241

Journal of Physiology (1990), 427, pp. 241-260 With 8 figures Printed in Great Britain

MECHANISM OF ACTION OF GABA ON INTRACELLULAR pH AND ON SURFACE pH IN CRAYFISH MUSCLE FIBRES

BY K. KAILA*, J. SAARIKOSKI AND J. VOIPIO From the Department of Zoology, Division of Physiology, University of Helsinki, Arkadiankatu 7, SF-OO100 Helsinki, Finland (Received 6 December 1989) SUMMARY

1. The mode of action of y-aminobutyric acid (GABA) on intracellular pH (pHi) and surface pH (pH.) was studied in crayfish muscle fibres using HW-selective microelectrodes. The extracellular HC03- concentration was varied (0-30 mM) at constant pH (7*4). 2. GABA (5 x 10-6-10-3 M) produced a reversible fall in pHi which showed a dependence on the concentrations of both GABA and HCO3-. The fall in pHi was associated with a transient increase in pH. and it was inhibited by a K+-induced depolarization. 3. In the presence of 30 mM-HCO3G, a near-saturating concentration of GABA (0 5 mM) produced a mean fall in pHi of 0 43 units. This change in pHi accounted for about two-thirds of the GABA-induced decrease (from -66 to -29 mV) in the sarcolemmal H+ driving force, while the rest was due to the simultaneous depolarization. 4. The apparent net efflux of HCO3 (j co.) produced by a given concentration of GABA was estimated on the basis of the instantaneous rate of change of pHi. In the presence of 30 mM-HCG3-, JHco. following exposure to 0 5 mM-GABA had a mean value of 8-0 mmol 1-1 min-'. Under steady-state conditions (at plateau acidosis), the intracellular acid load produced by 0 5 mM-GABA was about 25 % of that seen at the onset of the application. 5. The GABA-induced HCO3- permeability, calculated on the basis of the flux data, showed a concentration dependence similar to that of the GABA-activated conductance described in previous work. 6. The GABA-induced increase in pH. was immediately blocked by both a membrane-permeant inhibitor of carbonic anhydrase (acetazolamide, 10-6 M) and by a poorly permeant inhibitor (benzolamide, 10-6 M). 7. Application of acetazolamide (10-4 M) for 5 min or more produced a decrease of up to 60 % in the maximum rate of fall of pHi at GABA concentrations higher than 20 #M. 8. The recovery of the GABA-induced acidosis was associated with a fall in pH.. The recovery was completely blocked in solutions devoid of Na+ or of CO-, as well as *

MS 8124

To whom correspondence should be

addressed.

242

K. KAILA, J. SAARIKOSKI AND J. VOIPIO

by DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid, 10-5 M). This indicates that the maintenance of a non-equilibrium H' gradient at plateau acidosis and the recovery of pHi are attributable to Na'-dependent Cl--HCO3- exchange. 9. We conclude that the effects of GABA on pHi and pHs are due to electrodiffusion of HCO3- across postsynaptic anion channels. This leads to an influx of CO2 across the plasma membrane and thereby to a release of H' ions within the cell. In the presence of a functional intracellular carbonic anhydrase, the effect of GABA on pHi is rate-limited by the GABA-gated bicarbonate conductance, but following inhibition of the intracellular carbonic anhydrase, hydration of CO2 becomes rate-limiting if a significant fraction of the conductance is activated. The GABA-induced increase in pHs is catalysed by a carbonic anhydrase which has its active site at the extracellular surface of the plasma membrane. In view of the well-known sensitivity of various ion channels to intracellular and extracellular H' ions, it is possible that changes in postsynaptic pH similar to those described here play a role in the modulation of synaptic inhibition in nervous tissue. INTRODUCTION

