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.
| Identifer | oai:union.ndltd.org:pdx.edu/oai:pdxscholar.library.pdx.edu:open_access_etds-2303 |
| Date | 01 January 1991 |
| Creators | Xiong, Hui |
| Publisher | PDXScholar |
| Source Sets | Portland State University |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Dissertations and Theses |
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