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Ligand and modulator binding in the skeletal muscle ryanodine receptor

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Title: Ligand and modulator binding in the skeletal muscle ryanodine receptor
Author: Lobo, Paolo Antonio
Degree: Master of Science - MSc
Program: Biochemistry and Molecular Biology
Copyright Date: 2012
Issue Date: 2012-12-11
Publicly Available in cIRcle 2013-12-31
Publisher University of British Columbia
Abstract: Calcium (Ca²⁺) is required in the cytoplasm as a potent second messenger for a variety of vital physiological events in the cell. Its supply to the cytoplasm can either be from extracellular sources, or from intracellular sarcoplasmic reticulum stores, predominantly through the ryanodine receptors (RyRs). The latter are large, ~2 MDa homotetrameric channels that sense initial Ca²⁺ blips due to voltage-­‐gated calcium channel influx and respond by opening to result in physiologically significant concentration spikes. This action is known as calcium induced calcium release and is the major process by which an excitation signal is translated to a physical, muscular contraction. Cytoplasmic Ca²⁺ concentrations have to be very well regulated and must return to resting levels for subsequent contractions to occur. The importance of this with regard to the RyR can be seen in two different ways: Firstly, its large cytoplasmic bulk, a huge docking site for modulators, emphasizes the necessity for regulation, and secondly, mutations in the RyR can cause severe genetic diseases as a result of Ca²⁺ mishandling. Presented here, are preliminary structural and binding studies for several different RyR regulators, both physiological and pharmacological. Skeletal muscle RyR (RyR1) 1-­‐617 that contains the drug dantrolene’s supposed binding site was crystallized and its structure determined. Isothermal Titration Calorimetry (ITC) and co-­‐crystallization attempts have not confirmed the binding of dantrolene. The structure does however shed light on the physical involvement of phosphatases as modulators of the channel. Caffeine binding was detected successfully by ITC and attributed to RyR1 217-­‐ 536. A co-­‐crystal structure yielded a binding site in the construct that could not be knocked out by site-­‐directed mutagenesis according to ITC. Three types of modulators were shown to bind RyR1 4071-­‐4128 by ITC. 1) Ca²⁺, which affects the channel both positively and negatively, 2) magnesium ions, which inhibit the channel, and 3) an intrinsic ligand in RyR1: residues 4295-­‐4325; a peptide that shows affinity for Calmodulin, yet another modulator of RyR. The results provide insight into allosteric reactions as a result of RyR ligand or modulator binding.
Affiliation: Medicine, Faculty of
URI: http://hdl.handle.net/2429/43668
Scholarly Level: Graduate

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