Recent work on both vertebrate and invertebrate preparations has shown that, under physiological conditions, GABA-sensitive anion channels permit the passage of not only chloride, but also of bicarbonate (Inomata, Oomura, Akaike & Edwards, 1986; Kaila & Voipio, 1987 a; Bormann, Hamill & Sakmann, 1987; see also Kelly, Krnjevic, Morris & Yim, 1969). Since the transmembrane distribution of a weak-acid anion, such as HCO3-, is set by the H+ gradient (see Roos & Boron, 1981), a transmitter-activated bicarbonate conductance will have effects both on the electrical properties and on pH regulation in the postsynaptic cell. In a previous paper, we examined the postsynaptic electrical consequences of HCO3- permeability of GABA-gated channels (Kaila, Pasternack, Saarikoski & Voipio, 1989b). The present work focuses on the mechanism of action of GABA on intracellular pH (pHi) and surface pH (pH,) in the dactyl opener muscle of crayfish. The opener fibre has been a fruitful research model in the study of synaptic transmission in general and of synaptic inhibition in particular (for review, see Atwood, 1982; Roberts, 1986). An advantage that deserves comment in the present context is that the muscle fibres provide a GABA-sensitive preparation where an examination of transmembrane fluxes of acid equivalents, based on a simultaneous measurement of pHi and pHs, is not influenced by other cell types, such as glia. Furthermore, the inhibitory channels of the opener fibre show little desensitization (Takeuchi & Takeuchi, 1967), so that a maintained conductance can be activated in a concentration-dependent manner by bath-applied GABA. The results show that, in the presence of CO2-HCO3-, an electrogenic efflux of bicarbonate ions through GABA-gated channels gives rise to a net influx of CO2 and, thereby, to a fall in postsynaptic pHi which is linked to an increase in pHs. Experiments with inhibitors of carbonic anhydrase indicate that two types of this enzyme are involved in the GABA-induced pH changes: an intracellular carbonic anhydrase, and a carbonic anhydrase with its catalytic site at the extracellular surface of the sarcolemma. Inhibition of the intracellular carbonic anhydrase

pH CHANGES INDUCED BY GABA

243

produces a decrease in the rate of fall in pHi and inhibition of the extracellular carbonic anhydrase blocks the pHs transient. The recovery of the GABA-induced intracellular acidosis is mediated by Na'-dependent Cl--HCO3- exchange. In view of the sensitivity of various ion channels to changes in intracellular and extracellular H' (e.g. Takeuchi & Takeuchi, 1967; Moody, 1984), the present results raise the possibility that GABA-mediated inhibition may be influenced by postsynaptic changes in pHi and pH,. It is also possible that GABA-induced fluxes of acid equivalents play a role in activity-induced pH changes in nervous tissue (e.g. Kraig, Ferreira-Filho & Nicholson, 1983). Some preliminary reports on the present results have appeared (Kaila & Voipio, 1987a, b; Kaila, Pasternack, Saarikoski & Voipio, 1989c). METHODS

The experiments were done on the dactylopodite opener muscle of the crayfish Astacus astacus which was perfused at a rate of about 10 ml min-'. The experimental arrangement, as well as the methods used in the construction of and recording with conventional and Hf-selective intracellular microelectrodes, have been described before (Kaila, Mattsson & Voipio, 1989 a; Kaila et al. 1989 b). In some experiments, pHi was simultaneously measured from two cells in a preparation (e.g. Fig. 4). Surface pH was recorded with a He-selective electrode which had a shape resembling that of a patch-clamp electrode (tip inner diameter about 10 lum). The pH, electrode was gently pressed against the fibre under investigation. Solutions and drugs. The nominally C02-free standard solution contained (mM): NaCl, 200; KCl, 5-4; CaCl2, 7 0; MgC12, 2-6; HEPES, 10 (pH 7-4; adjusted with NaOH). In the HCO3--containing solutions, about 6, 12 or 30 mM-NaCl was substituted by NaHCO3 to yield a pH of 7-4 when equilibrated with air containing 1, 2 or 5% CO2. Sodium-free solutions were made by isomolar substitution of sodium by choline or by N-methyl-D-glucamine. Methanesulphonate was used to replace chloride in solutions with a low or zero Cl- concentration. In high-K+ solutions used to depolarize the membrane potential, an equal reduction was made in the Na' concentration and, in order to minimize changes in intracellular Cl- and in cell volume, the product [K+][Cl-] was kept constant by replacing Cl- ions by methanesulphonate. Three millimolar Mn2+ was added to block contractile activation during the depolarizations. GABA (Sigma), acetazolamide (Sigma) and benzolamide (donated by Lederle Laboratories, American Cyanamid Company) were added from stock solutions made in water. DIDS (4,4'diisothiocyanatostilbene-2,2'-disulphonic acid; Sigma) was dissolved directly into the saline. Calculation offluxes and permeabilities. The instantaneous GABA-induced HCO3- efflux (Je~0; mmol 1V1 min-') was estimated from Je = -dpHi/dt, (1) where /3 is the intracellular H+ buffering power of the muscle fibre. In the presence of CO2. /3 is the sum of the non-CO2 buffering power and of the CO2-HCO3- buffering power which equals 2-3 x [HCO3-]i (see Roos & Boron, 1981). The HCO3- permeability (PHco 3) was calculated on the basis of the net flux data using the constant-field equation (Goldman, 1943; Hodgkin & Katz, 1949):

