Two novel residues in M2 of the gamma-aminobutyric acid type A receptor affecting gating by GABA and picrotoxin affinity

An amino acid residue was found in M2 of gamma -aminobutyric acid (GABA) ty pe A receptors that has profound effects on the binding of picrotoxin to th e receptor and therefore may form part of its binding pocket. In addition, it strongly affects channel gating. The residue is located N-terminally to residues suggested so far to be important for channel gating. Point mutated alpha (1)beta (3) receptors were expressed in Xenopus oocytes and analyzed using the electrophysiological techniques. Coexpression of the alpha (1) s ubunit with the mutated beta (3) subunit beta (3)L253F led to spontaneous p icrotoxin-sensitive currents in the absence of GABA. Nanomolar concentratio ns of GABA further promoted channel opening. Upon washout of picrotoxin, a huge transient inward current was observed. The reversal potential of the i nward current was indicative of a chloride ion selectivity. The amplitude o f the inward current was strongly dependent on the picrotoxin concentration and on the duration of its application. There was more than a 100-fold dec rease in picrotoxin affinity. A kinetic model is presented that mimics the gating behavior of the mutant receptor. The point mutation in the neighbori ng residue beta (3)A252V resulted in receptors that displayed an about 6-fo ld increased apparent affinity to GABA and an about 10-fold reduced sensiti vity to picrotoxin.