Date of Award

5-1-2011

Degree Name

Doctor of Philosophy

Department

Pharmacology

First Advisor

Copello, Julio

Abstract

During skeletal muscle stimulation, there is a summation of local events of Ca2+ release from the sarcoplasmic reticulum, known as Ca2+ sparks. Ca2+ sparks originate from groups of skeletal ryanodine receptors (RyR1) that activate and close in synchrony. This synchrony allows for the rapid and massive release of Ca2+ from the sarcoplasmic reticulum to initiate contraction and, more important, would provide a mechanism to terminate Ca2+ release under conditions where independent RyR1 are normally active. RyR1 mutations can result in abnormal intracellular Ca2+ signaling that is associated with numerous skeletal muscle disorders including malignant hyperthermia and central core disease. Therefore, investigating the mechanisms that control RyR1 function can help identify how these mutations cause deleterious Ca2+ handling. Currently, most published research on RyR1s gating utilizes single RyR1 reconstituted into planar lipid bilayers to test isolated RyR1. However, in vivo, arrays of RyR1 function in synchrony. Attempts to reconstitute RyR1s into planar lipid bilayers result in experiments that contain multiple channels, which under specific conditions may gate in synchrony, also known as coupled gating. Coupled RyR1 gating was first reported by A. Marks' laboratory and attributed to FK-506 binding protein 12 (FKBP12) associating with neighboring RyR1s the stabilization of RyR1-RyR1 interactions that promote coupled gating. Previous studies suggested that ATP is required for coupled RyR1 gating; however, the mechanism by which ATP promotes the coordinated activity of RyR1s has not been elucidated and is the focus of this thesis. Therefore, my hypothesis is that the agonist action of ATP and FKBP12 bound to RyR1 are required for coupled RyR1 gating. In addition, new pharmacological tools are required to better understand coupled RyR gating. Thus, an additional goal is to identify pharmacological agents that modulate RyR1s in an innovative manner, i.e., help to uncover novel aspects of RyR1 gating and conduction. This investigation suggests that the adenosine based nucleotides, ATP, ADP and AMP, are agonists of RyR1s and promote coupled RyR1 gating in planar lipid bilayers. However, ADP and AMP were unable to maintain coupled RyR1 gating with physiological levels of Mg2+. This suggests that coupled gating would be impaired when the levels ATP decrease, as in muscle fatigue. When ATP was compared to other nucleotides (GTP, ITP, and TTP), the results suggest that the nucleotide agonist action on RyR1s is dependent on the phosphate groups and amino group on the nucleobase. As ATP is the most efficient nucleotide for coupled gating, I also investigated the indirect action of ATP to act as a kinase substrate or alter the cytoskeletal network. The addition of kinases, phosphatases and cytoskeletal modulators did not produce a significant disruption of coupled RyR1 gating. I also tested the role of addition of exogenous FKBP12 to RyR1s that gated independently or had partial coupling, but coupled gating was never improved. Also, the addition of high doses of rapamycin to remove FKBP12 from coupled RyR1 failed to functionally uncouple the channels. Finally, I attempted to find pharmacological agents that could aid in the understanding of coupled RyR1. Some agents were found to modulate RyR1s; however, I did not find a probe that would affect kinetics/conductance of RyR1s and was suitable for comparing coupled gating in bilayers with Ca2+ sparks in cells. Overall, coupled RyR gating is dependent on the physiological modulators ATP and Mg2+. This thesis represents a step forward in identifying the requirements for coupled RyR1 gating and understanding how RyR1s function in cells. Until an understanding of how these receptors communicate in cells is obtained, how different mutations alter the Ca2+ leak will continue to be quite difficult to study.

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