Stoichiometry of a recombinant GABAA receptor.
GABA is the main inhibitory neurotransmitter in the mammalian brain. The postsynaptic GABAA receptor/pore complex is presumed to be a pentamer typically composed of a combination of alpha, beta, and gamma subunits, although the stoichiometry remains controversial. We probed the stoichiometry of the GABAA receptor by site-directed mutagenesis of a conserved leucine (to serine) in the putative second membrane-spanning domain of the rat alpha 1(alpha L263S), beta 2(alpha L259S), and gamma 2(alpha L274S) subunit isoforms. Coexpression of wild-type and mutant subunits of each class (e.g., alpha and alpha L263S), along with their wild-type counter-parts (e.g., beta and gamma), in Xenopus laevis oocytes resulted in mixed populations of receptors with distinct GABA sensitivities. This is consistent with the interpretation that the leucine mutation increased the GABA sensitivity in proportion to the number of incorporated mutant subunits. The apparent number of incorporated subunits for each class (alpha, beta, and gamma) could then be determined from the number of components comprising the compound GABA dose-response relationships. Using this approach, we conclude that the recombinant alpha 1 beta 2 gamma 2 GABAA receptor is a pentamer composed of two alpha subunits, two beta subunits, and one gamma subunit.
Chang
Y
Y
Department of Physiology and Biophysics, University of Alabama at Birmingham 35294, USA.
Wang
R
R
Barot
S
S
Weiss
D S
DS
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D007930:Leucine / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes / Q000235:genetics; D051381:Rats; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D015345:Oligonucleotide Probes; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D005680:gamma-Aminobutyric Acid; D007930:Leucine