Abstract
Background: Mice carrying the GABAA receptor β3(N265M) point mutation, which renders receptors incorporating β3-subunits insensitive to many general anesthetics, have been used experimentally to link modulation of different receptor subtypes to distinct behavioral endpoints. Remarkably, however, the effect of the mutation on the susceptibility to modulation by isoflurane (a standard reference agent for inhalational vapors) has never been tested directly. Therefore, we compared the modulation by isoflurane of expressed α5β3(N265M)γ2L receptors with their wild type counterparts. Methods: Using whole-cell electrophysiological recording and rapid solution exchange techniques, we tested the effects of isoflurane at concentrations ranging from 80 μM to 320 μM on currents activated by 1 μM GABA. We measured drug modulation of wild-type α5β3γ2L GABAA receptors and their counterparts harboring the β3(N265M) mutation. Results: Currents elicited by GABA were enhanced two- to four-fold by isoflurane, in a concentration-dependent manner. Under the same conditions, receptors incorporating the β3(N265M) mutation were enhanced by approximately 1.5- to two-fold; i.e., modulation by isoflurane was attenuated by approximately one-half. Direct activation by isoflurane was also present in mutant receptors but also attenuated. Conclusions: In contrast to the complete insensitivity of β3(N265M) mutant receptors to etomidate and propofol, the mutation has only a partial effect on receptor modulation by isoflurane. Therefore, the persistence of isoflurane effects in mutant mice does not exclude a possible contribution of β3-GABAA receptors.
Highlights
The ionotropic γ-aminobutyric acid receptor (GABAAR) remains a target of considerable interest in understanding the mechanism of action of general anesthetics [1,2,3]
We found that GABAA receptors that incorporate the β3(N265M) mutation, co-expressed with α5 and γ2 subunits, are only partially insensitive to modulation by isoflurane
Experiments showing that mutations of the GABAAR β2 or β3-subunit that attenuate or eliminate their modulation by anesthetics confer resistance to anesthesia in behavioral studies provided important confirmation that GABAARs mediate the effects of these drugs [4,5]
Summary
The ionotropic γ-aminobutyric acid receptor (GABAAR) remains a target of considerable interest in understanding the mechanism of action of general anesthetics [1,2,3]. To link modulation of specific subtypes of GABAARs to the various endpoints of anesthesia, a fruitful experimental strategy has utilized mice carrying point mutations of individual receptor subunits that alter or eliminate anesthetic sensitivity; by comparing drug effects in wild-type versus mutant animals, the role of the specific receptor subtypes can thereby be tested. Mice carrying α1- or α2-subunit mutations weakly resisted isoflurane’s suppression of the righting reflex, but they did not differ from wild-type mice in tests of isoflurane-induced immobilization or memIontr.yJ. D amnesia, and only contribute modestly to immobility, may be unwarranted. 2020, 21, x FOR PEER REVIEW Int. J. Direct Activation of GABAA Receptors by Isoflurane. Application of isoflurane alone (i.e., in the absence of GABA) weakly activated wild-type 2Ar.2ep.cpeDpliirtcoeacrttsiAo(Fnctiigovufartisieoon3fl,ouforGpaeAnnBeAbaalAorRsn)e,ecae(psi.teor.re,spibnoyrttIhesdeofalpubrrseaevnnieocuesolyf G[1A9]B.
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