Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating.
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.
Engblom
A Christine
AC
Department of Pharmacology, The Royal Danish School of Pharmacy, Copenhagen 2100, Denmark.
Carlson
Berit X
BX
Olsen
Richard W
RW
Schousboe
Arne
A
Kristiansen
Uffe
U
D000595:Amino Acid Sequence; D000777:Anesthetics / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D004594:Electrophysiology; D015640:Ion Channel Gating / Q000187:drug effects*; D007700:Kinetics; D008969:Molecular Sequence Data; D010424:Pentobarbital / Q000494:pharmacology; D017354:Point Mutation; D015742:Propofol / Q000378:metabolism; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics*; D016415:Sequence Alignment; D018411:Spodoptera; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
D000777:Anesthetics; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D010424:Pentobarbital; D015742:Propofol