Roles for N-terminal Extracellular Domains of Nicotinic Acetylcholine Receptor (nAChR) {beta}3 Subunits in Enhanced Functional Expression of Mouse {alpha}6{beta}2{beta}3- and {alpha}6{beta}4{beta}3-nAChRs [Molecular Biophysics]

July 15th, 2014 by Dash, B., Li, M. D., Lukas, R. J.

Functional heterologous expression of naturally-expressed mouse α6*-nicotinic acetylcholine receptors (mα6*-nAChRs; where '*' indicates presence of additional subunits) has been difficult. Here we expressed and characterized wild-type (WT), gain-of-function, chimeric or gain-of-function chimeric nAChRs, 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) yield higher function than mα6mβ4-nAChRs. Function is not detected when mα6 and mβ2 subunits are expressed together or in the additional presence of hβ3 or mβ3 subunits. However, function emerges upon expression of mα6mβ2mβ3V9'S-nAChRs containing β3 subunits having gain-of-function, V9'S [valine(V)-to-serine(S)] mutations in transmembrane domain II and is further elevated 9-fold when hβ3V9'S subunits are substituted for mβ3V9'S subunits. 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" (E221 and F223), "E" (S144 and S148) and "β2-β3" (Q94 and E101) increases function of mα6mβ2*- (~2-3 fold) or mα6mβ4*- (~2-4 fold) nAChRs. EC50 values for nicotine acting at mα6mβ4*-nAChR are unaffaected by β3 subunit residue substitutions in loops C or E. Thus, amino acid residues located in primary (loop C) or complementary (loops β2-β3) interfaces of β3 subunits are some of the molecular impediments for functional expression of mα6mβ2β3- or mα6mβ4β3-nAChRs.
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