Point Mutation in the First Transmembrane Region of the β2 Subunit of the γ-Aminobutyric Acid Type A Receptor Alters Desensitization Kinetics of γ-Aminobutyric Acid- and Anesthetic-induced Channel Gating

A Christine Engblom,Berit X Carlson,Richard W Olsen,Arne Schousboe,Uffe Kristiansen

doi:10.1074/jbc.m111215200

A Christine Engblom, Berit X Carlson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m111215200

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Abstract

A conserved glycine residue in the first transmembrane (TM1) domain of the beta2 subunit has been identified to be involved with desensitization induced by gamma-aminobutyric acid (GABA) and anesthetics. Recombinant GABA(A) receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the beta2 subunit (G219F) was to slow the time constant (tau) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median tau values were 0.87 s (25-75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than alpha1beta2gamma2 (p < 0.01) and alpha1(G223F)beta2gamma2 (p < 0.05). For pentobarbital-induced currents (500 microm), the corresponding median tau values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than that for alpha1beta2gamma2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.

Highlights

Most volatile and intravenous anesthetics enhance the activity of ␥-aminobutyric acid type A (GABAA)1 receptors and directly activate this ligand-gated chloride ion channel in the absence of its endogenous ligand, GABA [1, 2]
In the previous study by Carlson et al (2000), the mutation of the TM1 glycine of the ␤2 subunit to the homologous residue, phenylalanine, in the ␳1 subunit, i.e. ␤2(G219F) was shown to affect receptor gating induced by both GABA and anesthetics [15]. This finding was consistent with the suggestion that the TM1 region may work together with TM2 for channel gating [16]. Because this TM1 glycine on the ␤ subunit is perhaps linked with the channel gating region of TM2, the present study tests the hypothesis that glycine 219 on the ␤2 subunit affects the conformational events of desensitization induced by GABA and/or anesthetics
␤2(G219F)-mutant Receptors and GABA-induced Currents— The present study examined the effect of a point mutation in the TM1 region of the ␣1 and ␤2 GABAA receptor subunits on the kinetics of GABA-mediated ClϪ currents

Summary

EXPERIMENTAL PROCEDURES

Site-directed Mutagenesis and Generation of Recombinant Baculoviruses—Point mutations were introduced into the cDNAs of rat ␣1 and ␤2 GABAA receptor subunits with an in vitro mutagenesis system (Altered Sites II, Promega). The determination of virus titer and the amount of recombinant baculovirus added for each infection was performed according to the protocol from the Invitrogen instruction manual, Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques. The Sf9 cell cultures were used in experiments after incubation with virus for 27–30 h They were placed in an artificial balanced salt solution (ABSS) composed of (in millimolar): NaCl 162.5, KCl 3.5, Na2HPO4 1.25, MgSO4 2, CaCl2 2, glucose 10, and HEPES 10. Statistics—Current data (peak currents, end currents) were normally distributed They were described using mean and standard error (S.E.) and compared with analysis of variance followed where relevant by a Tukey multiple comparison procedure.

RESULTS

TABLE II

DISCUSSION