Introducing a proline in the α1 M2-M3 linker relieves a molecular brake on channel activation in α1β2γ2 GABAA receptors

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels (pLGICs) essential for inhibitory synaptic transmission throughout the central nervous system. Despite progress in understanding their three-dimensional structure, the molecular basis for how neurotransmitter binding is transduced to ion channel gating remains poorly understood. Furthermore, relatively little is known about the contributions of distinct subunits to this coupling within typical heteromeric receptors. A highly conserved proline (site 1) in the M2-M3 linker of pLGIC subunits is involved in channel gating - e.g., P273 in the GABAAR β2 subunit. In GABAARs, only the β subunits have an additional proline in the M2-M3 linker (site 2) - e.g., β2(P276) - whereas all other subunits have a non-proline at the homologous site 2 position. Here, we investigate the functional contribution of proline at site 2 in distinct subunits of α1β2γ2 GABAARs. We expressed wild type or mutant α1β2γ2 GABAARs in Xenopus laevis oocytes and used two-electrode voltage clamp electrophysiology to record channel currents in response to GABA and/or other ligands. First, we introduced a proline at site 2 in α1 or γ2 subunits: α1(A280P) and γ2(S291P). Second, we replaced the site 2 proline in the β2 subunit with its homologous non-proline residue from α1 or γ2 subunits: β2(P276A) or β2(P276S). We show that α1(A280P) confers enhanced GABA-sensitivity and spontaneous unliganded channel activity, whereas γ2(S291P) has minor effects on channel activation. In contrast, β2(P276A) or β2(P276S) either had no effect or enhanced GABA-activation, respectively, indicating complex functional dependence on the side chain at site 2 in the β2 subunit. When in combination with other substitutions, the presence or absence of α1(A280P) was consistently correlated with enhanced GABA-sensitivity and spontaneous unliganded opening. Thus, introduction of a proline at site 2 in the α1 M2-M3 linker biases the channel towards an activated state and prevents it from remaining closed at rest.