Roles for N-terminal extracellular domains of nicotinic acetylcholine receptor (nAChR) β3 subunits in enhanced functional expression of mouse α6β2β3- and α6β4β3-nAChRs

Abstract

Functional heterologous expression of naturally expressed mouse α6∗-nicotinic acetylcholine receptors (mα6∗-nAChRs; where "∗" indicates the presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric, or gain-of-function chimeric nAChR subunits, sometimes as hybrid nAChRs containing both human (h) and mouse (m) subunits, in Xenopus oocytes. Hybrid mα6mβ4hβ3- (∼5-8-fold) or WT mα6mβ4mβ3-nAChRs (∼2-fold) yielded higher function than mα6mβ4-nAChRs. Function was not detected when mα6 and mβ2 subunits were expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerged upon expression of mα6mβ2mβ3V9′S-nAChRs containing β3 subunits having gain-of-function V9′S (valine to serine at the 9′-position) mutations in transmembrane domain II and was further elevated 9-fold when hβ3V9′Ssubunits were substituted for mβ3V9′Ssubunits. Studies involving WT or gain-of-function chimeric mouse/human β3 subunits narrowed the search for domains that influence functional expression of mα6∗-nAChRs. Using hβ3 subunits as templates for site-directed mutagenesis studies, substitution with mβ3 subunit residues in extracellular N-terminal domain loops "C" (Glu221and Phe223), "E" (Ser144and Ser148), and "β2-β3" (Gln94and Glu101) increased function of mα6mβ2∗- (′2-3-fold) or mα6mβ4∗(′2-4-fold)-nAChRs. EC50values for nicotine acting at mα6mβ4∗-nAChR were unaffected by β3 subunit residue substitutions in loop C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3 and E) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs.