikingly reduced the hyperpolarization-activated Cl- conductance (Fig. 2 A). This decrease was particularly marked for membrane potentials close to the threshold of activation of this conductance. The kinetics of activation of this conductance were also affected, becoming slower at very negative membrane potentials and more rapid around -50 mv (Fig. 2B). These effects were confirmed in three other similar experiments. Successive intracellular injections of Cl-. One could argue that the prolonged hyperpolarization used to lower the intracellular Cl- concentration may affect the Cl- current, independently of the observed change in EC,. Therefore a few experiments in which the intracellular Cl- concentration was directly modified were done, by successive intracellular ionophoretic injections ofcl- ions, through a double-barrelled micro-electrode which was introduced into the cell in addition to the two usual micro-electrodes. In the experiment illustrated in Fig. 3, a series of hyperpolarizing jumps, from -45 mv to -55, -65, -95 mv, was applied at three different

9 HYPERPOLARIZATION-ACTIVATED CV- CONDUCTANCE 285 A Ea(M) -45 F na los 250 r E 150[ j J 100p- 501 ol c V (mv) S U)n t t LOt X,~ _.. _. _. _1 5' ss S B f'-25t V (mv) V (mv) Fig. 3. Effects of direct modifications of the intracellular C1- concentration. The neurone was first loaded with Cl- ions by the leak from the KCl-filled intracellular micro-electrodes, bringing Ec, to a stable value of -45 mv. Then, after a series of hyperpolarizing pulses for -45 to -55, -65, -75, -85, -95 mv, Cl- ions were injected by ionophoresis (through a double-barrelled micro-electrode) bringing Eci first to -29 mv and then to -20 mv. The same series of hyperpolarizing pulses was repeated for these two levels of intracellular Cl-. A, current records during voltage jumps from -45 mv to -75 mv (upper row) or -85 mv (lower row) for the three intracellular Cl concentrations. B, measurement of the reversal potential by extrapolation of the ratio AI(-45 -_ V)/IAI( V -_ -45) (see eqn. (3)) for the two highest values of the intracellular Cl- concentration. (For EC, = -45 mv it was not possible to measure AI(V ).) Increasing the intracellular Cl- concentration shifts in the same way the reversal potential, Er, and the Cl- equilibrium potential, Ecl, which was measured as the reversal potential of the cholinergic Cl- response. Eci and Er are indicated by arrows. C, steady-state conductance g.s(v) calculated according to eqn. (4), for EC, = -45 mv (O), -29 mv (0) and -20 mv (ZO). D, voltage dependence of the kinetics of activation (or of deactivation upon return to V after a jump from -45 to -95 mv) for Eci = -45 mv (O), -29 mv (@) and -20 mv (c). About 1 h after the end of the second intracellular Cl- injection, Eci was back around -45 mv, and records very similar to the initial ones were obtained. intracellular Cl- concentrations: before the first C1- injection, when EC1 had stabilized at -45 mv; after a first injection which brought EC1 to -29 mv and then after a second injection, which brought EC1 to -20 mv. Fig. 3B shows the influence of the intracellular Cl- concentration on the reversal potential of these hyperpolarizationactivated inward currents. The reversal potentials were measured by extrapolation - I- 10o 5 2 D

10 286 D. CHESNO Y-MARCHAIS of the ratio AI( V)/AI(V--45), as in Fig. 1. Fig. 3C and D show the influence of the intracellular Cl- concentration on the voltage dependence of the steady-state conductance and on the voltage dependence of the kinetics of activation. The effects described in this Figure are consistent with those described in Fig. 2; this justifies the use of prolonged hyperpolarizations as a method to easily modify the intracellular Cl- concentration. AI 4$s 5 na B a b c d Fig. 4. Agonist-induced changes in the hyperpolarization-induced C1- relaxations. Records from two different experiments are reported. In A are shown the current traces recorded during hyperpolarizing jumps from -60 to -110 mv, before (left) and 5 min after (right) a 4 min perfusion duration with 20 /SM-suberyldicholine. During the application of suberyldicholine, the membrane potential was held at -60 mv. Suberyldicholine induced a large efflux of C1- ions (inward current of about 5 na) and the disappearance of the Clrelaxation. This effect was slowly reversible (not shown). In B are shown the current traces recorded during hyperpolarizing jumps from -50 to -100 mv before (a), 7 min (b) and 25 min (c) after an 8 min perfusion with 20 /M-suberyldicholine performed at -20 mv. This perfusion induced a large influx ofcl- ions into the neurone (outward current of about 15 na), and transiently facilitated the activation of the Cl- current. The record shown in (d) was obtained after a 8 min depolarization to -20 mv performed in the absence of suberyldicholine and, as the control record (a), it did not present any slow relaxation. The neurones used in these experiments had not been intentionally loaded with Cl- ions and the recording electrodes were filled with KS04. The fact that a small Cl- relaxation was nevertheless present in A may indicate that some Cl- had entered the cell at the introduction of the electrodes. Alteration of the intracellular Cl- concentration by cholinergic stimulation. The hyperpolarization-activated Cl- conductance may be modified by prolonged applications of high doses of suberyldicholine, a cholinergic agonist which induces an increase in conductance specific for Cl- ions in molluscan neurones (Ger & Zeimal, 1976; Kehoe, 1979). In the experiment of Fig. 4A, suberyldicholine was applied at a potential more hyperpolarized than EC1, thus inducing a large net efflux of Cl- ions from the neurone; this suppressed the hyperpolarization-activated Cl- relaxation which was initially present. In contrast, when suberyldicholine was applied while the potential was held above EC1 (at -20 mv), it induced a large net influx of Cl- ions into the neurone and facilitated the activation by hyperpolarization of the Cl- current (Fig. 4B). Maintaining the neurone at -20 mv in the absence of suberyldicholine did not have the same effect (Fig. 4B, d). Such effects most probably result from

