Identification of a [3H]muscimol photoaffinity substrate in the bovine gamma-aminobutyric acidA receptor alpha subunit.
An amino acid which appears to be involved in an agonist binding site of the gamma-aminobutyric acid type A (GABAA) receptor protein has been identified by photoaffinity labeling of affinity purified bovine receptor with [3H]muscimol and microsequencing of a chymotryptic fragment. Sequencing of a major purified labeled peptide gave the sequence Thr-Ile-Asp-Val-Phe with release of 3H label in the fifth cycle, corresponding to Phe. These 5 residues match the published sequence for the bovine alpha 1 subunit beginning at Thr61, following the chymotryptic substrate Tyr60. This sequence is found in the alpha 1-3 subunits of rat and cow and alpha 5 of rat, but not in any beta. Although previous evidence tentatively suggested that the beta subunit was the major [3H]muscimol-labeled subunit, homomeric expression studies have shown the potential for a functional gamma-aminobutyric acid binding site on all classes of subunits. The residue carrying the label, Phe65 on alpha 1, is conserved among all GABAA receptor alpha subunits, gamma 2, and delta. In addition, recent data indicated that a point mutation of Phe64-->Leu in rat alpha 1 subunit strongly decreased agonist and antagonist affinity when coexpressed with beta 2 and gamma 2 subunits in Xenopus oocytes (Sigel, E., Baur, R., Kellenberger; S., and Malherbe, P. (1992) EMBO J. 11, 2017-2023). The photolabeled Phe residue identified in this report is the bovine homolog to the Phe64 identified by the mutation studies of Sigel et al., to affect function in the rat GABAA receptor.
Smith
G B
GB
Department of Molecular and Medical Pharmacology, University of California, Los Angeles 90024.
Olsen
R W
RW
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D009118:Muscimol / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010777:Photochemistry; D051381:Rats; D018079:Receptors, GABA / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment
D000345:Affinity Labels; D010446:Peptide Fragments; D018079:Receptors, GABA; D009118:Muscimol