PROTON SENSITIVITY OF THE GABA(A) RECEPTOR IS ASSOCIATED WITH THE RECEPTOR SUBUNIT COMPOSITION

1. Modulation of GABA(A) receptors by external H+ was examined in cult ured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A), receptor s composed of combinations of alpha, beta 1, beta 2, gamma 2S and delt a subunits. 2. Changing the external pH from 7.4 reduced GABA-activate d currents in sympathetic neurones. pH titration of the GABA-induced c urrent was fitted with a pH model which predicted that H+ interact wit h two sites (pK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA( A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH t itration predicted the existence of two sites with pK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the lef t by low pH and non-competitively inhibited at high pH (> 7.4). 4. alp ha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sens itivity to pH, with predicted pK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whil st high pH caused an inhibition (pK(a) values of 6.6 and 9.9). The GAB A concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipation with sub unit and epitope-specific antisera to alpha 1, beta 1 and delta subuni ts demonstrated that these subunits could co-assemble in cell membrane s. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid app lication of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) cause d an increased peak and reduced steady-state current, with a correspon dingly increased rate of desensitization. 9. Modulation of GABA(A) rec eptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge d istribution on the zwitterionic GABA molecules. 10. In conclusion, thi s study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dep endent on the receptor subunit composition. The potential importance o f H+ sensitivity of GABA(A) receptors is discussed.