JHCO3 HCO3

-rFV/RTFPHCO3 (rHC3 ]1ie 3

e-FR

RT

rHCO3 Jo),

(2)

1

where r is the surface/volume ratio, V is the membrane potential and F, R and T have their usual meanings. Below, PHCO3 is expressed as a normalized value such that for a given fibre, the permeability obtained at 1-0 mm-GABA is taken as the maximum. The intracellular HCO3concentration was obtained from [HCO3- i = 10(pHi-PHo)[HCO3- o

K. KAILA, J. SAARIKOSKI AND J. VOIPIO

244

99% air 100 % air 1 % C02/6 mM-HC03 II

GAkBA (80 MM)

GABA (20 pM)

68

GABA (80 MM)

pHi 7.0 72L -60 [

Em (mV) -80 _

95 % air 5 % C02/30 mM-HC03 GABA (20 pM)

GABA (80 uM)

6.8

pHi 7-0 72

460

Em (mV) -80

-

30 min

Fig. 1. Effect of GABA (20 and 80 #M) on pHi in the nominal absence of C02-HC03and in solutions containing 6 and 30 mM-HC03-. The upper and lower parts are from a continuous recording. 100 % air GABA GABA (20_uM ) (8Bm )

6-9

95 % air 5 % C02/30

mM-HC03f

GABA (20 pM)

GABA (80 PM)

pH; 7B 1 73

_-

7.37

pHs 7L40 7.43 -70

Em (mV)

r -80

10 min Fig. 2. Effect of GABA (20 and 80 #M) on pH, and pH. in the nominal absence of CO2-HCO3- and in a solution containing 30 mM-HCO3-. The break in the trace is

40 min.

pH CHANGES INDUCED BY GABA

245

RESULTS

Dependence of the GABA-induced pHi changes on CO2-HCO3Figure 1 shows an experiment where the influence of GABA on pHi was examined in a nominally HC03--free medium and in solutions containing 6 and 30 mM-HCO3T. It is evident that the magnitude of the fall in pHi depends on the concentration of both GABA and bicarbonate. In the presence of 30 mM-HCOG-, application of 20 and 80 /LM-GABA produces an intracellular acidosis of 0 30 and 0 44 pH units, respectively. These changes in pHi are, by a factor of about two, larger than those seen at 6 mm-HC03-7 Following withdrawal of GABA, a complete recovery of pHi takes place. A slight fall in pHi (usually about 0 05 units or less) was frequently observed upon application of GABA in the absence of HCO3-. This effect is probably attributable to the fact that nominally bicarbonate-free solutions are not completely devoid of C02-HC03-. In addition, metabolic production of C02 and hence HCO3- may also contribute. Effect of GABA on surface pH The GABA-induced fall in pHi was invariably associated with a transient increase in surface pH. As shown in Fig. 2, application of GABA (20 and 80 /M) in a nominally HCO3--free solution has a small effect on pHr. In the presence of 30 mM-HCO3-, a given concentration of GABA produces a much larger change in pH. The maximum change in pH. caused by 0 5 mM-GABA in the presence of 30 mM-HCO3 had an amplitude of up to 0-1 pH units. As discussed below, the surface alkalosis produced by GABA is consistent with the assumption that the simultaneous fall in pHj is due to an efflux of HCO3- mediated by inhibitory anion channels. The recovery of the GABA-induced acidosis was associated with a transient fall in pH. (Fig. 2). This surface acidosis is attributable to extrusion of acid during pHi recovery (see below).

