Structure and dynamics of the zinc potentiation site of neuronal nicotinic acetylcholine receptors
Date of Award
Doctor of Philosophy (Ph.D.)
Molecular and Cellular Pharmacology
First Committee Member
Charles W. Luetje - Committee Chair
Neuronal nicotinic acetylcholine receptors (nAChRs) are allosterically modulated by zinc in a subunit dependent manner. alpha4beta4 nAChRs are potentiated by micromolar concentrations of zinc, whereas alpha3beta2 nAChRs are inhibited. Maximal potentiation of the alpha4beta4 receptor of approximately 5.5 fold is seen with 100muM zinc. Site-directed mutagenesis studies performed on alpha4beta4 revealed that mutation at 3 positions (alpha4D113, alpha4E59, and alpha4H162) significantly reduces zinc potentiation. This is based on the results from single mutant and double mutant screens. To further test the role of these residues, the Substituted-Cysteine Accessibility Method (SCAM) was employed using rat alpha4beta4 nAChRs expressed in Xenopus oocytes. A cysteine residue was introduced at each position and potentiation was measured before and after application of N-biotinoylaminoethyl methanethiosulfonate (MTSEA-biotin). Zinc potentiation was significantly reduced following treatment with MTSEA-biotin for all 3 identified residues. Thus, three lines of evidence support the direct role in mediating zinc potentiation for residues alpha4D113, alpha4E59, and alpha4H162.SCAM was also used to identify relative accessibility rates for MTSEA-biotin modification of alpha4E59C and alpha4H162C under different functional receptor states (resting, agonist-bound, and antagonist-bound). The MTSEA-biotin reaction rate for resting state receptors was slowed in the presence of zinc by approximately 3-fold for both residues studied. The ability of zinc to slow MTSEA-biotin access confirms that alpha4E59 and alpha4H162 are indeed positioned at the zinc potentiation site. Reaction rates for agonist-bound receptors experienced a dramatic slowing of approximately 19-fold with the presence of zinc. The increased ability of zinc to slow the MTSEA-biotin access rate indicates that zinc binds better when the receptor is agonist-bound. DHbetaE, a competitive antagonist, also improved the ability of zinc to impede MTSEA-biotin access. These studies revealed that zinc can bind to the resting state receptor. However, the binding of an agonist or a competitive antagonist results in a conformational change at the zinc site that improves zinc binding.
Biology, Neuroscience; Health Sciences, Pharmacology
Mihalak, Karla B., "Structure and dynamics of the zinc potentiation site of neuronal nicotinic acetylcholine receptors" (2006). Dissertations from ProQuest. 2419.