11 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE suberyldicholine-induced changes of the intracellular C1- concentration and from the very strong sensitivity of the hyperpolarization-activated C1- conductance to the intracellular Cl- concentration (Figs. 2 and 3). Effect of the extracellular Cl- concentration To lower the extracellular Cl- concentration, the external NaCl was replaced either by sodium isethionate (Fig. 5) or by mannitol (Fig. 6). In both situations, the amplitude of the slow Cl- relaxations could be either strongly or very slightly affected, depending on the voltage jumps which were applied. Fig. 5 shows the effects of the substitution of 480 mm-external Cl- ions by isethionate ions. The isethionate substitution increased by a factor 2-3 the amplitude of the 'on' relaxation induced by a jump from -40 to -50 mv (Fig. 5A), but did not increase the amplitude of the 'on' relaxation induced by a jump from -40 to -60 mv (Fig. 5B). The substitution also increased markedly the amplitude of the 'off' relaxations recorded at -40 mv. Whereas in control sea water these 'off' relaxations were nearly undetectable, they are clearly present in isethionate sea water. Such results were observed in all experiments, but the membrane potential range, below which the amplitude of the 'on' relaxation was no longer increased by the Cl- substitution, was different from one experiment to another and depended on the intracellular Clconcentration, becoming more and more depolarized as the intracellular Cl- concentration was increased. The substitution of external Cl- ions by isethionate also modified the time constant of activation of the Cl- conductance (Fig. 5B): r(-60) was 1-7 times larger in control than in isethionate sea water (see also Table 2). The substitution of Cl- by isethionate rapidly induced an inward current at the holding potential. This inward current may be a Cl- current. During prolonged perfusion with the isethionate solution, this inward current slowly decreased, and this decrease occurred simultaneously with a decrease of the instantaneous conductance, which is visible in Fig. 5. The nature of this slow decrease of the instantaneous conductance was not investigated. It is worth noting that it was not observed when the extracellular Cl- concentration was lowered by using mannitol instead of sodium isethionate. Fig. 6 illustrates the effects of replacing NaCl by mannitol. CoCl2 (10 mm) was added both in control and in mannitol sea water in order to avoid synaptic activity and to reduce the slow decrease of the intracellular Cl- concentration which may occur in low external Cl- solutions (shown to be reduced by Co2+ ions, Ascher et al. 1976). In Fig. 6A are shown the Cl- relaxations recorded during voltage jumps from -40 to -50 (a) or -70 (b) mv. The alterations produced by the mannitol substitution resemble those produced by the isethionate substitution: an increase of the amplitude of the 'on' relaxations for small hyperpolarizations, an increase of the amplitude of the 'off' relaxations and modifications of the kinetics. The r( V) curves offig. 6 B indicate that reducing the extracellular Cl- concentration shortened r at hyperpolarized membrane potentials but not at -40 mv: r(-70) was 2-1 times smaller in mannitol solution than in control, whereas r(-40) was 1-2 times larger in mannitol than in control. These results are confirmed by the experiments reported in Table

12 288 D. CHESNO Y-MARCHAIS Control Isethionate Wash 4 ~ ~ ~ *4- B na ~~~~~~~~~2s Fig. 5. Effects of the substitution of 480 mm-external Cl- ions by isethionate ions (see Table 1). The Cl- relaxations recorded during voltage jumps from -40 to -50 (A) or -60 (B) mv are shown in control (Cl-) sea water, after 9 min in isethionate sea water and 11 min after returning to Cl- sea water. The neurone was isolated by cutting the axon, in order to avoid changes in synaptic activity. Both the amplitude and the kinetics of the relaxations are dependent on the extracellular Cl- concentration (see text). The dashed lines indicate the steady-state current at -40 mv. TABLE 2. Sensitivity of the kinetics of activation of the Cl- current to the extracellular Clconcentration. The experiments reported here indicate that whatever the low-cl- extracellular solution was, lowering the extracellular Cl- concentration shortened r at hyperpolarized potential (V). In contrast, it lengthened T at depolarized levels (VH). r( VH) has been indicated only for the experiments in which it was reliably measurable T(V) (s) T(VH) (s) Low C1- solution VH(mV) V(mV) Control Low Cl- Control Low Cl- Isethionate % P7 1-2 Isethionate (three expts.) Mannitol P8 (three expts.) -85 1P % Mannitol (two expts.) Fig. 6C shows that the extrapolated reversal potential of the hyperpolarizationactivated current (Er) depends on the extracellular Cl- concentration; a 29 mv shift of Er was observed when reducing extracellular Cl- concentration from 630 mm (control value) to 150 mm (value in mannitol sea water). The Cl- equilibrium potential is expected to shift by 36 mv if the intracellular Cl- concentration is not affected. As the extrapolation was not very precise, and since a slight modification of the

13 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE 289 Control A a ~~~~~~~~04nA 10S ~, b 2 na 5 B 4 0 c 3 2~ ~~~~~~~ ' V (mv) V (mv) Fig. 6. Effects ofthe substitution ofthe external NaCl by mannitol. 10 mm-cocl1 was added to both solutions. A, Cl- relaxations recorded in control and mannitol solutions during voltage jumps from -40 to -50 (a) or -70 (b) mv. Although the current traces at -40 mv are presented at the same level in control and mannitol solutions, the replacement of NaCl by mannitol induced an inward current of about 1-3 na. B, voltage sensitivity of the time constant of activation of the Cl- current in control (0) and mannitol (@) solutions. C, measurement, by extrapolation of the ratio AI( V)/AI(V -v-40) (see eqn. (3)) of the reversal potential of the hyperpolarization-activated current in control (0) and mannitol (0) solutions. intracellular Cl- concentration may have occurred, the significance of the discrepancy cannot be readily assessed. In six similar experiments using either mannitol or sodium isethionate to replace NaCl, the reversal potential of the hyperpolarization-activated current was shown to be modified by lowering the extracellular Cl- concentration. These results support the Cl- specificity of the current under investigation. Furthermore, the fact that replacing extracellular Cl- ions by isethionate ions modifies Er, indicates that isethionate ions are not as permeant as Cl- ions. 10 PHY 342

14 290 D. CHESNO Y-MARCHAIS From these experiments, it is difficult to deduce whether or not the steady-state conductance was reduced by lowering the extracellular C1- concentration. The amplitude of an 'on' relaxation from VH to V does not give the steady-state conductance at V(g(I(V)) directly but gives the conductance increase occurring during the jump (gf.(v)-g9(vh)). Furthermore, lowering the external C1- concentration sometimes induced a lowering of the intracellular C1- concentration which may by itself strongly reduce the conductance. Control Nitrate Wash 3 min 20 min 10 min 30 min < _ 0*5 2snA Fig. 7. Effect ofreplacement ofextracellular Cl- ions by NO3- ions on the reversal potential of the cholinergic anionic response. The reversal of the response to a 2 s ionophoretic pulse of carbachol is shown, from the left to the right, in a control solution, 3 min and 20 min after complete substitution of external Cl- ions by NO3- ions, and 10 min and 30 min after returning to CI- sea water. The membrane potential values are indicated in mv. Since the pulses of carbachol were not applied at regular intervals, the amplitudes of the responses are not significant. In this experiment, the 'control solution' was not the usual control sea water, but contained 5 mm instead of 10 mm-cacl', in order to allow the comparison with the NO- sea water; furthermore, 1 mm-tea was present in both the Cl-, and the NO3- solutions. The results indicate (1) that NO3- ions are more permeable than Cl- ions through the cholinergic-activated anionic channels, and (2) that NO3- ions slowly enter the neurones. Effects of NO3- ions From measurements of the reversal potential of the cholinergic anionic response of the 'medial' cells of the Aplysia pleural ganglia, before, during and after substitution of the extracellular Cl- ions by NO3- ions, P. Ascher & A. Brisson (personal communication) had concluded that: (1) NO3- ions are more permeable than Cl- ions through the acetylcholine-activated anionic channels and (2) that extracellularly applied NO3- ions slowly penetrate into the neurones, thus replacing the intracellular Cl- ions. Fig. 7 illustrates an experiment similar to those which led to these conclusions, but which was performed on the A neurones of the cerebral ganglia. The reversal potential of the response to an ionophoretic pulse of carbachol was measured in the presence of 1 mm-tea (tetraethylammonium chloride), in order to eliminate the cholinergic K+ response (Kehoe, 1972; Ascher & Chesnoy-Marchais, 1982). In control, this reversal potential, EA, was around -61 mv. The substitution of the extracellular Clions by NO3- ions induced two effects, an immediate one and a progressive one. First, 3 min after the substitution, the reversal potential of the anionic cholinergic response, EA, was much more negative (around -79 mv). One can assume that at this time there is no NOW inside the cell and that the intracellular Cl- concentration