Voltage dependence of GABA-induced intracellular acidosis The influence of membrane potential on the GABA-induced acidosis was examined using variation of the extracellular K+ concentration. This method was adopted because in the presence of GABA it is not possible to impose a large uniform depolarization of the fibre using a point source of current, i.e. a microelectrode. Experiments of the kind depicted in Fig. 3 showed that a depolarization of the membrane potential inhibits the acidosis caused by GABA in the presence of 30 mmHCO3-m A similar voltage dependence of the fall in pHi caused by GABA was observed also in the presence of 6 and 12 mM-HC03-. The dependence on membrane potential of the GABA-induced fall in pHi gives further support to the assumption that the acidosis is due to electrodiffusion of HCO3- through postsynaptic anion channels. A noteworthy feature in the above experiments was that in the absence of GABA, pHi showed little dependence on voltage (Fig. 3). As will be discussed below, this

K. KAILA, J. SAARIKOSKI AND J. VOIPIO

246

GABA

pHi 7 41

7-35

Em (mV) 80-

[K+]o

5

37.8

3

5.4

3

(mM) 10 min

Fig. 3. Dependence on voltage of the fall in pHi caused by GABA (02 mM). A highpotassium solution (37T8 mM-K+) was used to depolarize the membrane potential while the product [K+][CI-] was kept constant. All solutions contained 30 mM-HC03-. 3 mM-Mn2+ was added to block contractile activation during the depolarizations. Note insensitivity of pHi to a change in Em in the absence of GABA. TABLE 1. Dissipation of the plasmalemmal electrochemical H+ gradient by a near-saturating concentration (0-5 mM) of GABA in the presence of 30 mM-HC03-

Em (mV)

pHi

EH (mV) Em-EH (mV)

Control -75 4+0063 7-24+0 03 -66 1 -9-3 GABA -628+074 681+006 -342 -286 Difference 12 6 -0 43 -24 9 37*5 The data represent mean values obtained from fourteen preparations under steady-state conditions. The primary measurement data (Em and pH,) have been given as mean + S.E. of mean. EHCO = EH (see text).

observation suggests that the resting (transmitter-insensitive) Cl- channels (Dudel & RUdel, 1969) are not significantly permeable to HCO3-. GABA-induced dissipation of the transmembrane H' electrochemical gradient In addition to its influence on pHi, an efflux of HCO3- through GABA-gated channels produces a depolarization (Kaila et al. 1989 b). From a thermodynamic point of view it is obvious that both the fall in pHi and the depolarization contribute to the GABA-induced dissipation of the plasmalemmal H+ electrochemical gradient. It is worth pointing out here that in the presence of C02, the electrochemical gradients and equilibrium potentials of HCO3- and H+ are equal (EHCO3= EH; see Kaila & Voipio, 1990). Table 1 gives a quantitative summary of the electrical and chemical components of the H+ gradient that prevail across the plasmalemma in the absence and presence of GABA (0-5 mM) in a solution containing 30 mM-HCO3-. The data show that under steady-state conditions, a near-saturating concentration of GABA produces a 37.5 mV decrease (from -66-1 to -28-6 mV) in the H+ electrochemical gradient.

