Acetylcholine and Alcohol Sensitivity of Neuronal Nicotinic Acetylcholine Receptors: Mutations in Transmembrane Domains Cecilia M. Borghese, Deeba N. Ali, Virginia Bleck, and R. Adron Harris Background: The effect of n-alcohols on glycine and -aminobutyric acid type A receptors depends on two specific amino acids (AAs) located in the transmembrane domains (TM) 2 and 3. Our aim was to assess whether the corresponding AAs in the neuronal nicotinic acetylcholine receptor (nAChR) also formed a binding pocket for alcohols. Methods: We made single AA substitutions in the homologous sites in rat neuronal nAChR 2 and 4 (L261 and L283) and expressed them in Xenopus laevis oocytes in combination with 4 wild type. The effect of different n-alcohols was studied in 4(L261A)4 and 4(L283A)4 nAChRs. The effect of ethanol, propanol, and octanol on acetylcholine (ACh) responses was studied in 2(L261X)4 and 2(L283X)4 nAChRs. Results: Most of the mutations in the 2 subunit, in either the 261 or the 283 position, induced changes in ACh sensitivity and increased alcohol action, but none was able to reduce ethanol potentiation. In 4(L283A)4, enhancement of potentiation by short-chain alcohols was observed, as well as a change from inhibition to potentiation for long-chain alcohols. The exposure of the AAs was assessed through the action of a charged thiol-specific reagent on 2(L261C)4 and 2(L283C)4, and these experiments suggest that the AA in TM2 is located in a water-accessible position, whereas the AA in TM3 is inaccessible. However, a noncharged thiol-specific reagent did not affect either ACh responses or ethanol effect on 2(L261C)4. Conclusions: The AAs located at positions 261 and 283 of the 2 and 4 nAChR subunits do not seem to form a binding pocket for alcohols. Additional studies are required to determine whether alcohols act on a site near these AAs or on sites unrelated to the TM2-TM3 site found in glycine and -aminobutyric acid type A receptors. Key Words: Nicotinic Acetylcholine Receptor, Ligand-Gated Ion Channel, Transmembrane Domain, Alcohol, Xenopus Laevis Oocyte. T HE NICOTINIC ACETYLCHOLINE receptor (nAChR) is the prototype for a superfamily of ligand- gated ion channels (Corringer et al., 2000) that includes inhibitory [glycine (Gly), -aminobutyric acid type A (GABA A ), and GABA receptors] as well as excitatory receptors (nAChRs and 5-hydroxytryptamine 3 receptors). These receptors have a pentameric structure, whereby the five subunits are arranged in a quasisymmetric distribution around a central pore (Unwin et al., 1988). Each subunit presents a large extracellular amino-terminal domain, fol- lowed by four transmembrane (TM) domains and a short extracellular carboxyl-terminus (Fig. 1C); the five TM2s (one from each subunit) line the central channel. The subunits that form the neuronal nAChRs are 2 to 9 and 2 to 4, and recently 10 was added to the list (Elgoyhen et al., 2001, Lustig et al., 2001). Some neuronal nAChR subunits can form homomeric receptors (e.g., 7), whereas others can form only heteromeric receptors (e.g., 2, 4). The muscle nAChR subunit composition has been defined, but the exact stoichiometry for the neuronal nAChR has not been determined yet; the evidence so far points to a receptor composed by two and three (Boorman et al., 2000; McGehee and Role, 1995). Although the structure of the Torpedo nAChR has been established at 4.6 Å resolu- tion (Miyazawa et al., 1999), the precise nature of the TM domains remains controversial. Whereas there is compel- ling evidence that TM2 is essentially -helix in muscle-type nAChR, the other TM domains seem to present -strand structure or a mixed / topology (Corringer et al., 2000). Alcohol actions on this superfamily have been the object of numerous studies because these receptors are consid- ered primary candidates among the proteins that may pos- From the Waggoner Center for Alcohol and Addiction Research, The University of Texas at Austin, Austin, Texas. Received for publication March 19, 2002; accepted September 16, 2002. This study was supported by NIAAA, NIGMS, the Texas Commission on Alcohol and Drug Abuse, and the Waggoner Center for Alcohol and Addic- tion Research. Reprint requests: Reprint requests: R. Adron Harris, PhD, The University of Texas at Austin, Waggoner Center for Alcohol and Addiction Research, 1 University Station A4800, Austin, TX 78712-0159; Fax: 512-232-2525; E- mail: harris@mail.utexas.edu This work was presented in part at the Society for Neuroscience 30th Annual Meeting, November 4 –9, 2000, New Orleans, LA. DOI: 10.1097/01.ALC.0000042012.58231.D9 0145-6008/02/2612-1764$03.00/0 ALCOHOLISM:CLINICAL AND EXPERIMENTAL RESEARCH Vol. 26, No. 12 December 2002 1764 Alcohol Clin Exp Res, Vol 26, No 12, 2002: pp 1764–1772