Spontaneous activity, singly bound states and the impact of alphaPhe64 mutation on GABAR gating in the novel kinetic model based on the single-channel recordings.

GABA receptor is the primary mediator of inhibition in the adult mammalian brain. Our recent studies revealed that a classic gating scheme for GABAR needed to be updated with an intermediate step (flipping) and that the αPhe64 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 αβγ receptors and αPhe64 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 αPhe64 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 GABAR 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 αPhe64 mutation affects not only the flipping but also the opening/closing transitions indicating its generalized impact on the receptor gating.