pH CHANGES INDUCED BY GABA

247

Sixty-six per cent (24-9 mV/37 5 mV) of the dissipation of the gradient is attributable to the mean fall of pHi of 0 43 units (which corresponds to a - 24-9 mV shift in EH), while the rest is due to the depolarization. The data in Table t also indicate that in the presence of 30 mM-HC03-, the GABAinduced fall in pHi is associated with a mean decrease of the intracellular bicarbonate concentration from 20 8 to 7-2 mm (this corresponds to a fall in the intracellular HCO3- activity from 15-2 to 5-3 mM). Concentration dependence of the GABA-induced HCO3- efflux and HCO3permeability In order to examine the concentration dependence of the GABA-induced intracellular acid load, we quantified the underlying net HCO3 flux (JeCO) by measuring the instantaneous changes in pHi produced by brief (30-90 s) exposures of the preparation to GABA in the concentration range 5 x 10-6 to 10-3 M. The JHCO was estimated on the basis of the initial rate of change in pHi (dpH1/dt) caused by a given concentration of GABA (eqn (1); Methods). Sufficient time was allowed between each application of GABA to permit full recovery of pHi to its control steady-state level. In order to obtain an estimate of the intracellular H+ buffering power of the opener fibre, we measured the change in pHi produced by a change from a nominally C02free perfusion solution to one equilibrated with 5% CO2 (see Roos & Boron, 1981). These measurements were made in sodium-free solutions to block transmembrane movements of acid equivalents other than those related to passive movements of CO2. They yielded a mean value of 238 + 0-47 mequiv 11 (pH unit)-1 (± S.E. of mean; n = 10) for the non-CO2 buffering power of the opener fibres. This value was used in the calculation of HCO3- fluxes by means of eqn (1). In the standard solution containing 30 mM-HCO3 , JHC attained its maximum value of 8 03 + 0-36 mmol 11 min-' (± S.E. of mean; n = 18) following the application of a near-saturating concentration of GABA (0 5 mM). Figure 4A shows the results of a single continuous experiment, where the dependence of JHCO3 on [GABA] was examined at two concentrations of bicarbonate, first at 12 mM-HCO3- and then at 30 mM-HCO3-. In this experiment, pHi was simultaneously measured in two cells. It is evident that JH shows a dependence on the concentration of both GABA and HCO3-. Using the constant-field flux equation (eqn (2), Methods), the values of JH can be converted into HCO3- permeabilities (PHco3). In these calculations, the value of JHCO3 measured for a given cell in the presence of 30 mM-HCO3- and 10 mM-GABA was taken as 100%. In Fig. 4B, the data shown in Fig. 4A have been replotted in this manner in order to illustrate the dependence of PHCO3 on GABA concentration. It should be noted that following the conversion of flux values to permeabilities, the data points obtained at the two HCO3- concentrations conform to a single sigmoidal concentration-response curve. An important feature of the flux-based permeability values in Fig. 4B is that they show a dependence on GABA concentration which is similar to that of the GABAactivated conductance (see Kaila et al. 1989b). In five experiments of the kind depicted in Fig. 4, the Hill coefficients ranged from 14 to 17 with a half-maximum

K. KAILA, J. SAARIKOSKI AND J. VOIPIO

248 A

10 r

E

-6.)_

5

ox 11

0

100

[GABA] (pM)

B

100 r

A

; 50

0

u

A

0

10

100

1000

[GABA] (pM) Fig. 4. A, dependence on [GABA] of the instantaneous net efflux of HCO3- (JHco ). The data are based on a simultaneous recording of pHi in two cells at 12 and 30 mM-AC03-. B, dependence of HCO3- permeability (PHcO ) on [GABA]. Values of PHCO3 have been calculated (eqn (2)) on the basis of the flux data in A. The permeability values are normalized such that for both cells the estimate of PHCO obtained in the presence of 30 mM-HC03- and 1P0 mM-GABA has been taken as 100 %.