15 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE has not been modified. The shift of EA therefore indicates that NO3- ions are about two times more permeable than Cl- ions through the carbachol-activated anionic channels. According to the Hodgkin-Katz-Goldman equation giving the reversal potential of an ionic current as a function of the concentrations and permeabilities of the ions carrying this current (Hodgkin & Katz, 1949), we have: in control, EA = 58 log immediately after the substitution, EX = 58 log PC1[01i] PNO3[NO3aie' As [Cl-ie and [NOi-]e were equal, the shift of the reversal potential is: EA-EA = 58 log PN0. Secondly, during the next minutes following the perfusion with the extracellular NO3- solution, the reversal potential of the cholinergic anionic response was not stable, becoming more and more depolarized. After 20 min in NO3- sea water the reversal was around-68 mv (Fig. 7). This indicates that NO3- ions had slowly entered the cell. The situation was reversible during wash such that there was also an immediate effect and a progressive one. The effects on the hyperpolarization-activated current of the total replacement of extracellular Cl- ions with NO3- ions are shown in Fig. 8. A few seconds after this substitution, the hyperpolarization-induced slow 'on' relaxation was clearly reduced (Fig. 8A, b). After about 20 min in NO0- sea water, this relaxation was no longer detectable (Fig. 8A, c). On washing in control sea water, it slowly recovered its initial value (not shown). By using (as in Fig. 7) the anionic response to ionophoretic pulses of carbachol to follow the entry of NO3- ions, it was seen that the disappearance of the relaxation occurred only when NO3- ions had largely replaced Cl- ions inside the neurone. The progressive disappearance of the hyperpolarization-induced slow relaxations in NO3- sea water was observed in three similar experiments. The absence of any hyperpolarization-activated inward current after a long time in NO0- sea water may indicate that NO3- ions are completely impermeant through the hyperpolarization-activated pathway, but it could also indicate simply that intracellular Cl- ions are required for activation of this pathway. Even in control sea water, the Cl- current is not activated by hyperpolarization if the intracellular Clconcentration is low. If intracellular NO3- ions were unable to substitute for intracellular Cl- ions on the Cl- binding site which is implicated in the mechanism of activation of the current, one could imagine that NO3- ions permeate through the hyperpolarization-activated pathway but that the pathway could not be activated by hyperpolarization when NO3- ions have replaced the intracellular Cl- ions. Furthermore, intracellular NO3- ions injected into the neurone by ionophoresis completely blocked the large hyperpolarization-activated Cl- current which was observed before the injection of NO3- (Fig. 9). Certainly, the effects of extracellular

16 292 D. CHESNO Y-MARCHAIS a b C A Cl--NO3- K + id_ l s 4 s 2 na Cl--NO3-Kf Fig. 8. Blockade of the hyperpolarization-activated C1 current by external N03- ions. A, current records during hyperpolarizing pulses from -50 to -100 mv, in control sea water (a), 30 8 (b) and 35 min (c) after complete substitution of external C1- ions by N08-. B, responses to an ionophoretic pulse of carbachol, of 400 ms duration, applied 15 s after the voltage jumps. The slow response to carbachol is the K+ response; the initial response, which was nearly at equilibrium in control sea water (Fig. 8B, a), is the C1- response. This anionic initial response became a very large outward response just after the substitution of external Cl- by N03- (Fig. 8B, b); it then decreased (Fig. 8B, c), until it eventually reversed (not shown). These changes of the cholinergic anionic response at -50 mv result from modifications of the reversal potential of this response (Fig. 7) and indicate that NO3- ions slowly penetrate into the cell. The change of the current trace at -50 mv, which occurred immediately after the substitution of Cl- by NO3- (compare records a and b), does not seem to result from an effect of NO3- ions on the Cl- current. Indeed, it has been observed that replacement of Cl- by NO3- may induce an outward current even when, in control sea water, the Cl- current could not be activated by hyperpolarization. N03- ions therefore seem to induce additional effects. and intracellular NO3- ions require further study to be understood, but they may be very useful to block the hyperpolarization-activated C1- current. Effects of external and internal ph Increasing the external ph reduces the hyperpolarization-activated Cl- conductance. This is illustrated in Fig. IOA which shows the Cl- relaxations induced by hyperpolarizing pulses at ph 7-6 and at ph 9 0. The relaxation induced at ph 9 0 by a voltage jump from -50 to -120 mv, looks very much like the relaxation induced at ph 7-6 by the voltage jump from -50 to -100 mv (the duration of these jumps were, however, not identical). This suggests that increasing the external ph does not actually block the hyperpolarization-activated Cl- conductance, but rather shifts its activation curve along the voltage axis. This is confirmed by Fig. lob and C which give, both at ph 7-6 and at ph 9 0, the voltage dependences of the Cl- conductance, g98( V) and of the time constant of activation T( V). Although these curves are not very precise (the relaxations were not quite exponential in this experiment), it is clear that increasing the external ph not only affected the conductance, but also modified the kinetics. The activation became more rapid at -60 mv and slower at -100 mv. These effects did not result from

