Mutation at the putative GABA A ion‐channel gate reveals changes in allosteric modulation

We have mutated a conserved leucine in the putative membrane‐spanning domain to serine in human GABA A β2 and investigated the actions of a number of GABA A agonists, antagonists and modulators on human α1β2ΔL259Sγ2s compared to wild type α1β2γ2s GABA A receptors, expressed in Xenopus oocytes. The mutation resulted in smaller maximum currents to γ‐aminobutyric acid (GABA) compared to α1β2γ2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation significantly decreased the GABA EC 50 (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 EC 50 . In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on α1β2ΔL259Sγ2s receptors, with different degrees of maximum inhibition. The effects of a range of allosteric modulators, including benzodiazepines and anaesthetics were examined on a submaximal GABA concentration (EC 20 ). Compared to wild type, none of these modulators potentiated the EC 20 response of α1β2ΔL259Sγ2s receptors, however they all directly activated the receptor in the absence of GABA. 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.British Journal of Pharmacology (1999) 127 , 1349–1358; doi: 10.1038/sj.bjp.0702687