The second transmembrane domain (TMD2) of the Cys-loop family of ligand-gated ion channels forms the channel pore. The functional role of the amino acid residues contributing to the channel pore in neuronal nicotinic α3 receptors is not well understood. We characterized the contribution of TMD2 position V7′ to channel gating in neuronal nicotinic α3 receptors. Site-directed mutagenesis was used to substitute position α3 (V7′) with four different amino acids (A, F, S, or Y) and coexpressed each mutant subunit with wild-type (WT) β2 or β4 subunits in Xenopus oocytes. Whole-cell voltage clamp experiments show that substitution for an alanine, serine, or phenylalanine decreased by 2.3-6.2-fold the ACh-EC 50 for α3β2 and α3β4 receptor subtypes. Interestingly, mutation V7′Y did not produce a significant change in ACh-EC50 when coexpressed with the β2 subunit but showed a significant approximately two-fold increase with β4. Similar responses were obtained with nicotine as the agonist. The antagonist sensitivity of the mutant channels was assessed by using dihydro-β-erythroidine (DHβE) and methyllycaconitine (MLA). The apparent potency of DHβE as an antagonist increased by ∼3.7- and 11-fold for the α3β2 V7′S and V7′F mutants, respectively, whereas no evident changes in antagonist potency were observed for the V7′A and V7′Y mutants. The V7′S and V7′F mutations increase MLA antagonist potency for the α3β4 receptor by ∼6.2- and ∼9.3-fold, respectively. The V7′A mutation selectively increases the MLA antagonist potency for the α3β4 receptor by ∼18.7-fold. These results indicate that position V7′ contributes to channel gating kinetics and pharmacology of the neuronal nicotinic α3 receptors.
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