Mutation at the putative GABAA ion-channel gate reveals changes in allosteric modulation

1 We have mutated a conserved leucine in the putative membrane-spanning domain to serine in human GABA(A) beta 2 and investigated the actions of a number of GABA(A) agonists, antagonists and modulators on human alpha 1 beta 2 Delta L259S gamma 2s compared to wild type alpha 1 beta 2 gamma 2s GABA(A) receptors, expressed in Xenopus oocytes. 2 The mutation resulted in smaller maximum currents to gamma-aminobutyric acid (GABA) compared to alpha 1 beta 2 gamma 2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation significantly decreased the GABA EC50 (110 fold), and reduced desensitization. Muscimol and the partial agonists 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) and piperidine-4-sulphonic acid (P4S) also displayed a decrease in EC50 3 In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on alpha 1 beta 2 Delta L259S gamma 2s receptors, with different degrees of maximum inhibition. 4 The effects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC20) Compared to wild type, none of these modulators potentiated the EC20 response of alpha 1 beta 2 Delta L259S gamma 2s receptors, however they all directly activated the receptor in the absence of GABA. 5 To conclude, the above-mutation resulted in receptors which exhibit a degree of spontaneous activity, and are more sensitive to agonists. Benzodiazepines and other agents modulate constitutive activity, but positive modulation of GABA is lost. The competitive antagonists bicuculline and SR95531 can also act as allosteric channel modulators through the same GABA binding site.