Multiple Modes for Conferring Surface Expression of Homomeric β1 GABAA Receptors

John R Bracamontes,Joe Henry Steinbach

doi:10.1074/jbc.m801292200

John R Bracamontes, Joe Henry Steinbach

Open Access

https://doi.org/10.1074/jbc.m801292200

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Abstract

The gamma-aminobutyric acid type A (GABA(A)) receptor assembles from individual subunits to form ligand-gated ion channels. Human (h) beta3 subunits assemble to form homomeric surface receptors in somatic cells, but hbeta1 subunits do not. We have identified three distinct sets of amino acid residues in the N-terminal extracellular domain of the hbeta1 subunit, which when mutated to the homologous residue in hbeta3 allow expression as a functional homomeric receptor. The three sets likely result in three modes of assembly. Mode 1 expression results from a single amino acid change at residue hbeta1 Asp-37. Mode 2 expression results from mutations of residues between positions 44 and 73 together with residues between positions 169 and 173. Finally, mode 3 results from the mutations A45V and K196R. Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit. These mutations do not, however, have a major effect on the surface expression of heteromeric receptors which include the alpha1 subunit.

Highlights

Studies have been made of the assembly of glycine and GABAA receptors, elucidating regions directing the protein interactions that underlie receptor assembly and revealing specific sequences involved in those interactions
Examination of homology-based structural models indicates that many of the residues are unlikely to be involved in physical inter-subunit interactions, suggesting that a major alteration is stabilization of an assembly competent form of the subunit
The results indicate additional regions of the extracellular domain, which are important in conferring competence for assembly, and suggest that intrasubunit interactions can be of critical importance

Summary

EXPERIMENTAL PROCEDURES

CDNA Constructs—A cDNA construct for the h␤1 subunit, GenBankTM accession number X14767, was obtained from David Weiss (University of Texas, San Antonio), originally cloned by Paul Whiting (23); and h␤3, accession number M82919, was obtained from Geoffrey White, (Neurogen, Bradford, CT). Each subunit combination was transfected into 5 wells of a 24-well plate, of which three were for ELISA and two for assaying total protein. The 2nd day after transfection, each well was aspirated and blocked with 0.5 ml of 4% milk phosphate-buffered saline (MPBS: 4% powdered milk, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.3) for 30 min at room temperature. 0.5 ml of MPBS containing secondary antibody was added to the wells for ELISA and incubated for 1 h at room temperature. Pentobarbital was dissolved in external solution and applied by a multichannel perfuser (SF-77B; Warner Instruments, Hamden, CT) The goal of these experiments was to examine the function of receptors on the cell surface, rather than to assay levels of expression. Cells expressing high levels of transfected receptors were identified using beads with coupled anti-FLAG antibody (20). Were done using the three-dimensional molecule viewer of the nized on the surface as when h␤3F alone was expressed (99%; Vector NTI Advance version 10.0.1 software (Invitrogen)

RESULTS

Findings

Homomeric expressiond

DISCUSSION