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The Journal of Neuroscience, January 1, 2001, 21(1):67-74
Structure and Dynamics of the GABA Binding Pocket: A Narrowing
Cleft that Constricts during Activation
David A.
Wagner and
Cynthia
Czajkowski
Department of Physiology, University of Wisconsin, Madison,
Wisconsin 53706
Photo-affinity labeling and mutagenesis studies have identified
several amino acids that may contribute to the ligand binding domains
of ligand-gated ion channels. These types of studies, however,
only generate a one-dimensional, static description of binding site
structure. In this study, we used the substituted cysteine
accessibility method not only to identify binding pocket residues but
also to elicit information about binding site dynamics and structure.
Residues surrounding the putative loop C ligand binding domain of
the GABAA receptor ( 2V199 to
2S209) were individually mutated to cysteine, and
the mutant subunits were coexpressed with wild-type  1
subunits in Xenopus oocytes.
N-biotinylaminoethyl methanethiosulfonate (MTSEA-biotin)
reacts with cysteines introduced at positions G203, S204, Y205, P206,
R207, and S209. This accessibility pattern is not consistent with
either an -helix or -strand. Instead, G203-S209 seems to form a
water-accessible extended coil, whereas V199-T202 appears to buried in
the protein or membrane. Coapplication of either GABA or the
competitive antagonist SR-95531 significantly slows MTSEA-biotin
modification of cysteines introduced at positions S204, Y205, R207, and
S209, demonstrating that these residues line and face into the GABA
binding pocket. MTSEA-biotin reaction rates reveal a steep
accessibility gradient from G203-S209 and suggests that the binding
pocket is a deep narrowing cleft. Pentobarbital activation of the
receptor significantly slows MTSEA-biotin modification of cysteines at
S204, R207, and S209, suggesting that the binding site may constrict
during gating.
Key words:
GABA; GABAA receptor; binding site; substituted cysteine accessibility method; cysteine mutagenesis; agonist efficacy; protein structure
Copyright © 2001 Society for Neuroscience 0270-6474/01/21167-08$05.00/0
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