Call number: LD2668 .T4 1978 K58 / Master of Science

Muscle contraction and relaxation are controlled by the intracellular free Ca²⁺ concentration. The sarcoplasmic reticulum (SR) is an intracellular membrane system which regulates this internal free Ca²⁺ concentration. Responding to an electrical excitation of the cell surface membrane, the SR releases Ca²⁺ through a specific Ca²⁺ release channel, thus elevating the Ca²⁺ concentration inside muscle cell and causing the muscle to contract. Subsequent sequestration of Ca²⁺ by the SR Ca²⁺ pumps restores the resting state of the muscle cell. This research focuses on the Ca²⁺ release channel from skeletal muscle SR. The planar lipid bilayer technique was used to study the channel at the single channel level. The SR Ca²⁺ release channel was identified and isolated via its interaction with specific sulfhydryl oxidizing agents. This protein of a molecular mass of 106 kDa was then incorporated into a planar lipid bilayer membrane (BLM). In an asymmetrical Ca²⁺ solution, the channel protein demonstrates a single channel conductance of 107 ± 13 pS and a permeability ratio of Ca²⁺ versus Tris⁺ of 7.4 ± 3.3. In a symmetrical 250 mM NaCl solution, the channel protein displays a large single channel conductance of 400 ± 20 pS, and a weak voltage-dependence. The channel is activated by millimolar ATP and inhibited by micromolar ruthenium red. Nanomolar concentrations of ryanodine modify the channel by changing it from a rapidly gating full conductance state to a long-lived subconductance state. These results demonstrate that the isolated 106 kDa protein channel has properties similar to those observed following fusion of SR vesicles to a BLM. The bilayer system was also used to examine the effect of Ag⁺ on the SR Ca²⁺ release channel. Ag⁺ (0.2-1. 0 μM ) activates the SR Ca²⁺ release channel. Activation by Ag⁺ does not require the presence of Ca²⁺, Mg²⁺, or ATP. Ag⁺ activates the channel by increasing the open probability Po. Ag⁺ activation is always followed by a spontaneous inactivation. The channel is still sensitive to ruthenium red inhibition after exposure to Ag⁺. Isolated SR vesicles were fused to a BLM to study the effect of the photooxidizing dye, rose bengal, on the gating characteristics of the reconstituted SR Ca²⁺ release channel. Rose bengal activates the Ca²⁺ release channel in the presence of light by increasing the channel open probability and leaving the single channel conductance unchanged. This photoactivation is independent of the myoplasmic Ca²⁺ concentration, and can be achieved from either side of the membrane. In addition, the effect is inhibited by addition of 10-20 μM ruthenium red. When modified to its subconducting state by ryanodine, subsequent addition of rose Bengal reactivates the channel to a rapidly fluctuating full conducting state. These studies carried out at the single channel level utilizing the planar lipid bilayer technique have not only enhanced our understanding of the Ca²⁺ release mechanism of skeletal muscle SR, but also provided information about the toxic effects on biological membrane systems caused by heavy metals and oxidizing agents.

Calcium ions are important mediators in the mechanism of contraction and relaxation of muscle fibers. Depolarization of sarcolemma and transverse tubule causes an increase of myoplasmic ca2+ concentration which induces contraction of the myofibrils. In skeletal muscle fibers, the intracellular Ca2+ concentraton is regulated by an extensive membrane system, the sarcoplasmic reticulum (SR). Ca2+-release from SR is initiated by depolarization of the transverse tubule via a process referred to as excitation-contraction coupling. The Ca2+ - release channel located in the junctional SR plays an important role in this mechanism.

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Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 116-121).

Thesis (M.S.)--Catholic University of America. / Includes bibliographical references.

Thesis (Ph. D.)--University of Chicago, 1933. / Lithoprinted. "Private edition, distributed by the University of Chicago libraries, Chicago, Illinois." Includes bibliographical references.

Thesis (M.S.)--University of Wisconsin--Madison. / Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 37-41).

Thesis (M.S.)--Catholic University of America. / Includes bibliographical references.

No description available.