Charged residues in the GABA-a receptor intracellular domain impact channel activation and desensitization.

Abstract

Opening and closing of GABA receptor (GABAR) channels regulate inhibitory signaling in the brain. Mechanisms underlying channel open, closed, desensitized transitions remain unclear. Although GABAR cryo-EM structures provide details about its extracellular and transmembrane domains, little is understood about its intracellular domain (ICD) and its role in regulating channel gating. We constructed a GABAR homology model based on the 5HT-type-3 receptor, which revealed a helix (MX) at the C-terminal end of M3 that is lysine rich in the α1 (6 lysines) and γ2 (8 lysines) subunits. Charged residues in the α and γ subunits were mutated to alanine. Effects on GABA EC50 were measured using two-electrode voltage clamping of oocytes expressing α1β2γ2L WT and mutant GABARs. αK327A significantly increased GABA EC50 6-fold in comparison to WT and significantly reduced GABAR macroscopic desensitization. Extent of current desensitization after 10 seconds in saturating GABA (10 mM) was 6.86 ± 2.28% (n=7) versus WT (23.7 ± 7.29%, n=10). Neutralizing charge at αK328, K330 and K335 had no functional effects. γK328A, γ2K330A and γ2K332A significantly increased GABA EC50 by 2 to 3 fold. γ2K330A reduced GABAR desensitization (extent after 10 seconds 7.36 ± 2.37%, n=2). Our data indicate structural perturbations in the α and γ MX regions of the GABAR ICD alter GABAR channel activation and desensitization. Interestingly, in our GABAR structural model, α1R312 (in MX) interacts with α1R249 (M1-M2 linker). Single charge reversals at these positions increased GABA EC50 and the rate of GABA desensitization. A charge swap (R312E - E249R) rescued receptor function to WT suggesting a functional electrostatic interaction and that these residues form a salt bridge, providing support for our homology model.