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

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Abstract
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+-selective microelectrodes. The extracellular HCO3- concentration was varied (0-30 mM) at constant pH (7.4). 2. GABA (5 .times. 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 pHs and it was inhibited by a K+-induced depolarization. 3. In the presence of 30 mM-HCO3-, 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- JHCO3e) 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-HCO3-, JHCO3e following exposure to 0.5 mM-GABA had a mean value of 8.0 mmol l-1 min-1. 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 pHs 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 pH1 at GABA concentrations higher than 20 .mu.M. 8. The recovery of the GABA-induced acidosis was associated with a fall in pHs. The recovery was completely blocked in solutions devoid of Na+ or of Cl-, as well as 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.

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