Roles for nicotinic acetylcholine receptor subunit large cytoplasmic loop sequences in receptor expression and function

To evaluate possible physiological roles of the large cytoplasmic loops (C2) and neighboring transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits, we generated novel fusion constructs in which human nAChR α4, β2, or β4 subunit C2 or C2 and neighboring sequences were replaced by corresponding sequences from the mouse serotonin type 3A (5-HT _3A) receptor subunit. Following stable expression in human SH-EP1 cells, we found that extensive sequence substitutions involving third and fourth transmembrane domains and neighboring "proximal" C2 sequences (e.g., β2 H322-V335 and V449-R460) did not allow functional expression of nAChR containing chimeric subunits. However, expression of functional nAChR was achieved containing wild-type α4 subunits and chimeric β2 (β2χ) subunits whose "nested" C2 domain sequences K336-S448 were replaced with the corresponding 5-HT_3A subunit sequences. Whereas these findings suggested indispensable roles for M3/M4 transmembrane and/or proximal C2 sequences in α4β2-nAChR function, nested C2 sequences in the β2 subunit are not essential for functional receptor expression. Ligand-binding analyses also revealed only subtle differences in pharmacological profiles of α4β2-nAChR compared with α4β2χ-nAChR. Nevertheless, there was heightened emergence of agonist-mediated self-inhibition of α4β2χ function, greater sensitivity to functional blockade by a number of antagonists, and faster and more complete acute desensitization of α4β2χ-nAChR than for α4β2-nAChR. These studies are consistent with unexpected roles of nested C2 sequences in nAChR function.