17 HYPERPOLARIZATION-ACTI VATED Cl- CONDUCTANCE 293 A B 1~~~~~~~ 0S- 2 na l0s C 7- ~~~~D Fig. 9. Effect of an intracellular injection of NO3- ions. In addition to the two usual voltage-clamp micro-electrodes, a double-barrelled micro-electrode was introduced into the neurone to allow intracellular ionophoretic injection of NO3- ions. One barrel was filled with KNO3 (1 M), the other with KS04 (0-5 M). A braking current of 10 na was applied between the two barrels, except during the injection, which was performed by applying a current of opposite polarity (50 na for 15 min). The neurone had been loaded with C1- ions, by the leak from the voltage-recording micro-electrode filled with KCl. The current traces recorded during hyperpolarizing jumps from -50 to -80 mv are shown, before the intracellular NO3- injection (A), during the injection (after 5 min injection) (B), 1 min after the end of the injection (C), and 1 h 10 min later (D). The Cl- relaxations were suppressed by the injection of NO3- (B and C). They slowly recovered after the end of the injection (D). The difference between the kinetics of the Cl- relaxations obtained before (A) and long after (D) the injection of NO3- was not investigated; it may result from the persistence of NO3- ions inside the neurone and/or from a modification of the intracellular Cl- concentration. a lowering of the intracellular C1- concentration since Ec1 was -34 mv at ph 7-6 and -32 mv at ph 9-0 in the experiment presented in Fig. 10. Similar results were obtained in four other similar experiments. Reducing the external ph has opposite, although less pronounced, effects: the r(v) curve was shifted to the right and the conductance was slightly increased (Fig. 11). Whereas the shift of the r( V) curve in the hyperpolarized range was a very reproducible result (shift of mv (5) (mean + s.e. of the mean (n)) when reducing the ph from 7-6 to 6 0), the increase in conductance was much more difficult to detect. In most cases the Cl- current slowly decreased during perfusion with the extracellular acid solution, and then did not recover its control value during wash. This may be explained if the intracellular medium becomes progressively more acid during the perfusion with the extracellular acid solution (see below). Reducing the internal ph by intracellular injection of H+ ions did markedly reduce the Cl- relaxations induced by hyperpolarizing jumps from -40 (or -50) mv to -90 (or -100) mv (not shown) in neurones which were loaded with C1-. This was observed in two independent experiments by using an intracellular double-barrelled microelectrode (one barrel was filled with K2SO4 0 5 M, the other with H2S04 1 M; injection of H+ ions (5-10 na during 1-2 min)).

18 294 D. CHESNOY-MARCHAIS ph ph A 2 na 5 na los B 120 _05 ~ '5 U~~~~~~~~~~~~~~~~~~~~~~~~~~ - at 1 I U 0 L p( V(mV) V (mv) Fig. 10. Effects of high external ph. Fig. 10A shows the records obtained either at ph 7-6 (upper line) or at ph 9-0 (lower line) during hyperpolarizing pulses from -50 to the various potentials indicated. Fig. lob and C show the curves g.(v) and r(v) obtained from such records, either at ph 7-6 (filled symbols) or at ph 90 (open symbols). The steady-state Cl- conductance, g5.(v), was calculated according to eqn. (4). Ec, was measured at both phs as the reversal potential of the cholinergic Cl- response. It was -34 mv at ph 7-6 and -32 mv at ph 9-0. For some membrane potentials the Clconductance was so small that it was not possible to measure the time constant of the 'on' relaxation (this was the case at -50 mv at ph 7-6 and at -60 and -70 mv at ph 9-0). In such a case, r was measured as the time constant of the 'off' relaxation observed when returning from -100 mv to the test potential V. This Figure shows that increasing the extracellular ph affects both the hyperpolarization-activated Clconductance and its kinetics of activation. VH was -50 mv. Effects of depolarizing pre-pul8eb The C1- current induced by hyperpolarizing jumps (from -45 to -75 mv) was larger when the jumps were preceded by a train of depolarizing pulses (from -45 to +15 mv) (Fig. 12A). However, the kinetics of the Cl- relaxations were not modified by the depolarizing pre-pulses. Since it is known that depolarizing pulses activate an entry of Ca2+ ions into neurones, and that this entry can be blocked by extracellular Co2+ ions, the experiment of Fig. 12A was repeated in the presence of Co2+. Fig. 12B shows that in the presence of Co2+ ions there was no longer any effect

19 HYPERPOLARIZATION-ACTIVATED Cl_ CONDUCTANCE r A 'or B 30F U) c S 201 J -a t OL _L -100 ii I I I I V (mv) V (mv) Fig. 11. Effects of low external ph. A, steady-state conductance in control sea water at ph 7-6 (-), at ph 6-0 (O), and back at ph 7 6 after wash of the ph 600 sea water (0). B, T(V) curve at ph 7-6 (-) and 60 (0). After wash, the x(v) curve was very similar to the control one. Ecl, measured as the reversal potential of the cholinergic Cl- response, was -40 mv, before, during and after perfusion with the ph 60 sea water. -50 mv. VH was a b c A I 1 na 20 s Fig. 12. The hyperpolarization-activated Cl- current is increased by a short train of depolarizing pulses. The current traces which were recorded during hyperpolarizing jumps from -45 to -75 mv are shown before (a), 3 min after (b) and 13 min after (c) a train of depolarizing pulses (pulses of 20 ms duration every 20 ms during 10 s) from -45 to + 15 mv in control sea water (A) and after lowering the intracellular Cal'+ concentration from 10 to 1 mm and replacing Mg'+ ions by Col+ ions (B). In control sea water, the hyperpolarization-activated Cl- current was increased by the train of depolarizing pulses (it was 1-8 fold larger in (b) than in (a) and (c)). In the Co'+ solution, the train ofdepolarizing pulses did not affect the Cl- current. After wash with control sea water, the depolarizing pulses again induced an increase of the Cl- current (not shown).

20 296 D. CHESNOY-MARCHAIS ofthe depolarizing pulses on the Cl- relaxations. These results were confirmed in seven similar experiments. (The Co2+-containing solution was either 1 mm-cacl2 and 60 (or 50) mm-coc12 or 1 mm-cacl2, 30 mm-mgcl2 and 30 mm-cocl2.) Moreover, in a neurone which had not been loaded with Cl- ions, hyperpolarization (from -60 to -100 mv) which initially did not induce any slow Cl- relaxation could activate one if preceded by a train of depolarizing pulses to +5 mv (Fig. 13). This A B C D 1 na Fig. 13. A train of depolarizing pulses may allow the activation of the C1- current by a previously non-activating hyperpolarization. The current traces recorded during hyperpolarizing jumps from -60 to -100 mv are shown before (A), 20 s after (B), 6 min after (C) and 16 min after (D) a train of depolarizing pulses from -60 to +5 mv (pulses of 100 ms duration every 100 ms during 10 s). The neurone had not been loaded with Cl-. Part of the extra noise occurring during the hyperpolarization is an artifact. effect was not obtained in the presence of Co2+ ions and was confirmed in five experiments, one of which was performed on an isolated cell body. The effects of depolarizing pulses were reversible in a few minutes (Figs. 12A, c and 13C and D). They do not seem to result from a modification of the intracellular Cl- concentration since EC, was not found to be affected by short depolarizing pulses. The fact that the effects of short depolarizing pulses disappeared in a low Ca2+- and Co2+-containing sea water suggests that these effects may result, either directly or indirectly, from an increase of the intracellular Ca2+ concentration. In a few experiments, intracellular ionophoretic injections of EGTA were performed into neurones previously loaded with Cl- ions. Such injections often reduced the hyperpolarizationactivated Cl- current. However, this effect was difficult to interpret since in some cases, a parallel change of EC, was also measured, indicating a decrease of the intracellular Cl- concentration. The origin of this second effect is not clear but it may be the result of an osmotic entry of water. Extracellular Ca2+ ions do not seem to be necessary to the activation of the Clconductance by hyperpolarization. When the 10 mm-cacl2 normally present in the control sea water had been replaced by 10 mm-mgcl2, and 1 mm-egta had been added, it was possible to record large hyperpolarization-induced Cl- relaxations, which were even larger than in control sea water. These results do not favour the hypothesis that the depolarizing pre-pulses act by increasing the intracellular Ca2+ concentration. Another explanation is suggested by the recent demonstration that depolarizing pulses may induce an increase of the intracellular ph of molluscan neurones and that this effect no longer occurs in the presence of extracellular Co2+ ions (Thomas & Meech, 1982). As it has been found

