Spontaneous activity, singly bound states and the impact of alpha 1 Phe64 mutation on GABA A R gating in the novel kinetic model based on the single-channel recordings

Abstract GABA A receptor is the primary mediator of inhibition in the adult mammalian brain. Our recent studies revealed that a classic gating scheme for GABA A R needed to be updated with an intermediate step (flipping) and that the α 1 Phe64 mutation at the GABA binding site affects this transition. However, description of flipping at the single-channel level remains incomplete. In particular, its role in singly-bound and spontaneous activity remains unknown. We have performed thus single-channel recordings over wide range of agonist concentration for wild-type α 1 β 2 γ 2L receptors and α 1 Phe64 mutants. For WT receptors we observed relatively frequent brief spontaneous openings which were also present at low [GABA]. However, closed times distributions for spontaneous activity and at low [GABA] were clearly different indicating that a proportion of short-lived openings were due to liganded, most likely singly bound receptors. Increasing [GABA] resulted in prolongation of bursts and increased occurrence of bursts with long openings and short closures. Mutations of α 1 Phe64 residue dramatically affected the open and closed time distributions at high and saturating [GABA], especially in the case of cysteine mutants. However, this mutation weakly affected spontaneous or singly bound activity. Model fitting of our single-channel data led us to propose a novel and, to our knowledge, most complete GABA A R kinetic model in which flipping occurs in singly and doubly bound states. However, spontaneous activity did not reveal involvement of flipping. Moreover, we report that α 1 Phe64 mutation affects not only the flipping but also the opening/closing transitions indicating its generalized impact on the receptor gating.