Amino acids lining the channel of the gamma-aminobutyric acid type A receptor identified by cysteine substitution.
The binding of gamma-aminobutyric acid (GABA) to gamma-aminobutyric acid type A (GABAA) receptors triggers the opening of an anion-selective channel. To identify amino acid residues that line the channel, we combined cysteine mutagenesis and covalent chemical modification. We mutated, one at a time, four consecutive residues (268-271) in the M2 membrane-spanning segment of the rat GABAA receptor alpha 1 subunit to cysteine and expressed the mutant alpha 1 subunits, together with either the beta 1 subunit or the beta 1 and gamma 2 subunits, in Xenopus oocytes. We probed the susceptibility of the cysteine substitution mutants to covalent modification by charged, sulfhydryl reagents added extracellularly. We assumed that among the residues in membrane-spanning segments, only those lining the channel would be susceptible to modification by polar reagents and that such modification would irreversibly alter conduction. We infer that the residues Thr-268 and Ile-271 are exposed in the channel in both the open and closed states but that Leu-269 and Ser-270 are not exposed. The susceptibility of Thr-268 and Ile-271 in the closed state implies that the gate must be closer to the cytoplasmic end of the channel than Thr-268.
Xu
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M
Center for Molecular Recognition, Columbia University, New York, New York 10032.
Akabas
M H
MH
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis*; D000818:Animals; D003545:Cysteine / Q000235:genetics*; D007473:Ion Channels / Q000737:chemistry*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D018079:Receptors, GABA / Q000737:chemistry* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D000596:Amino Acids; D007473:Ion Channels; D018079:Receptors, GABA; D003545:Cysteine