Tryptophan scanning mutagenesis in TM4 of the GABA A receptor α1 subunit: implications for modulation by inhaled anesthetics and ion channel structure

Abstract

Previous studies have shown that amino acid residues in trans-membrane (TM) segments 1, 2 and 3 of the α subunit are critical for the enhancement of GABA A receptor function by inhaled anesthetics. In this study we used tryptophan (Trp) scanning mutagenesis between Ile 406 and Asn 417 in the α1 subunit to determine the effects of Trp substitution in the fourth transmembrane segment (TM4) on receptor gating and anesthetic modulation. Wild-type and mutant α1 subunits were transiently expressed in HEK 293 cells with wild-type β2 and γ2s subunits and GABA-activated currents were recorded using whole-cell voltage clamp. The potentiation by three inhaled anesthetics (isoflurane, halothane and chloroform) of responses elicited by a submaximal concentration of GABA were also examined. EC 50 values for GABA at the mutant receptors were in the range 4–60 μM (wild-type=20 μM), indicating that Trp substitution can alter the apparent affinity of the receptor for GABA positively or negatively, dependent on position. The variation of the calculated EC 50 value for GABA exhibited an interesting periodicity, with the cycle length for each repeat corresponding to approximately 3.6 amino acids. These data are consistent with an α-helical structure for the TM4 segment of the α subunit. Several of these Trp point mutations altered the ability of one or more of the three inhaled anesthetics to modulate receptor function; four of the 12 mutations abolished receptor modulation by one or more of the anesthetics tested. These data are consistent with a role for these residues at the extracellular end of TM4 in anesthetic modulation of GABA A receptors.