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Yang, Zheng Fang

University of British Columbia

Heartbeats and locomotion require muscle contraction, which is governed by the transport of calcium ions across membrane compartments within cells. Excitation-contraction coupling (ECC) is an essential process that connects extracellular signals to intracellular ion channels. Cells from excitable tissue have specialized ultrastructure in the form of transverse (T) tubules, where the plasma membrane comes into proximity with the sarcoplasmic reticulum (SR), an intracellular calcium reservoir. Voltage-gated calcium channels (CaV) in the plasma membrane and ryanodine receptors (RyR) in the SR membrane coordinate calcium release during ECC. Auxiliary proteins regulate and maintain the channels within the junctional membrane complex (JMC), including the junctophilin (JPH) proteins. JPH proteins form a bridge between membranes and have direct interactions with calcium channels; the skeletal isoform JPH1 and the cardiac isoform JPH2 are essential for muscle ECC. Mutations in JPH2 lead to severe diseases including hypertrophic cardiomyopathy (HCM). In this thesis, the goals are to elucidate the structures of JPH proteins using X-ray crystallography, study the interaction between JPH and CaV using a combination of X-ray crystallography and ITC, and study the lipid-binding function of JPH using protein-lipid overlay assays. The structures of JPH1 and JPH2 were determined at resolutions of 1.31 Å and 2.35 Å, respectively. ITC data showed that JPH1/JPH2 binds to the C-terminal domain of skeletal CaV1.1 with an affinity of 1-2 μM, and the structure of JPH2 in complex with the CaV1.1 binding site was determined at a resolution of 2.03 Å. Over 80 variants were mapped onto the JPH2 structure, most of which are HCM-causing mutations, and a subset of mutations target the JPH/CaV interaction site. Co-crystallization and ITC experiments showed no binding between JPH and short-chain phospholipids. Protein-lipid overlay assays demonstrate that high ionic strength disrupts the interaction between JPH and phospholipids, and clusters of positive residues on the surface of JPH affect lipid-binding.

Text

2023-06-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/81852

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/