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Calcium regulation of calcium transport by sarcoplasmic reticulum

University of British Columbia

The sarcoplasmic reticulum (SR) of skeletal muscle is an intracellular membraneous network that, through the cyclical release and re-uptake of Ca²⁺ into and from, respectively, the cytoplasmic space, regulates myofilament shortening and, therefore, muscle contraction. SR derived from the terminal cisternae (HSR) demonstrates the property of Ca²⁺-induced Ca²⁺ release. Upon attainment of a threshold intralumenal Ca²⁺ load, application of a small pulse of extralumenal Ca²⁺ stimulates the release of a pool of intralumenal Ca²⁺ via the ligand gated Ca²⁺ permeable pore of the Ca²⁺ release channel/ryanodine receptor complex. It was hypothesised that intralumenal Ca²⁺ regulates the opening of the release channel. HSR vesicles were purified from skeletal and cardiac muscle by a novel technique. Structural characterisation of these membranes demonstrated an enrichment of harvested fractions in the Ca²⁺ release channel and the intralumenal Ca²⁺ binding protein, calsequestrin. In radiometric studies, skeletal HSR vesicles were shown to bind ryanodine with high capacity at both low and high affinity sites, with 2 fold stimulation of Ca²⁺ accumulation by the polyorganic cation Ca²⁺ channel blocker, ruthenium red. HSR vesicles passively loaded Ca²⁺. Passive loading of HSR vesicles with Ca²⁺ was found to be non-linearly dependent upon the concentration of Ca²⁺ within the loading medium. This suggested the presence of 2 intralumenal Ca²⁺ binding sites with different affinities for Ca²⁺. A spectroscopic dual-wavelength assay of Ca²⁺ release was developed that took advantage of peculiar spectral properties of the metallochromic sensitive dye Antipyrylazo III. In the presence of mM MgATP and mM Mg2+ the initial fast phase of HSR Ca²⁺ was well resolved. Evidence was presented that initial rapid uptake was associated with high affinity binding to an intralumenal compartment. Ca²⁺ -induced Caz+ release was shown to occur with a threshold loading of intralumenal Ca²⁺. The intralumenal Ca²⁺ threshold for Ca²⁺-induced Ca²⁺ release was decreased in the presence of ryanodine. Ryanodine induced Ca²⁺ release was also dependent upon the amount of intralumenal Ca²⁺. Ryanodine was also shown to inhibit sustained Ca²⁺-induced Ca²⁺ release by apparent inhibition of the binding of Ca²⁺ to intralumenal sites. These results suggested that junctional state transitions of the Ca²⁺ channel and calsequestrin were interdependent. Purified mM and mM Ca²⁺ activated neutral protease isoforms selectively cleaved the Ca²⁺ channel into 410 and 150kDa peptides with limited proteolysis. This was demonstrated in both HSR vesicles and the purified Ca²⁺ release channel. A novel 88kDa protein was also shown to be fragmented by both CANP isoforms. The identity of this prominent HSR associated protein remains obscure. CANP fragmentation of HSR protein elevated passive and active 4^Ca²⁺ loading in vesicles. This indicated that selective structural modification of the cytoplasmic portion of the release channel modified the comformational states of a intralumenal Ca²⁺ binding compartment in HSR vesicles. In spectroscopic studies, CANP proteolysis of HSR proteins increased the sensitivity to Ca²⁺ and ryanodine-induced Ca²⁺ release through decreases in the required intralumenal Ca²⁺ threshold for release. These functional alterations coincided with apparent single site cleavage of the release channel. Further proteolysis of the initial 410 and 150kDa peptides was without further significant effect upon function. Based upon the hypothesis that primary sequences rich in proline (P), glutamate (E), aspartate (D), serine (S) and threonine (T) (PEST regions) are recognition sites for CANP binding to substrates, a search for PEST regions within the Ca²⁺ channel was undertaken. It was tentatively proposed that two PEST regions near the N-terminal of the Caz release channel may represent sites close to the CANP cleavage site. The results of this work were discussed in relation to a possible role of Ca²⁺-induced Ca²⁺ release in regulating the patterning of Ca²⁺ cytosolic transients. The frequency and amplitude of cytosolic Ca²⁺ transients appear to be important in regulating protein expression. The requirement of intralumenal Ca²⁺-induced Ca²⁺ release may be a means by which the cyclical uptake and release of Ca²⁺ during muscle relaxation and contraction can be coordinated. This coordination may define the patterning of cytosolic Ca²⁺ transients. The increased sensitivity to Ca²⁺-induced Ca²⁺ release by HSR after CANP treatment may represent a means by which the patterning of cytosolic Ca²⁺ transients can be altered to effect changes in protein synthesis.

Text

2011-01-27

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http://hdl.handle.net/2429/30880

Interdisciplinary Studies

University of British Columbia

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