Mutations at the M2 and M3 Transmembrane Helices of the GABA(A)Rs α(1) and β(2) Subunits Affect Primarily Late Gating Transitions Including Opening/Closing and Desensitization

[Image: see text] GABA type A receptors (GABA(A)Rs) belong to the pentameric ligand-gated ion channel (pLGIC) family and play a crucial role in mediating inhibition in the adult mammalian brain. Recently, a major progress in determining the static structure of GABA(A)Rs was achieved, although precise molecular scenarios underlying conformational transitions remain unclear. The ligand binding sites (LBSs) are located at the extracellular domain (ECD), very distant from the receptor gate at the channel pore. GABA(A)R gating is complex, comprising three major categories of transitions: openings/closings, preactivation, and desensitization. Interestingly, mutations at, e.g., the ligand binding site affect not only binding but often also more than one gating category, suggesting that structural determinants for distinct conformational transitions are shared. Gielen and co-workers (2015) proposed that the GABA(A)R desensitization gate is located at the second and third transmembrane segment. However, studies of our and others’ groups indicated that other parts of the GABA(A)R macromolecule might be involved in this process. In the present study, we asked how selected point mutations (β(2)G254V, α(1)G258V, α(1)L300V, and β(2)L296V) at the M2 and M3 transmembrane segments affect gating transitions of the α(1)β(2)γ(2) GABA(A)R. Using high resolution macroscopic and single-channel recordings and analysis, we report that these substitutions, besides affecting desensitization, also profoundly altered openings/closings, having some minor effect on preactivation and agonist binding. Thus, the M2 and M3 segments primarily control late gating transitions of the receptor (desensitization, opening/closing), providing a further support for the concept of diffuse gating mechanisms for conformational transitions of GABA(A)R.