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

Abstract

1 We have mutated a conserved leucine in the putative membrane-spanning domain to serine in human GABA A b2 and investigated the actions of a number of GABA A agonists, antagonists and modulators on human a1b2DL259Sg2s compared to wild type a1b2g2s GABA A receptors, expressed in Xenopus oocytes. 2 The mutation resulted in smaller maximum currents to g-aminobutyric acid (GABA) compared to a1b2g2s receptors, and large leak currents resulting from spontaneous channel opening. As reported, this mutation signi®cantly 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 . 3 In addition to competitively shifting GABA concentration response curves, the antagonists bicuculline and SR95531 both inhibited the spontaneous channel activity on a1b2DL259Sg2s receptors, with di erent degrees of maximum inhibition. 4 The e ects 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 a1b2DL259Sg2s 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.

Tonically Active GABAA Receptors in Hippocampal Pyramidal Neurons Exhibit Constitutive GABA-Independent Gating

“…Spontaneous gating of recombinant GABA A receptors can be enhanced by introducing mutations in the transmembrane 2 domain (Chang et al, 1996;Thompson et al, 1999). In agreement with our findings using native and recombinant GABA A receptors, gabazine has a negligible efficacy relative to bicuculline as an inhibitor of spontaneously active GABA A receptors containing mutations that induce constitutive gating (Chang and Weiss, 1999;Thompson et al, 1999).…”

An Engineered Glutamate-gated Chloride (GluCl) Channel for Sensitive, Consistent Neuronal Silencing by Ivermectin

“…1A). Weak agonist-induced signals were the result of elevated base-line fluorescence (data not shown), suggesting an increased level of background conductance, which has been reported for some leucine 9Ј mutations in other Cysloop receptors (13,20,(22)(23)(24)(25). The L9ЈF mutant retained a maximal response comparable with that of WT-XFP.…”

“…A number of studies on both cationselective (nicotinic acetylcholine and serotonin 5-HT 3 ) and anion-selective (GABA and glycine) Cys-loop receptors have demonstrated that mutation of a highly conserved pore-lining residue, leucine 9Ј, to one of several other amino acids can dramatically increase agonist sensitivity through effects on channel gating (13)(14)(15)(16)(17)(18)(19)(20)(21). This gain-of-function effect is evident in the dose-response relation, in which channel activation occurs with lower concentrations of agonist.…”

Study on the structure and insecticide sensitivity of the RDL GABA receptor^#

“…1). [3][4][5][6][7][8][9][10] Thus, the development of cyclodiene-resistant pests and a worldwide outbreak of fipronil-resistant pests are causing problems. Fipronil is a second generation NCA, but A2′S and A2′G mutations provide a low level of cross-resistance to fipronil.…”