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NEUROPHARMACOLOGY

Roles for Nicotinic Acetylcholine Receptor Subunit Large Cytoplasmic Loop Sequences in Receptor Expression and Function

Division of Neurobiology (Y.-P.K., L.X., J.B.E., R.J.L.) and Neurology (L.Z., J.W.), Barrow Neurological Institute, Phoenix, Arizona

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 4, 2, or 4 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., 2 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 4 subunits and chimeric 2 (2) 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 42-nAChR function, nested C2 sequences in the 2 subunit are not essential for functional receptor expression. Ligand-binding analyses also revealed only subtle differences in pharmacological profiles of 42-nAChR compared with 42-nAChR. Nevertheless, there was heightened emergence of agonist-mediated self-inhibition of 42 function, greater sensitivity to functional blockade by a number of antagonists, and faster and more complete acute desensitization of 42-nAChR than for 42-nAChR. These studies are consistent with unexpected roles of nested C2 sequences in nAChR function.

Address correspondence to: Dr. Yen-Ping Kuo, Department of Neurobiology, Barrow Neurological Institute, 350 West Thomas Road, Phoenix, AZ 85013. E-mail: ykuo{at}chw.edu.

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