The β Subunit Determines the Ion Selectivity of the GABAA Receptor

Marianne L Jensen,Daniel B Timmermann,Tina H Johansen,Arne Schousboe,Thomas Varming,Philip K Ahring

doi:10.1074/jbc.m205645200

Abstract

The gamma-aminobutyric acid, type A (GABA(A)) receptor is a chloride-conducting receptor composed of alpha, beta, and gamma subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1-M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1-M2 loop of the beta(3) subunit to the corresponding amino acids of the alpha(7) nicotinic acetylcholine subunit rendered the GABA(A) receptor cation-selective upon co-expression with wild type alpha(2) and gamma(2) subunits. Similar mutations in the alpha(2) or gamma(2) subunits did not lead to such a change in ion selectivity. This suggests a unique role for the beta(3) subunit in determining the ion selectivity of the GABA(A) receptor. The pharmacology of the mutated GABA(A) receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn(2+) and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC(50) = 1.3 microm) compared with the wild type receptor (EC(50) = 6.4 microm) and a loss of desensitization to GABA of the mutant receptor.

Highlights

Introduction of theBsu36I site in the hGABAAR ␤3 subunit led to the I25ЈV mutation, which turned out to affect receptor function
Wild type and mutated GABAARs were characterized with respect to ligand binding properties and by electrophysiological experiments
The permeability of the gluconate ion has been shown to be ϳ10% of the chloride permeability in GABAARs [4]; using this value, the expected shift in reversal potential is ϩ43 mV according to the Nernst equation

Summary

EXPERIMENTAL PROCEDURES

The GABAA receptor subunits ␣2, ␤3, and ␥2L were cloned from human hippocampus poly(A)ϩ mRNA (Clontech) using reverse transcriptase-PCR. HindIII and Bsu36I sites at other positions within the cDNAs were eliminated without affecting the amino acid sequence. The synthetic M2 domains were digested with HindIII and Bsu36I and ligated with the cDNA of a GABAAR subunit. Introduction of the Bsu36I site in the hGABAAR ␤3 subunit led to the I25ЈV mutation, which turned out to affect receptor function. This was reversed using the oligonucleotide: ␤3 V25ЈI, 5Ј-GAGACCTTGCCTAAGATCCCCTATGTCAAAGCC-3Ј. Binding was performed with 0.5, 1, 2, 3, or 6 nM [3H]flumazenil (87 Ci/mmol, PerkinElmer Life Sciences) in triplicate in a final volume of 550 ␮l containing 50 –100 ␮g of protein, and nonspecific binding was determined in the presence of 1 ␮M clonazepam (Roche Molecular Biochemicals). The pellet was resuspended in 20 mM KH2PO4 buffer (pH 7.4) containing 200 mM KCl

A Mutated Heteromeric GABAA Receptor with Cation Selectivity

RESULTS

DISCUSSION