21 H YPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE that decreasing the internal ph reduces the Cl- conductance (see p. 293), it is possible that depolarizing pulses increase this conductance by increasing the internal ph. Whatever their exact interpretation, the effects of depolarizing pre-pulses may have a physiological significance. Fig. 13 shows that a short but strong depolarization may allow the activation by hyperpolarization of the Cl- current even if the neurone 297 A a ~ ~ ~ ~~~bc B 10 na b a Fig. 14. Blocking effects of intracellular DIDS. In the two experiments illustrated here, a double-barrelled intracellular micro-electrode was used to inject DIDS by ionophoresis into the neurone. One barrel was filled with DIDS (0-02 M), the other with K2SO4 (0-02 M). Voltage-recording electrodes filled with KCl were used to load the neurones with C1-. A, current records during hyperpolarizing jumps from -50 to -70 mv, before (a), during (b) the injection of DIDS (after application of 80 na for 4 min), and 2 min after the end of the injection (80 na, 8 min) (c). B, current records in another neurone, during hyperpolarizing jumps from -50 to -80 mv, before the injection of DIDS (a), and 2 min after the end of the ionophoretic injection of DIDS (60 na, 15 min) (b). In addition to the reduction (A) or blockade (B) ofthe Cl- relaxation, intracellular DIDS induced an outward current shift at the holding potential and an increase of the instantaneous conductance. had not been loaded with Cl-. This suggests that the Cl- current, which was mostly studied under 'artificial' conditions (after loading of the cells with Cl-), may play a role. under more 'physiological' conditions, as, for example, in cells which are spontaneously firing. Pharmacological properties DIDS (4,4'-diisothiocyanostilbene-2,2'-disulphonic acid) and SITS (4-acetamino- 4'-isothiocyanostilbene-2,2'-disulphonic acid) are two inhibitors of several anion transport systems (Passow, Fasold, Jennings & Lepke, 1982). When these drugs were applied in the external solution (up to 0-1 mm for SITS and up to 1 mm for DIDS), they did not modify at all the Cl- conductance under study. On the other hand, intracellular injections of DIDS (by ionophoresis) did block this C1- conductance (Fig. 14). In addition, the injection of DIDS progressively induced an increase in membrane conductance. This effect was not investigated. Other inhibitors of some anion-transport systems were tested extracellularly: furosemide (up to 1 mm; see Burg, Stoner, Cardinal & Green, 1973), penicillin

22 298 D. CHESNO Y-MARCHAIS (sodium benzylpenicillinate, up to 10 mm; see Hochner, Spira & Werman, 1976) and niflumic acid (up to 20/SM; see Cousin & Motais, 1979) had no effect on the Cl- current. Higher doses of niflumic acid seemed toxic, inducing a large increase in membrane conductance. Effect of external C8+ ions Extracellular Cs+ ions (above 0 5 mm) block a variety of slow hyperpolarizationactivated inward currents. Three well-studied examples ofsuch currents are considered as non-selective cationic currents: the current labelled 'if' in heart cells (Brown & Cs a b c d A -~-W--G--i--l 20 s B --,l HL 110nA (A) 5 na (B) 4 s Fig. 15. Effect of 50 mm-extracellular Cs+ ions. Suppression of these effects by addition of 10 mm-coc~l in the external solution. A solution containing 50 mm-cs+ ions (replacement of 50 mm-nacl by 50 mm-csci) was applied either in the absence (A) or in the presence (B) of 10 mm-cocl,. (The 10 mm-cocl, was added to the control and Cs+-substituted solutions without any change in CaCl, or MgCl,.) Records (a), (c) and (d) show the current observed during hyperpolarizing pulses from -50 to -100 mv, in the absence of Cs+ ions (a), 2 min (c) and 3 min (d) after substitution of 50 mm-nacl by CsCl. Records (b) show the effects of this substitution on the current traces recorded at the holding potential. (Note that the time scale is 5 times less expanded in (b) than in (a), (c) and (d).) In the absence of Co2+ ions (A), Cs+ ions affected the current trace at the holding potential (b). There was an increase in membrane conductance and the slow relaxation observed during the jump to -100 mv was modified (c). These effects were only transient so that records (d) and (a) are very similar. In the presence of Co2+ ions (B), Cs+ ions did not have any effect. Di Francesco, 1980; Di Francesco & Ojeda, 1980), the current labelled 'iq in hippocampal pyramidal neurones (Adams & Halliwell, 1982; Halliwell & Adams, 1982), and the current labelled 'ih' in isolated rods of the salamander (Bader, Bertrand & Schwartz, 1982). The effects of Cs+ ions on the slow hyperpolarization-activated current of Aplysia neurones were therefore studied. It was found that 1-10 mm-cs+ ions did not modify this current, thus confirming that the hyperpolarization-activated C1- current under study is different from other known hyperpolarization-activated inward currents such as if, tq, ih. However, as shown on Fig. 15A, higher doses of Cs+ did affect the current records in a complex way. When 50 mm-na+ was replaced by 50 mm-cs+, the current trace at the holding potential was clearly altered: an inward current developed (Fig. 15A, b) and then slowly decreased. Comparison of the records (c) and (a) clearly shows that this inward current corresponded to an increase in membrane conductance (the instantaneous current change occurring during the jump from -50 to -100 mv is