effect occurring at 50-70 fM. This lends further support for the conclusion that the HC03- efflux is mediated by the same channels which underlie the electrical effects of GABA (cf. Takeuchi & Takeuchi, 1967; Kaila et al. 1989b). Role of carbonic anhydrase in the action of GABA on pHi and pH, As will be explained in the Discussion, the proximate cause of the GABA-induced fall in pHi and increase in pH. is a net influx of CO2 caused by a fall in the intracellular CO2 concentration, which, in turn, is due to the channel-mediated loss of internal HCO3-m To have an effect on pH, CO2 has to be hydrated to produce

pH CHANGES INDUCED BY GABA

249

carbonic acid, a process which in various cells is catalysed by the enzyme carbonic anhydrase (Maren, 1984; Droz & Kazimierczak, 1987). In order to find out whether carbonic anhydrase is involved in the pHi and pH5 changes produced by GABA, we examined the effects of acetazolamide and Benzolamide

Acetazolamide

GABA

7.0-

pHi

7.1 E 7.35

pHs 740E

7-45 L-i -60Em (mV) -70 [ 2 min

Fig. 5. Influence of benzolamide (10-6 M) and acetazolamide (10-4 M) on the effects of 0 5 mM-GABA on pH, and pH. in a solution containing 30 mM-HCO3-. Acetazolamide has been present for 6 min before the application of GABA. After each exposure to GABA, the preparation was superfused with control solution for about 45 min to allow full recovery of pHi.

benzolamide. Benzolamide is a very acidic (pKa = 3 2) sulphonamide and therefore poorly permeant across cell membranes, while acetazolamide (pKa = 7 4) is expected to be effective in both the extracellular and intracellular compartments (see Maren, 1984). As shown in Fig. 5, a brief application of benzolamide (10-6 M) completely blocked the transient increase in pH5 but it had no inhibitory effect on the rate of the GABA-induced fall in pHi. The effect of benzolamide on pH, was immediate, that is, the pH. transient was blocked in experiments where GABA and benzolamide were applied simultaneously. An immediate, selective inhibition of the pH5 transient caused by GABA was also produced by 10-6 M-acetazolamide. Application of a higher concentration (10-4 M) of acetazolamide for a few minutes gave rise to a considerable slowing down of the GABA-induced intracellular acidosis (Fig. 5). The maximum effect of acetazolamide was achieved following application of about 5 min. A consistent observation, evident also in Fig. 5, was that in the presence of inhibitors of carbonic anhydrase, the GABA-induced acidosis was closely paralleled by a slight decrease in pH8. We did not investigate this effect in detail, but it may be due to extrusion of H+ on Na+-dependent

K. KAILA, J. SAARIKOSKI AND J. VOIPIO

250

Cl--HCO3- exchange (see Thomas, 1984). Unlike fluxes of CO2 and HCO3-, movements of H' ions are expected to produce pH changes at the surface membrane which are enhanced rather than blocked upon inhibition of carbonic anhydrase activity.

Figure 6 shows the influence of 100 /IM-acetazolamide (applied for more than 8 min) on the maximum rate of fall of pHi induced by GABA at various Control

a

0-1 _

E

Acetazolamide D

I 0-05

10

100

1000

[GABA] (gM) Fig. 6. Influence of 10' M-acetazolamide on the dependence of -dpHi/dt on [GABA], measured in the presence of 30 mM-HCO3-. The data are from a single continuous experiment.

concentrations. It is clearly evident that acetazolamide produces a flattening of the dependence of -dpHi/dt on [GABA], so that the maximum value observed in the presence of acetazolamide is much smaller than that observed under control conditions. On average, the suppression of - dpHi/dt by 0-1 mM-acetazolamide was 59 + 6-3 % (+ S.E. of mean; n = 4) at 1 0 mM-GABA. In contrast to this, acetazolamide had no discernible effect on - dpHi/dt at low concentrations (5-10 aM) of GABA. These observations suggest that under control conditions, the effect of GABA on pHi is rate-limited by the GABA-gated bicarbonate conductance, but following inhibition of the intracellular carbonic anhydrase, hydration of CO2 becomes ratelimiting if a significant fraction of the conductance is activated. The above observations strongly suggest that the muscle fibre is equipped with two functionally different types of carbonic anhydrase: one which is intracellular, and one which has its catalytic site on the extracellular surface. The latter one is readily accessible to inhibitors irrespective of their membrane permeability. Further evidence supporting the above conclusion came from experiments (not illustrated) which showed that application of benzolamide (at a concentration of lo-6M and higher) immediately blocked the large changes (0-7-0-8 units) in pH, which were caused by a change from a nominally C02-free perfusion solution to one

