Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.
GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.
Taylor
P M
PM
The Medical Research Council Laboratory of Molecular Cell Biology, Department of Pharmacology, University College, London WC1E 6BT, United Kingdom.
Connolly
C N
CN
Kittler
J T
JT
Gorrie
G H
GH
Hosie
A
A
Smart
T G
TG
Moss
S J
SJ
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007668:Kidney; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D010424:Pentobarbital / Q000494:pharmacology; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D011963:Receptors, GABA-A / Q000737:chemistry* / Q000235:genetics* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D014162:Transfection; D014407:Tumor Cells, Cultured; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D046911:Macromolecular Substances; D020033:Protein Isoforms; D011963:Receptors, GABA-A; D011993:Recombinant Fusion Proteins; D005680:gamma-Aminobutyric Acid; D010424:Pentobarbital