Background: The effect of n-alcohols on glycine and gamma-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 alpha2 and alpha4 (alphaL261 and alphaL283) and expressed them in Xenopus laevis oocytes in combination with beta4 wild type. The effect of different n-alcohols was studied in alpha4(L261A)beta4 and alpha4(L283A)beta4 nAChRs. The effect of ethanol, propanol, and octanol on acetylcholine (ACh) responses was studied in alpha2(L261X)beta4 and alpha2(L283X)beta4 nAChRs.
Results: Most of the mutations in the alpha2 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 alpha4(L283A)beta4, 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 alpha2(L261C)beta4 and alpha2(L283C)beta4, 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 alpha2(L261C)beta4.
Conclusions: The AAs located at positions 261 and 283 of the alpha2 and alpha4 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 gamma-aminobutyric acid type A receptors.