pH CHA-NVGES IINDUICED BY GABA

251

which was equilibrated with 5% CO2 (30 mM-HCO3-). The blocking action of benzolamide on the pH, transient was not associated with a change in the rate of the C02-induced fall in pHi. A low concentration (10'6 M) of acetazolamide also had an immediate inhibitory effect on the CO2-induced changes in pHll with little effect on the rate of change of pHi. However, application of 10' M-acetazolamide for 5 min or more produced a marked suppression of the rate of fall of pHi. Attainment of steady-state pHi at plateau acidosis and the mechanism of recovery Acid extrusion during plateau acidification The present data show that application of GABA in the presence of bicarbonate does not lead to a complete dissipation of the plasmalemmal H' electrochemical gradient (cf. Table 1). Therefore, the attainment of a steady-state pHi level at plateau acidosis must depend on active extrusion of acid. At steady state, the GABAimposed acid load and the active extrusion of acid are of equal magnitude. The magnitude of these equal but opposite fluxes can be estimated by removing GABA and measuring the instantaneous rate of change of pHi at the onset of the recovery. Measurements of this kind showed that in the presence of 30 mm-HCO3- the steadystate efflux of bicarbonate due to GABA (05 mM) was 21-28% (mean 253%, n = 8) of that seen at the start of the exposure in the same preparations. This result is in good agreement with eqn (2) which predicts that, if the GABA-induced HCO3permeability at plateau acidosis is identical to that prevailing at the start of the application, then the decrease in the intracellular HC03- concentration and the associated depolarization should produce a decrease to about 35 % in J co. The fact that the predicted steady-state value of JHco is somewhat higher than that observed is attributable to a slight desensitization of the GABA-gated channels during the prolonged exposure which is required for the attainment of the plateau acidosis. Recovery of the GABA -induced acidosis Experiments such as those in Fig. 7A showed that the recovery of the GABAinduced fall in pHi was completely blocked in an Na'-free solution. Similarly, no recovery was observed in the absence of Cl- (cf. Fig. 5 in Kaila et al. 1989b). These observations suggest that the recovery is attributable to Na'-dependent Cl--HCO3exchange. This conclusion was supported by the observation that DIDS (10 IuM) fully blocked the recovery of the GABA-induced fall in pHi (Fig. 7B). We found that DIDS had an inhibitory effect also on the GABA-induced efflux of HCO3-. At a concentration of 0 5 mM-GABA, 10 and 100 ,uM-DIDS inhibited JHCO3 by about 20 and 80 %, respectively. Since the inhibition of JHCO3 was paralleled by a comparable inhibition of the GABA-induced depolarization, the effect of DIDS on the efflux of HCO3- is probably due to a blocking effect of the drug on the anion channels (cf. White & Miller, 1979; Hanrahan, Alles & Lewis, 1985).

HCO3- permeability of the resting membrane A substantial fraction of the conductance of resting crustacean muscle fibres is attributable to transmitter-insensitive Cl- channels (Dudel & Rudel, 1969). This raises the question whether an efflux of HCO3- mediated by these channels might

K. KAILA, J. SAARIKOSKI AND J. VOIPIO

252

impose an intracellular acid load in the presence of CO2-HCO3-. Such a constant acid load has been detected in frog muscle fibres (Abercrombie, Putnam & Roos, 1983; Putnam, Roos & Wilding, 1986), which are known to have a high resting Cl- conductance (Hodgkin & Horowicz, 1959). However, as stated above, the fact A

GABA 0 Na'

