Title:
Production of ryanodine receptor calcium release channel ATP-binding site mutants
Production of ryanodine receptor calcium release channel ATP-binding site mutants
Author(s)
Cutter, Catarina Santos
Advisor(s)
Spencer, Chrissy
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Abstract
Ryanodine receptors (RyRs) are a class of mammalian ion channels which are the primary efflux pathways for the release of Ca2+ from the sarcoplasmic reticulum. They play a critical role in muscle excitation-contraction coupling (ECC). Because it is the largest known ion channel, the mechanisms for its activation are not fully understood. ATP is a well characterized channel activator. However, its mechanism of activation has not been determined and the importance of ATP regulation of RyRs in vivo is not clear. In 2016, des George, et al. published a structure of RyR1 with ATP bound. The adenosine group of ATP is contained within a hydrophobic cleft while the triphosphate tail is extended and interacts with positively charged residues. The goal of this study was to identify residues important for ATP binding to the channel. Site-directed mutagenesis of the receptor was used to substitute specific residues in order to change their size and or charge. After transfection with recombinant DNA, HEK293 cells were harvested for isolation of microsomal membranes. Two of the largest hydrophobic residues of the cleft were replaced with alanine with the goal of drastically reducing or abolishing ATP binding to RyR1. The selected mutations F4960A and L4985A were expected to impair channel activation by both ATP and adenosine. After initial verification of wild type channel expression in HEK293 cells, later transfections with wild type and mutant RyR1 DNA failed to produce detectable amounts of protein. Low DNA transfection efficiency combined with the low yield of microsomal membrane likely contributed to the inability to detect channels in these preparations. Optimizing DNA transfections and scaling up the cell culture may increase the likelihood of successful protein production.
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Date Issued
2023-01-18
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Text
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Undergraduate Thesis