Changes in intracellular calcium represent an essential signal in excitation contraction and stimulus-response coupling. The objective of our laboratory has been directed towards identifying intracellular calcium-binding proteins in order to understand the mediation of the intracellular calcium signal. This revised renewal is focused towards elucidating the physiological function of the 67 kDa calcimedin (annexin VI). The annexins are a family of seven structurally related calcium- dependent phospholipid binding proteins. Our current results demonstrate that muscle tissues have the highest concentrations of annexin VI. Anti-annexin VI co-localizes with the calcium-pump, suggesting an association with the sarcoplasmic reticulum (SR). In addition, fractionation studies of SR demonstrate co-purifiiation of annexin VI with terminal cisternae. We propose four specific aims necessary to define the physiological function of annexin VI: (1) Binding constants for phospholipids and calcium will be determined in order to to evaluate the physiological significance of regulation; (2) Anti-annexin VI antibodies will be co-localized with the calcium-pump, calsequestrin and the ryanodine-sensitive calcium release channel; (3) Reconstitution studies of HSR proteins and fractions will be performed and in planar lipid bilayers in order to further resolve the regulatory role of annexin VI in calcium-dependent calcium release. Photoaffinity labeling studies will be employed to identify annexin VI target proteins in isolated terminal cisternae; and (4) The expression of annexin VI will be selectively depressed through the use of permeable, stable anti-sense RNA S-oligo nucleotides in cultured mouse muscle cells. The phenotypes examined will be rates of contraction/relaxation and dynamics of calcium release/uptake. Information on the function of annexin VI in skeletal muscle will be obtained by correlating localization studies in this highly ordered tissue with regulation of calcium release/uptake in a well-defined organelle, the SR, and with alteration in a highly differentiated phenotype, contraction. Our studies will provide definitive evidence for the physiological role of annexin VI in maintaining intracellular calcium homeostasis.
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