7.0rpHi 7-2h

7.4 L..

Em (mV)

-60

m

-75_ 5 min B

GABA

GABA

GABA

DIDS 6.6

6.8 pHi

7.0 7.2 L -60

Em (mV)

7

-80

L

10 min

Fig. 7. The recovery of the GABA-induced acidosis is blocked in the absence of Na' and in the presence of DIDS. A, dependence on Na' of the recovery of the acidosis induced by 0 5 mM-GABA in the presence of 30 mM-HCO3-. B, inhibition by DIDS (10-5 M) of the recovery of the acidosis induced by 0 5 mM-GABA in the presence of 30 mM-HCO3-. The initial part of the experiment in B shows that following a brief application of GABA in the absence of DIDS, a full recovery of pHi takes place.

that in the absence of GABA, variation of the membrane potential had little effect on pHi argues against a significant efflux of bicarbonate through resting Cl- channels (see Fig. 3). Further evidence which supported the above conclusion was gained from experiments which showed that under steady-state conditions in a solution

pH CHANGES INDUCED BY GABA

253

containing 30 mM-HCO3-, complete replacement of Na+ by choline for a period of up to 40 min did not produce a detectable change in pHi. Since secondary active transport of H+ in crayfish muscle fibres is strictly dependent on the presence of Na+ (Galler & Moser, 1986), a conductive acid load imposed in the absence of sodium should be observable as a fall in pHi. It is evident, therefore, that HCO3- efflux mediated by resting Cl- channels does not have an influence on pH regulation in the present preparation. It may be worthwhile noting that the above results also exclude the presence of Na+-HC03- co-transport in the plasma membrane of the opener muscle (see Boron & Boulpaep, 1983; Deitmer & Schlue, 1989). DISCUSSION

This work shows that in the presence of HCO3-, GABA produces a large fall in postsynaptic intracellular pH in the crayfish dactyl opener fibre. The fall in pHi is attributable to a net transmembrane movement of acid equivalents which has a maximum value of about 10 mequiv 11 min-'. Mechanism of the GABA -induced fall in pH1 Evidence for net efflux of HC03- through GABA -gated channels The postsynaptic intracellular acidosis caused by GABA is most probably due to an electrogenic efflux of HCO3- mediated by inhibitory anion channels. This conclusion is supported by several lines of evidence: (i) The HCO3- permeability calculated on the basis of the instantaneous change in pHi caused by GABA shows a concentration dependence which is similar to that of the GABA-activated conductance. (ii) The GABA-induced fall in pHi is linked to an increase in surface pH which indicates that it is caused by a transmembrane movement of acid equivalents and not by intracellular net production of HW. (iii) In agreement with the assumption that the movement of acid equivalents is due to electrodiffusion of HCO3- through transmitter-sensitive anion channels, the GABA-induced fall in pHi is inhibited by a depolarization of the membrane potential. (iv) The acidosis is paralleled by a depolarization which is consistent with a conductive efflux of HCO3-. Voltage-clamp experiments have, indeed, shown that GABA activates an inward current carried by HCO3- (Kaila et al. 1989b). (v) The GABA-induced acidosis and the accompanying depolarization are blocked by picrotoxin (Kaila & Voipio, 1987 a; Kaila et al. 1989 b), a non-competitive antagonist of GABA in the present preparation (Takeuchi & Takeuchi, 1969).

Prerequisite for the acidosis: open buffer system It is important to point out that a channel-mediated down-hill movement of a weak-acid anion (such as bicarbonate) is stoichiometrically equivalent to a net inward movement of H+ if and only if the acid and its conjugate anion constitute an open H+ buffer system, i.e. the neutral form of the acid (or its anhydride, e.g. C02) has to be permeant across the cell membrane. This can be explained as follows.

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Mechanism of action of GABA on intracellular pH and on surface pH in crayfish muscle fibres.

1. The mode of action of gamma-aminobutyric acid (GABA) on intracellular pH (pHi) and surface pH (pHs) was studied in crayfish muscle fibres using H(+...
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