Date of Award

2022

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Vera Moiseenkova-Bell

Abstract

Calcium homeostasis is a highly regulated process in the body. Transient receptor potential vanilloid 5 (TRPV5) is a calcium selective ion channel that is responsible for the regulation of calcium reabsorption in the kidney. TRPV5 has been shown to be the essential for maintaining this calcium homeostasis and is implicated in disorders including hypercalciuria and kidney stone disease. However, little is known about the structural basis for mechanisms of TRPV5 regulation. Therefore, we aimed to address questions surrounding TRPV5 structure and regulation by solving cryo-electron microscopy (cryo-EM) structures of TRPV5 under various conditions. Specifically, we focused on regulation by low pH and phosphorylation by protein kinase A (PKA) that inhibit and activate TRPV5 respectively. Employing cryo-EM, we were able to structurally evaluate TRPV5 under several low pH conditions along with the activating phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The structures resolved conformational changes elicited by low pH conditions. Next, we solved the cryo-EM structures of TRPV5 to investigate the effect of PKA phosphorylation. TRPV5 was investigated using a phosphomimic mutation to simulate TRPV5 phosphorylation. Overall, the phosphorylation had little effect on channel structure, but we found that phosphorylation decreases calmodulin (CaM) mediated inactivation. These findings provide a better picture of TRPV5 modulation and will be helpful in the development of therapeutics aimed to address calcium centric pathologies.

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