Reduced effect of propofol at human {alpha}1{beta}2(N289M){gamma}2 and {alpha}2{beta}3(N290M){gamma}2 mutant GABA(A) receptors.
BACKGROUND
Propofol is an i.v. anaesthetic commonly used during general anaesthesia and intensive care. It is known that the second transmembrane segment of the beta subunit in the GABA(A) receptor is an important target for the effects of propofol; however, this has not been investigated in human receptors. The aim of this study was to investigate the effect of propofol on human beta2 and beta3 GABA(A) subunits with point mutations corresponding to the N265M mutation in the rat beta2 and beta3 subunits.


METHODS
Asparagine-to-methionine replacement at amino acid position 289 and 290 (N289M and N290M) in the beta2 and beta3 GABA(A) receptor subunits, respectively, was accomplished by site-directed mutagenesis. Thereafter, subunits for three human wild-type (alpha1beta2gamma2, alpha2beta2gamma2, and alpha2beta3gamma2) and two mutant GABA(A) receptor channels [alpha1beta2(N289M)gamma2 and alpha2beta3(N290M)gamma2] were introduced into Xenopus oocytes and studied with two-electrode voltage clamp.


RESULTS
The mutant receptors left-shifted the GABA concentration-response curve. In comparison with the wild-type receptors, both the positive modulatory and the agonistic effects of propofol were strongly reduced in potency and amplitude at both mutated GABA(A) channels.


CONCLUSIONS
We demonstrate that N289M or N290M mutation in human GABA(A) beta2 and beta3 subunits increases sensitivity to GABA, which is in contrast to the corresponding rat N265M mutation. Furthermore, the N289M and N289M mutations reduce both the potentiation of GABA-induced currents and the direct effect of propofol on channels incorporating either of the mutated subunits, which confirms earlier findings concerning the corresponding mutation in rat receptors and knock-in mice.
Jonsson Fagerlund
M
M
Department of Anesthesiology and Intensive Care Medicine, Karolinska University Hospital, Stockholm, Sweden. malin.jonsson.fagerlund@ki.se
Sjödin
J
J
Krupp
J
J
Dabrowski
M A
MA
D000595:Amino Acid Sequence; D018686:Anesthetics, Intravenous / Q000494:pharmacology*; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005260:Female; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017354:Point Mutation; D015742:Propofol / Q000494:pharmacology*; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D018686:Anesthetics, Intravenous; C495710:GABRB2 protein, human; C546273:GABRB3 protein, human; C546270:Gabrb3 protein, mouse; C546276:Gabrb3 protein, rat; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D015742:Propofol