23 HYPERPOLARIZATION-ACTI VATED Cl- CONDUCTANCE much larger in (c) than in (a)). The substitution of 50 mm-na+ by Cs+ also induced a change of the slow relaxation recorded during the jump to -100 mv, and, in some cases, it might seem that the slow Cl- relaxation has been blocked by Cs+ (see record (c)); but this apparent blockade resulted from the superposition of the Cl- relaxation and of an inverse Cs+-induced slow relaxation (such inverse relaxations have been observed in the presence of Cs+ in cells which did not present the Cl- relaxation in control sea water (not shown)). The effects of high doses of Cs+ were only transient. The record obtained in the presence of Cs+, but 8 min after the application of Cs+ (Fig. 15A, d), is very similar to the control record (Fig. 15A, a). 299 A 2 B na 2s na <~ ~ ~~~~~~ ~0-20 L V (mv) Fig. 16. The Cl- relaxations persist after substitution of extracellular and intracellular monovalent cations by Cs+. A cell-body which had been Cs+-loaded by treatment with nystatin (see Methods) was perfused in the Na+-free and K+-free isotonic Cs+ sea water. The intracellular micro-electrodes were filled with CsS04 (0 5 m) in order not to reintroduce Na+ or K+ ions inside the cell. A, current records obtained during hyperpolarizing jumps from -45 mv to -55, -65, -75 and -85 mv. These jumps induced slow relaxations very similar to the usual Cl- relaxations. The relaxations were exponential. B, voltage dependence of the time constant of these relaxations. Since high doses of Cs+ ions may depolarize nerve endings and thus induce an increase of the spontaneous or synaptic liberation of neurotransmitters, it was of interest to look at the effects of Cs+ in the presence of C02+ ions, which are known to block the release of neurotransmitter. Fig. 15B shows that when 10 mm-cocl2 were added both in control sea water and in the Cs+-containing solution, there was no longer any effect of Cs+. Results similar to those illustrated in Fig. 15 were observed in four other similar experiments; however, the current which developed at the holding potential when applying the Cs+ solution was either inward or outward. Furthermore, it was observed that the effects of the substitution of 50 mm-na+ by 50 mm-cs+ were blocked by addition, in the control and Cs+-solutions, of 1 mm-( + )- tubocurarine, which in Aply8ia blocks a variety of responses to neurotransmitters (Ascher & Kehoe, 1975). These results support the hypothesis that the effects of high doses of Cs+ are due to a release of neurotransmitters. This would also explain why the effects of Cs+ were transient. Finally, Fig. 16 shows that the hyperpolarization-activated Cl- current persists after complete substitution of intracellular and extracellular monovalent cations by Cs+ ions. In order to replace the intracellular monovalent cations by Cs+, the ganglion was treated with nystatin in a Cs+-loading solution; then, after washing out the

24 300 D. CHESNO Y-MARCHAIS nystatin, the ganglion was perfused with an isotonic Na+-free, K+-free, Cs+ sea water (see Methods). In order to avoid the effects of a possible Cs+-induced increase of the spontaneous release of neurotransmitters, the cell bodies were isolated by cutting the axons before recording. The records in Fig. 16A were obtained in isotonic Cs+ sea water during hyperpolarizing jumps from -45 mv in such an isolated Cs+-loaded cell body. This cell body was also Cl--loaded and EC1, measured as the reversal potential of the cholinergic Cl- response, was around -5 mv. (The high intracellular C1- concentration may result both from the fact that Cl- ions are not completely impermeant across the nystatin ionophore (Russell et al. 1977), and from a possible leak of extracellular Cl- ions into the neurone during cutting of the axon.) The hyperpolarizing jumps induced slow relaxations which were very similar to the C1- relaxations usually observed in control conditions. The relaxations shown on Fig. 16A were exponential and the voltage dependence of their time constant is illustrated on Fig. 16B. This r(v) curve is very similar to the curves which were obtained from control Cl- relaxation. It is bell-shaped and has a slope of about e/13 mv in the hyperpolarized range. Two reasons may explain why the relaxations of Fig. 16 were more rapid than the other Cl- relaxations shown: the temperature was higher in this experiment (28 TC instead of C) and the intracellular Cl- concentration was higher than usual (see Figs. 2B and 3D). DISCUSSION Comparison of the hyperpolarization-activated Cl- conductance of Aplysia neurones with other 'inward rectifying' (or hyperpolarization-activated) conductances Chloride vs. cationic inward rectifying conductances. Several kinds of non-clrectifications have been described. The instantaneous K+ inward rectification of frog muscle is well known (Adrian, 1969). In starfish egg, there exist both an instantaneous K+ inward rectification and a time-dependent hyperpolarization-activated increase in K+ conductance (Hagiwara, Miyazaki & Rosenthal, 1976). Time-dependent inward rectifications involving non-selective cationic channels have also been reported. The it current of the rabbit sino-atrial node (Brown, Di Francesco & Noble, 1979) has been shown to be a non-selective cationic current (Di Francesco & Ojeda, 1980). More recently, a similar current has been described in Purkinje fibres (Di Franceso, 1981), in isolated rods of the salamander (ih'; Bader et al. 1982; see also Fain, Quandt, Bastian & Gerschenfeld, 1978) and in hippocampal neurones ('iq'; Adams & Halliwell, 1982; Halliwell & Adams, 1982) (see Halliwell & Adams, 1982, for a recent review). The inward-rectifying current described in the present paper and in the previous one (Chesnoy-Marchais, 1982) is neither a K+ nor a non-selective cationic current. It can be very large at the K+ equilibrium potential. It is neither affected by complete substitution of external Na+ ions by Tris, nor by addition of Cs+ ions (1-50 mm) in the external solution. It persists after complete substitution by Cs+ of the Na+ and K+ ions of the extracellular and intracellular solutions (Fig. 16). It is a voltage-gated Cl- current. Its reversal potential (which was measured by extrapolation) was always found very close to the Cl- equilibrium potential and was affected by modifications of the extracellular (Fig. 6) and intracellular (Fig. 3) Cl- concentration. Furthermore,

25 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE 301 prolonged activation of this current may lower the intracellular Cl- concentration (see legend of Fig. 2). The hyperpolarization-activated inward current presented in this paper is therefore clearly distinct from the K+ inward rectifier and from ifh and iq; rather it is a hyperpolarization-activated Cl- current. Cs+ ions may be used in order to differentiate this Cl- current from the other inward-rectifiers. NO3- ions may also be used for this purpose. Anomalous rectification of molluscan neurones. The 'anomalous' (or 'inward') rectification of molluscan neurones was first described by Tauc & Kandel (1964) (see also Kandel & Tauc, 1966 and Marmor, 1971). A priori, this phenomenon may be due to the activation, by hyperpolarization, of several different conductances. The Cl- conductance presented in the present paper was certainly implied when KCl-filled micro-electrodes were used. It is much less likely that the non-cl- conductances slowly activated by hyperpolarization which are described on other preparations (the K+ one (Hagiwara et al. 1976) or the cationic one underlying if, iq, ih (see above)) may contribute to the 'anomalous rectification' of molluscan neurones. Indeed, in Aplysia neurones, when the Cl- current described here is not activated (when the intracellular Cl- concentration is too low (see e.g. Fig. 13A), or after blockade of the Cl- current by, for example, NO3- (Figs. 8A, c and 9C) or DIDS (Fig. 14B, b)), hyperpolarizing jumps do not induce any slow increase in inward current (see also Marty & Ascher, 1978). Finally, the existence, in molluscan neurones, of an instantaneous K+ inward rectifier remains to be investigated. The experiments reported in the present paper do not clarify this point. Some of the 'inward rectifying' I-V curves obtained on the A neurones (by hyperpolarizing jumps of a few hundred milliseconds duration) exhibit a negative slope, most likely due to the inward current described by Wilson & Wachtel (1974). It was therefore not possible to conclude that the observed rectification resulted from an increase in K+ conductance, rapidly induced by hyperpolarization, rather than from the superposition, above EK, of two currents of opposite polarities: a non-rectifying K+ outward current and the inward current responsible for the negative resistance observed in this range. Neither would an apparent blockade of the inward rectification of the I-V curve by extracellular Cs+ ions (or by other cations) demonstrate the existence of a K+ inward rectifier since Cs+ ions might block in a voltage-dependent way a non-rectifying K+ conductance. Other hyperpolarization-activated Cl- currents. Very few examples of voltage-gated chloride currents are known. However, the hyperpolarization-activated Cl- current found in Aplysia neurones may also exist in other preparations. It has been reported that crayfish musclefibres show an inward rectification which may be abolished by reducing the intracellular Cl- concentration (by prolonged exposure to a low-cl- extracellular solution). This rectification was therefore attributed to the activation by hyperpolarization of a Cl- current (Reuben, Girardier & Grundfest, 1962; Ozeki, Freeman & Grundfest, 1966). It was also reported that this rectification was blocked by increasing the extracellular ph (Reuben et al. 1962) and persisted in the presence of extracellular Cs+ ions (Ozeki et al. 1966). These results suggest that the hyperpolarization-activated Cl- conductance of Aplysia neurones, which is reduced by high external ph (Fig. 10) and is insensitive to Cs+ ions, may also exist in crayfish muscle fibres.

