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CELLULAR AND MOLECULAR

Functional Properties of Homomeric, Human 7-Nicotinic Acetylcholine Receptors Heterologously Expressed in the SH-EP1 Human Epithelial Cell Line

Divisions of Neurology, Barrow Neurological Institute, Phoenix, Arizona (L.Z., J.W.) and Neurobiology (Y.-P.K., A.A.G., J.-H.P., M.S.P., K.M.S., R.J.L.), Barrow Neurological Institute, Phoenix, Arizona

7-Nicotinic acetylcholine receptors (7-nAChRs) are broadly distributed in the central nervous system, where they play important roles in chemical and electrical signaling, and perhaps in neurite outgrowth, synaptic plasticity, and neuronal death/survival. To help elucidate their normal and pathophysiological roles, we have heterologously expressed human 7-nAChR in transfected SH-EP1 human epithelial cells. Reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses demonstrate expression of human 7 subunits as messenger RNA. Patch-clamp recordings exploiting a novel strategy to prevent functional rundown of whole-cell peak current responses to repeated acute challenges with nicotinic agonists show successful expression of functional 7-nAChR that mediate inward currents characterized by rapid phases of activation and inactivation. Concentration-response curves show that nicotine, acetylcholine, and choline are efficacious agonists at human 7-nAChRs. Current-voltage relationships show inward rectification for agonist-induced currents. Human 7-nAChRs exhibit some sensitivity to 7-nAChR antagonists -bungarotoxin (Bgt) or methyllycaconitine (MLA) when applied coincidentally with agonist, but much higher affinity block occurs when cells and 7-nAChRs are pre-exposed to antagonists for 2 min before challenge with agonist. Both Bgt and MLA are competitive inhibitors of 7-nAChR function. Whole-cell current peak amplitudes and half-times for inactivation of 7-nAChR functional responses to nicotine are dramatically reduced in the absence of extracellular Ca²⁺, suggestive of high Ca²⁺ permeability of the 7-nAChR channel. Thus, heterologously expressed human 7-nAChR in mammalian cells have properties of native 7-nAChR or of 7-nAChR heterologously expressed in other systems and serve as excellent models for studies of molecular bases of 7-nAChR function.

Address Correspondence to: Dr. Jie Wu, Division of Neurology, Barrow Neurological Institute, 350 West Thomas Rd., Phoenix, AZ 85013-4496. E-mail: jwu2{at}chw.edu

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