Effects of B2-Subunit Cytoplasmic-Loop Mutations A-4 Subunit Gain of Function Mutations on CNS Nicotinic Acetylcholine Receptors
PublisherThe University of Arizona.
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AbstractThe effects of the β2-365AAQA368 cytoplasmic-loop mutant subunit and the α4L9’s gain-offunction mutant subunit on CNS Nicotinic Acetylcholine Receptors were determined in this experiment. Four HSP β2-365AAQA368 mutants in pCEP4 were transfected into human SH-EPI cells. These constructs were screened for function through both an 3H-Epibatidine in situ binding assay and a 86-Rb+ efflux assay. The gain of function mutant α4L9’s position 5 (β3β2Aα6/3β2α4L9’s-pSHE) was injected into Xenopus laevis oocytes and tested for function via a Two-Electrode-Voltage Clamp apparatus. The β2-365AAQA368 mutant caused a rightward shift of the EC50 relative to the wild-type. Three monoclones from the β2- 365AAQA368 mutant 86-Rb+ efflux assay showed at least some activity: clones 212, 117, and 213. The maximum function achieved was 1023 scpm (0.9%) in clone 213, but this was not enough function for drug discovery work. The gain of function mutant α4L9’s position 5 caused a leftward shift of the EC50 relative to the wild-type. The mutation also increased the amount of function of the receptor by at least 1.5 fold.
Degree ProgramHonors College