Modulation of neuronal nicotinic acetylcholine receptors by zinc
Date of Award
Doctor of Philosophy (Ph.D.)
Molecular and Cellular Pharmacology
First Committee Member
Charles W. Luetje, Committee Chair
The effect of extracellular application of zinc on rat neuronal nicotinic acetylcholine receptors was investigated. alpha2, alpha3, and alpha4 subunits were expressed with beta2 and beta4 subunits in Xenopus oocytes and were studied under two-electrode voltage clamp. Co-application of zinc with low concentrations of acetylcholine resulted in differential effects based on receptor subunit composition. The response to acetylcholine of alpha3beta2 receptors was blocked by zinc. In contrast, alpha2beta2, alpha2beta4, alpha3beta4, alpha4beta2, and alpha4beta4 nAChRs exhibited biphasic modulation by zinc with potentiation of the acetylcholine response occuring at 1--100 muM zinc and block at higher concentrations. Zinc co-application with ACh resulted in currents which were as high as five fold greater than ACh alone in alpha4beta4 nAChRs. Cadmium was also able to potentiate alpha4beta4 nAChRs but was half as efficacious as zinc. Minimal potentiation occurred with nickel co-application.Specific amino acid residues responsible for potentiation of alpha4beta4 nAChRs by zinc were located. Both potentiation and block were voltage independent, suggesting a site of action outside of the transmembrane domains. By using diethylpyrocarbonate to modify histidine residues, potentiation was abolished while leaving the acetylcholine response intact. Varying extracellular pH modulated the degree of zinc potentiation, further supporting a role for histidine residues. Chimeric subunits identified areas important to potentiation by zinc. Candidate histidine, cysteine, and glutamate residues were subjected to mutagenesis. Four mutations decreased potentiation: H61N and H162G on the alpha4 subunit and D195C and H469Y on the beta4 subunit. These residues are found at the alpha/beta subunit interfaces neighboring those of the agonist binding site.The mechanism by which zinc potentiates nAChRs does not appear to be solely due to an increase in agonist binding affinity, as zinc is able to further potentiate a saturated ACh response. The rate of desensitization was unaffected by the presence of zinc. Open channel blocker experiments demonstrated that the number of active receptors is not changed by zinc. Single channel recordings showed that single channel amplitude is not increased by zinc; however, open probability was increased, mainly due to an increased burst duration. These results reveal a new mode of neuronal nAChR modulation.
Hsiao, Bernard, "Modulation of neuronal nicotinic acetylcholine receptors by zinc" (2002). Dissertations from ProQuest. 1843.