26 D. CHESNO Y-MARCHAIS TheCl- conductance of frog muscle fibres is affected in a complex way by modifications of the extracellular ph (Warner, 1972). It was reported that hyper- conductance at normal and high polarizing jumps induce a rapid decrease of theclexternal ph, but, in contrast, a slow increase of the Cl- conductance at low external ph. One way to explain this last result is to suppose that, in addition to theclconductance described at normal and high ph, the frog muscle membrane possesses a hyperpolarization-activated Cl- conductance, similar to that found in Aplysia neurones and markedly increased by acidification of the external solution. The high sensitivity to external acidification of the frog hyperpolarization-activated Clconductance may be mediated in part through the increase of the internal Clconcentration which, in muscle fibres, is indirectly induced by external acidification (Bolton & Vaughan-Jones, 1977) - an effect which does not occur in Aplysia. The Cl- conductance of theisolated toadskin has been shown to be strongly dependent on the transepithelial potential and it has been suggested that 'hyperpolarizing' (which, in this system, means increasing the spontaneous transepithelial potential) does activate a Cl- conductance (Bruus, Kristensen & Larsen, 1976; Larsen & Kristensen, 1978; Larsen, 1982). Although the authors could not establish whether the activation of the Cl- conductance resulted from a change in membrane potential or from a change in the concentration of an intracellular ion, which would participate in the activation of the Cl- conductance, it is tempting to propose that a voltage-gated Cl- conductance, similar to that found in Aplysia neurones, may account for the rectification of the Cl- conductance of the toad skin. In particular, it is interesting to note that this rectification was not detected if external Cl- ions were replaced by NO3- ions (Kristensen & Larsen, 1978). More recently, voltage-gated Cl- channels have been found and extensively studied in artificial bilayers of phospholipids in which vesicles extracted from the electric organ of Torpedo had been incorporated (White & Miller, 1979; Miller & White, 1980). The vesicles, which were introduced only on one side of the bilayer (the 'cis' side), were asymmetrically oriented into the bilayer so that the observed Cl- conductance was activated by applying a negative voltage to the 'Ci8' face of the bilayer. The incorporation procedure does not tell directly which face ('Ci8' or 'trans') corresponds to the intracellular face of the native membrane. But, if we suppose that it is the 'Ci8' side, the properties of the observed Cl- channels become strikingly similar to those of the hyperpolarization-activated Cl- conductance of Aplysia neurones: (1) voltage jumps induced slow relaxations of the total current corresponding to the slow activation or deactivation of the Cl- channels; (2) the steady-state conductance was strongly voltage-dependent and saturated at highly negative 'cis' potentials; (3) the elementary conductance (which could be directly measured in this system) was nearly voltage-independent (the analogous result of the present paper is the approximate linearity of the instantaneous I-V curves); (4) whereas DIDS and SITS did not affect at all the Cl- current when applied on the 'trans' side, they strongly blocked it from the 'Ci8' side (White & Miller, 1979) (compare with the present results, p. 297); (5) increasing the ph on the 'trans' side, as well as reducing the ph on the 'cis' side reduced the Cl- conductance (fig. 11 of Miller & White, 1980); (6) NO3- ions appeared to be impermeant through these Cl- channels (table 2 of Miller & White, 1980). The voltage-gated Cl- current described by White & Miller and the current described in the present paper may therefore be the same current.

27 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE * *.0 x sie a s 05_ 7 S: 6 11.io 0-5 -_ 0 I_ I I I " V (mv) I V (mv) Fig. 17. Fit of the g..(v) and r(v) curves by a two-state model. The points correspond to the experimental data already presented in Fig. 2 (-). The curves have been plotted by the computer after determination of the parameters a0, flo, a and b as explained in the text. The values of these parameters are the following: a-= 1P5 x 1O-3 s-l fio = 50 s-l; a = mv-' and b = mv-1. The correlation coefficients of the linear fits of the curves ln a( V) and ln fl( V) were respectively and Voltage dependence of the Cl- conductance. The hyperpolarization-activated Clconductance and its kinetics of activation or deactivation are both voltage-dependent. may be explained by a simple two-state model The two relations g.5(v) and r(v) supposing (1) that the membrane contains N elementary units, which may be, either in an 'activated state', X*, corresponding to a conductance y, specific for Cl- ions, or in a 'resting state', X, corresponding to a conductance zero and (2) that the transition rates between these two states (a( V) and fl( V)) are voltage-dependent: x 1 a(v) 4(V) For simplicity, and also because the instantaneous I-V curve was almost linear in the usual voltage range (Chesnoy-Marchais, 1982; see also Figs. 1 C, 3B and 6C), y is assumed to be independent of voltage. With such a simple model, the hyperpolarization-activated Cl- conductance g55( V) is given by eqn. (6): or gss(v) = Nxyx x*. a(v) gss( V) = g55max o(v) ± y( V) (5) (6) (6a)

