Membrane potential fluorescence: A rapid and highly sensitive assay for nicotinic receptor channel function

Seven cell lines expressing native and transfected nicotinic receptor subtypes were evaluated functionally by using fluorescent assays based on membrane potential and calcium dynamics with no-wash dye systems. Both assays provided the same rank orders of potency for (+/-)-epibatidine, 2S-(-)-nicotine, 7R,9S-(-)cytisine, and 1,1-dimethyl-4-phenylpiperazinium in a cell line expressing rat alpha3beta4 receptors. Nicotinic antagonists mecamylamine and dihydro-beta-erythroidine inhibited responses in both assays. Both agonist and antagonist activity were assessed within the same experiment. Agonists seemed more potent in the membrane potential assay than in the calcium assay, whereas the converse was true for antagonists. The membrane potential assay afforded robust responses in K-177 cells expressing human alpha4beta2 receptors, in IMR-32 and SH-SY5Y cells expressing human ganglionic receptors, and in TE-671 cells expressing human neuromuscular receptors. These lines gave weak to modest calcium responses. Moreover, membrane potential responses were obtained in cell lines expressing rat alpha4beta2 and alpha4beta4 receptors, which were devoid of calcium responses. Thus, membrane potential serves as a sensitive measure of nicotinic activity, and the resulting depolarization may be as important as calcium in cell signaling.