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

Area of Science:

• Neuroscience
• Pharmacology
• Cell Biology

Background:

• Nicotinic acetylcholine receptors (nAChRs) are crucial in the central and peripheral nervous systems.
• Functional assays are essential for characterizing nAChR subtypes and their responses to ligands.
• Existing fluorescent assays often require washing steps, complicating high-throughput screening.

Purpose of the Study:

• To functionally evaluate seven cell lines expressing various nicotinic receptor subtypes.
• To compare the sensitivity and utility of membrane potential and calcium flux assays for nAChR characterization.
• To assess both agonist and antagonist activities of nicotinic compounds in a single experimental setup.

Main Methods:

• Utilized "no-wash" fluorescent assays measuring membrane potential and intracellular calcium dynamics.
• Tested four reference ligands: epibatidine, nicotine, cytisine, and DMPP, across different cell lines.
• Employed nicotinic antagonists mecamylamine and dihydro-beta-erythroidine to validate assay responses.

Main Results:

• Both membrane potential and calcium assays yielded consistent rank orders of potency for tested agonists.
• Membrane potential assays showed robust responses in cell lines expressing human alpha 4 beta 2, ganglionic, and neuromuscular nAChRs.
• Calcium assays provided weak to modest responses in these lines, and no response in cells expressing rat alpha 4 beta 2 and alpha 4 beta 4 receptors, where membrane potential assays were effective.

Conclusions:

• Membrane potential serves as a highly sensitive indicator of nicotinic receptor activity, particularly for certain nAChR subtypes.
• The membrane potential assay is advantageous due to its "no-wash" format and broad applicability across diverse nAChR-expressing cell lines.
• Nicotinic receptor-mediated depolarization, reflected by membrane potential changes, may play a significant role in cellular signaling pathways, comparable to calcium dynamics.