28 304 D. CHESNO Y-MARCHAIS The time constants of activation and deactivation are equal and are given by: 7r(V) = (7 ta(v)+f(v) (7) Fig. 17 shows how such a model fits the data. To obtain the theoretical curves, I first used the experimental data to calculate a(v) and fl(v) for each potential V: g..max was deduced from the plateau of the g.s(v) curve (g..max - and fl( V) were then calculated as respectively 9Sg(V) /T(V) and (1 988(V)T(V) g88max g88max 150 ns); a(v) Then, it was determined that these values of a( V) and fl( V) could be considered as exponential functions of V: a = aoxexp (-av). (8) i =fl#oxexp (by). (9) The values of the parameters oo, flo, a and b, were deduced from the linear fit of the curves ln a( V) and In l( V). Finally, from these values, and from eqns. (6), (7), (8) and (9), the theoretical curves 988( V) and r( V) were calculated. The fit was done for nine independent experiments. The values found for a and b were not very different from one experiment to another. They did not appear to depend on the intracellular Cl- concentration. The mean values obtained for a and b were: a(mv-1) = 0-089±0-005 (9) (mean +s.e. of the mean (n)), b(mv-1) = 0-060±0-005 (9), which corresponds to an e-fold change for mv for a, and for mv for b. In contrast, the values of ao and flo were very variable. They appeared to depend in particular on the intracellular Cl- concentration: when the intracellular Clconcentration was increased, ao increased and flo decreased (see below). Effects ofthe intracellular Cl- concentration. The sensitivity ofthe hyperpolarizationactivated Cl- current to the intracellular Cl- concentration is very pronounced and it was usually necessary to load the neurones with Cl- ions in order to detect this current. Increasing the intracellular Cl- concentration markedly increased the steady-state conductance and strongly modified the kinetics of activation (Figs. 2 and 3). These two effects cannot be explained simply by the diffusion properties which are expressed by the Goldman equation (Goldman, 1943). This equation can explain partly why the maximum conductance (gsmax) was sometimes increased by increasing the intracellular Cl- concentration, but it can neither explain the very large increase in conductance observed near the threshold of activation, nor the Cl- dependence of the kinetics of activation. These effects indicate that increasing the intracellular Clconcentration induces both (a) an increase of a (accounting in particular for the fact that, at hyperpolarized potentials, increasing the intracellular Cl- concentration shortens r) and (b) a decrease of fi (accounting for the fact that, in the right part of the r( V) curve, increasing the intracellular Cl- concentration lengthens r).

29 HYPERPOLARIZATION-ACTIVATED Cl- CONDUCTANCE 305 The sensitivity of a and flto the intracellular Cl- concentration may be explained by introducing a binding site for intracellular Cl- ions both on the 'resting' and on the 'activated' forms of the elementary units responsible for the current. One of the schemes (equivalent to scheme (5)), which could account for the data is presented below as an example: K K* (10) XI -~ / ~ XI* I represents intracellular Cl- ions. In this scheme, the equilibrium between the 'resting' free and bound forms, x and xi (dissociation constant K) and the equilibrium between the 'activated' free and bound forms x* and xi* (dissociation constant K*) are supposed to be much faster than the steps of activation and deactivation. Furthermore, it is supposed that the activation occurs mainly from the bound 'resting' form, while the deactivation occurs mainly from the free 'activated' form. The transition rates from xi to xi* and from x* to x are called respectively a' and fi'. This scheme is equivalent to scheme (5) where X is the total amount of 'resting' forms (x + xi) and X* is the total amount of activated forms (x* + x*i) with: c8(v) + '] (11) fl(v) 1 +([Clj]/K*)* (12) The voltage dependence of a and / may result from the voltage dependence of a', fi', K and/or K* and the location of the binding sites (inside or outside the ionic pathway) is not specified. From eqns. (11) and (12) it is clear that this scheme may qualitatively account for the effects of the intracellular Cl- concentration on a and fia and therefore on g9( V) and r(v) see eqns. (6a) and (7)). The effects of intracellular Cl- ions on the hyperpolarization-activated Cl- conductance resemble the effects of extracellular K+ ions on the anomalous K+ conductance of the starfish egg (Hagiwara et al. 1976; Hagiwara & Yoshii, 1979). In both cases, the steady-state conductance, g.8(v), depends markedly on the concentration of the ions which carry the current (extracellular K+ ions in the case of the inward K+ current of the egg, intracellular Cl- ions here). Furthermore, this concentration does not affect very much the curve gs8( V-Er)/g..max, Er being the reversal potential of the response (EK in one case, Ec1 in the other) (Figs. 2 and 3 in the present paper). Several models have been proposed to explain the influence of the ions on the steady-state conductance (see Ciani, Krasne, Miyazaki & Hagiwara, 1978). However, these models are not presented from a kinetic viewpoint. The sensitivity of the hyperpolarization-activated Cl- conductance to the intracellular Cl- concentration may have a physiological role. As this concentration is

30 306 D. CHESNO Y-MARCHAIS increased, smaller and smaller hyperpolarizations activate the C1- conductance, and, if the membrane is held at a hyperpolarized level, for a long enough period, the C1- efflux resulting from the activation of the Cl- conductance may lower the intracellular Cl- concentration. The strong sensitivity of the Cl- current to the internal Cl- concentration also has some practical implications. For example, when this current may complicate the problem under investigation, it would be better to avoid intracellular microelectrodes filled with KC1. It was also shown (Fig. 4) that an agonist which induces itself a direct increase in Cl- conductance may, in addition, modify the voltagedependent Cl- conductance. Such effects may be of importance for the interpretation of large Cl- responses to high doses of agonist in cells which are not internally perfused. Effects of the extracellular Cl- concentration. As shown in Figs. 5 and 6A, modification of the extracellular Cl- concentration does affect the amplitude of the Cl- current but not always in a very pronounced way, especially if the hyperpolarization is strong. This may be partly explained by the fact that modifications of the reversal potential are more influential near the reversal potential but probably also results from the extracellular Cl- concentration sensitivity of the g,8(v) curve, which remains to be studied. The time constant of activation of the current has also been shown to be dependent on the extracellular Cl- concentration, the r(v) curve is shifted to the right by decreasing the extracellular Cl- concentration. This effect is clearly different from the effect of the intracellular Cl- concentration where the r(v) curve was shifted to the left by decreasing the intracellular Clconcentration (Figs. 2 and 3). This difference indicates that extracellular and intracellular Cl- ions do not bind in the same way and that in order to account for the effects of the extracellular Cl- concentration additional binding sites for the external Cl- ions, or additional conformations which would bind to these ions, should be introduced in scheme (10). Such modified schemes become very complex, even if they remain equivalent to scheme (5). Furthermore, many schemes may be a priori proposed and more data are required to choose a precise model. Note added in proof. It was reported recently (from spectrophotometric measurements of Ca2+ activities) that calcium ions are not complexed by nitrate ions, and that nitrate ions may interfere with the responses of Ca2+-sensitive electrodes (Dani, Sanchez & Hille, 1983). Thus, the corrections which had been made in order to adjust the Ca2+ activities in control and nitrate solutions (see Methods) may not have been appropriate. However, as expected from the fact that extracellular Ca2+ ions do not affect the Cl- current studied very much, the results presented above (pp ) were confirmed by an additional experiment where the control and nitrate solutions contained respectively 10 mm-cacl2 and 10 mm-ca (NO3)2. I acknowledge the support of the Universite Pierre et Marie Curie, of the C.N.R.S. (LA ) and of the D.G.R.S.T. (81 E 1382). I am very grateful to Prof. P. Ascher for his contributions to the orientation and presentation of this work. I thank Linda Nowak for her comments on the manuscript.

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