Abstract
Neuronal nicotinic acetylcholine receptors (nAChRs) are potential targets for a wide variety of general anesthetics. We recently showed that alpha(4)beta(2) nAChRs are more sensitive than alpha(4)beta(4) receptors to the gaseous anesthetics nitrous oxide and xenon. The present study examines chimeric and point mutant rat nAChRs expressed in Xenopus oocytes and identifies a single amino acid residue (beta(2)-Val(253) or beta(4)-Phe(255)) near the middle of the second transmembrane segment (TM2) that determines gaseous anesthetic sensitivity. Mutations of this residue in beta subunits and the homologous residue of alpha(4) subunits (alpha(4)-Val(254)) showed that this position also determines sensitivities of nAChRs to acetylcholine, isoflurane, pentobarbital, and hexanol. In contrast, these mutations did not affect actions of ketamine. The positively charged sulfhydryl-specific reagent methanethiosulfonate ethylammonium reacted with a cysteine introduced at alpha(4)-Val(254) or beta(2)-Val(253), and irreversibly reduced anesthetic sensitivities of nAChRs. Propyl methanethiosulfonate is an anesthetic analog that covalently binds to a TM2 site of gamma-aminobutyric acid(A) and glycine receptors and irreversibly enhances receptor function. However, propyl methanethiosulfonate reversibly inhibited cysteine-substitution mutants at alpha(4)-Val(254) or beta(2)-Val(253) of nAChRs, and did not affect anesthetic sensitivity. Thus, residues alpha(4)-Val(254) and beta(2)-Val(253) alter channel gating and determine anesthetic sensitivity of nAChRs, but are not likely to be anesthetic-binding sites.
Highlights
During the past several decades, a consensus has emerged that general anesthetics act on one or more superfamilies of ligand-gated ion channels that include glutamate, ␥-aminobutyric acidA (GABAA),1 glycine, nicotinic acetylcholine, and 5-hydroxytryptamine3 receptors [1, 2]
We showed that nicotinic acetylcholine receptors (nAChRs) composed of 2 subunits were more sensitive than those composed of 4 subunits to gaseous and volatile anesthetics as reported previously [7, 11], and to pentobarbital and hexanol
These results, together with a distinct distribution of  subunits in the central nervous system [3,4,5,6], suggest that differences in sensitivity between 2 and 4 subunits for gaseous anesthetics and barbiturates may be of clinical significance
Summary
Construction of Chimeras and Single Amino Acid Mutants—The rat nAChR subunit cDNAs in several expression vectors, ␣2 and 2 in pSP65 vector (pSP65␣2 and pSP652, respectively), ␣3 and ␣4 in pSP64 vector (pSP64␣3 and pSP64␣4, respectively), and 4 in pBluescript SKϪ vector (pBSK4) were provided by Dr Charles W. To create a SpeI restriction enzyme site, silent mutations were introduced into pSP652 at amino acid residues position 224 –225 (numbering of the mature 2 subunit protein [16]) and into pBSK4 at residue position of 226 –227 (numbering of the mature 4 subunit protein [17]) by using the QuikChange mutagenesis kit (Stratagene, La Jolla, CA). Anesthetics and Neuronal nAChR Mutants the 1.1-kb ClaI-HindIII fragment from pBSK4 were ligated to yield pSP652-298-4. Electrophysiological Recording—Oocytes expressing wild-type and mutant nAChRs were placed in a rectangular chamber (approximately 100 l volume) and perfused (2 ml/min) with Ba2ϩ-Ringer’s solution to minimize the effects of secondarily activated Ca2ϩ-dependent ClϪ currents (115 mM NaCl, 2.5 mM KCl, 1.8 mM BaCl2, and 10 mM HEPES, pH 7.4) containing 1 M atropine sulfate. This concentration of dimethyl sulfoxide did not affect current responses of nAChRs
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