INWARD CURRENT CAUSED BY SODIUM-DEPENDENT UPTAKE OF GABA IN THE CRAYFISH STRETCH-RECEPTOR NEURON

1. A two-microelectrode current-voltage clamp and Cl--selective microelectrodes were used to examine the effects of gamma-aminobutyric acid (GABA) on membrane potential, current and intracellular Cl- activity (a(Cl)i) in the crayfish stretch receptor neurone. All experimental solutions were CO2-HCO3- free. 2. GABA (500-mu-M) produced a mono- or biphasic depolarization (amplitude less-than-or-equal-to 10 mV), often with a prominent initial depolarizing component followed by a transient shift to a more negative level. In some neurones, an additional depolarizing phase was seen upon washout of GABA. Receptor desensitization, being absent, played no role in the multiphasic actions of GABA. 3. The pronounced increase in membrane conductance evoked by GABA (500-mu-M) was associated with an increase in a(Cl)i which indicates that the depolarization action was not due to a current carried by Cl- ions. 4. The currents activated by GABA under voltage clamp conditions were inwardly directed when recorded at the level of the resting membrane potential, and they often revealed a biphasic character. The reversal potential of peak currents activated by pulses of 500-mu-M-GABA (E(GABA)) was 9-12 mV more positive than the reversal potential of the simultaneously measured net Cl- flux (E(Cl)). E(Cl), was 2-7 mV more negative than the resting membrane potential. 5. E(GABA) (measured using pulses of 500-mu-M-GABA) was about 10 mV more positive than the reversal potential of the current activated by 500-mu-M-muscimol, a GABA agonist that is a poor substrate of the Na+-dependent GABA uptake system. 6. In the absence of Na+, the depolarization and inward current caused by 500-mu-M-GABA were converted to a hyperpolarization and to an outward current. Muscimol produced an immediate outward current both in the presence and absence of Na+. 7. Following block of the inhibitory channels by picrotoxin (100-200-mu-M), the depolarizing effect of 500-mu-M-GABA was enhanced and the transient hyperpolarizing shifts were abolished. 8. In the presence of picrotoxin, GABA (greater-than-or-equal-to 2-mu-M) produced a concentration-dependent monophasic inward current which had a reversal potential of +30 to + 60 mV. This current was inhibited in the absence of Na+ and by the GABA uptake blocker, nipecotic acid. Unlike the channel-mediated current, the picrotoxin-insensitive current was activated without delay also at low (2-10-mu-M) concentrations of GABA. 9. Brief (less-than-or-equal-to 10 s) pulses of GABA at a low concentration (less-than-or-equal-to 100-mu-M) produced only a small increase in conductance, and the reversal potential of the GABA-activated current obtained in this manner was close to that seen in the presence of picrotoxin. In contrast to this, the reversal potential of the current activated by pulses of 50-mu-M-muscimol was identical to that observed at 500 -mu-M. 10. The present results indicate that a sodium-dependent electrogenic GABA uptake mechanism has a direct influence on the current and voltage responses evoked by GABA in the crayfish stretch receptor neurone. The current component attributable to uptake makes E(GABA) significantly more positive than E(Cl) and explains the mono- and biphasic depolarizing actions of GABA. When measured using low concentrations and short pulses of GABA, the preferential activation of the uptake mechanism leads to an estimate of E(GABA) which is close to the reversal potential of the uptake current.