Identification of N-terminal Extracellular Domain Determinants in Nicotinic Acetylcholine Receptor (nAChR) α6 Subunits That Influence Effects of Wild-type or Mutant β3 Subunits on Function of α6β2*- or α6β4*-nAChR

Despite the apparent function of naturally expressed mammalian alpha 6*-nicotinic acetylcholine receptors (alpha 6*-nAChR; where * indicates the known or possible presence of additional subunits), their functional and heterologous expression has been difficult. Here, we report that coexpression with wild-type beta 3 subunits abolishes the small amount of function typically seen for all-human or all-mouse alpha 6 beta 4*-nAChR expressed in Xenopus oocytes. However, levels of function and agonist potencies are markedly increased, and there is atropine-sensitive blockade of spontaneous channel opening upon coexpression of alpha 6 and beta 4 subunits with mutant beta 3 subunits harboring valine-to-serine mutations at 9'- or 13'-positions. There is no function when alpha 6 and beta 2 subunits are expressed alone or in the presence of wild-type or mutant beta 3 subunits. Interestingly, hybrid nAChR containing mouse alpha 6 and human (h) beta 4 subunits have function potentiated rather than suppressed by coexpression with wild-type h beta 3 subunits and potentiated further upon coexpression with h beta 3(V9)'(S) subunits. Studies using nAChR chimeric mouse/human alpha 6 subunits indicated that residues involved in effects seen with hybrid nAChR are located in the alpha 6 subunit N-terminal domain. More specifically, nAChR h alpha 6 subunit residues Asn-143 and Met-145 are important for dominant-negative effects of nAChR h beta 3 subunits on h alpha 6h beta 4-nAChR function. Asn-143 and additional residues in the N-terminal domain of nAChR h alpha 6 subunits are involved in the gain-of-function effects of nAChR h beta 3(V9)'(S) subunits on alpha 6 beta 2*-nAChR function. These studies illuminate the structural bases for effects of beta 3 subunits on alpha 6*-nAChR function and suggest that unique subunit interfaces involving the complementary rather than the primary face of alpha 6 subunits are involved.