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Ryanodine receptors in calcium signaling pathways

Author

Yiming Li, University of the Pacific

Date of Award

2008

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Pharmaceutical and Chemical Sciences

First Advisor

David Thomas

First Committee Member

Timothy Smith

Second Committee Member

Miki Park

Abstract

Calcium (Ca²⁺) plays an important role as a second messenger, transmitting the message of arrival of stimuli such as hormones and neurotransmitters to the intracellular system that carries out the cellular response to the stimulus. The universality of Ca²⁺ as an intracellular messenger depends on its enormous versatility. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of Ca²⁺ being highly toxic.

Ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP₃Rs) are Ca²⁺ -release channels located on intracellular membranes of the endoplasmic reticulum (ER)/sarcoplasmic reticulum (SR) that perform essential functions as key targets of hormone/neurotransmitter action to initiate intracellular Ca²⁺ signals. The purpose of this project was to study the role of RyR₂ in Ca²⁺ signaling in the NG115-401L neuronal cell line. siRNA transfection methods were employed to knockdown RyR₂ expression levels in NG115-401L cells. We used reverse transcription and real-time PCR to evaluate RyR2 gene expression in transfected/untransfected cells. We also evaluated cytosolic Ca²⁺ changes induced by RyR activators or regulators, using fura-2 AM as the Ca²⁺ indicator. Successful RyR₂ gene knockdown allowed us to carry out some initial experiments to characterize the specific roles played by the RyR₂ receptor isoform. We examined cell responses to FK-506 under the condition of RyR₂ knockdown, finding that RyR₂ appears to confer selectivity to this response. Finally, the effects of siRNA transfection and FK-506 treatment on NG115-401L cell growth were evaluated. These experimental results may contribute to future studies of RyR₂, and help develop novel treatments for RyR₂-base d dysfunctional diseases.

Pages

82

Recommended Citation

Li, Yiming. (2008). Ryanodine receptors in calcium signaling pathways. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/710