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The role of Ryanodine receptors in neuronal calcium signaling

Author

Rui Cui, 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⁺) is a universal second messenger controlling a wide variety of cellular reactions and adaptive responses. All the versatility of a Ca²⁺ signaling requires that the concentration of Ca²⁺ ions in the cytoplasm be highly regulated. Generation of Ca²⁺ mobilizing signals in cells involves regulation by multiple components controlling Ca²⁺ release from the internal stores, Ca²⁺ influx across the plasma membrane, elicitation of Ca²⁺ sensitive processes and finally the removal of Ca²⁺ from the cells.

Inositol-1, 4, 5-trisphosphate receptors (IP₃Rs) and ryandine receptors (RyRs) are the most studied Ca²⁺ release channels located on the internal stores. Previous studies have shown ryanodine receptors (RyRs) play a key role in the process of Ca²⁺ signaling participating in the oscillatory patterns of controlling the release of Ca²⁺ from ER and regulating the influx of Ca²⁺ by coupling with plasma Ca²⁺ channels. Although recent progress deciphered the behavior and function of RyRs in regulation of Ca²⁺ signal, it still remains mysterious in understanding the molecular mechanism of its regulation and its connection with plasma membrane Ca²⁺ channels in neuronal cells. Here this study aimed to utilized the most cutting-edge RNA interference techniques, along with well-characterized pharmacological regulators of RyRs, to better characterized the role of RyRs is our neuronal cell line model NG115-401L.

Our first main goal of this project was to develop an effective protocol that could selectively knockout or knockdown expression levels of the RyR1 gene in NG115-401L cells. After testing different siRNA primers including their combination with different transfection reagent, the result shows a significant silencing effect to the RyR1 mRNA expression levels. In the second part of this study, we used a group of pharmacological agents with well-known regulatory actions on RyRs to characterize the functional roles of the RyRs expressed in NG115-401L cells. All four agonists which are ryanodine, caffeine, CMC and PCB 95 show their abilities to activate the RyRs, increase [Ca²⁺]_iand induce the influx of Ca²⁺ via SOC. After transfected NG115-401L cells by siRNA, the Ca²⁺ release and influx signals were highly diminished suggesting RyR1 gene was successfully knocked down and the successfully knocked down and the Ca²⁺ mobilization mediated by RyR1 was decreased greatly. Finally in order to study the effects of the regulation of Ca²⁺ by RyR modulators and RyR gene knockdown on cell growth patterns and cell viability, the NG115-401L cells were exposed to various concentrations of RyR regulators and siRyR1 primer for different time periods. The siRNA transfection showed the least effect on cell growth, as compared with pharmacological agents that modulate RyR function. Considering we achieved high levels of gene knockdown and its low cytotoxity, our result suggests that siRNA silencing for RyRs may become a promising gene therapeutic target in the future.

Pages

114

Recommended Citation

Cui, Rui. (2008). The role of Ryanodine receptors in neuronal calcium signaling. University of the Pacific, Thesis - Pacific Access Restricted. https://scholarlycommons.pacific.edu/uop_etds/705