Calcium plays an essential role in the regulation of excitation-contraction coupling. Alterations in the regulation of intracellular levels of calcium is the molecular basis of many muscle diseases including muscular dystrophy and malignant hyperthermia. The objective of this proposal is to identify the cellular role of annexin VI in regulating the ryanodine-sensitive calcium-release channel found in the terminal cisternal of skeletal muscle sarcoplasmic reticulum. The annexins are a family of structurally related proteins which bind phospholipids in a calcium-dependent manner. Our current studies demonstrate annexin VI to be localized in skeletal muscle sarcoplasmic reticulum. We have also shown annexin VI to be a potent regulator of the reconstituted ryanodine-sensitive calcium-release channel. The proposed studies are designed to define the physiological function of annexin VI in skeletal muscle and in non-muscle cells. Differential extraction of cultured muscle cells will be used with antibodies against sarcoplasmic reticulum marker proteins (calsequestrin, calcium-release channel and calcium-pump) to resolve the cellular compartment of annexin VI. These studies will be complemented with immunogold EM. Reconstitution studies will be performed in planar lipid bilayers in order to identify regulatory proteins associated with annexin VI. These results will be complemented with photoaffinity labeling experiments designed to identify annexin VI binding proteins. The expression of annexin VI will be selectively suppressed with anti-sense RNA oligo nucleotides in muscle and non-muscle cells. We will the determine whether reduced annexin VI levels alters cellular function in vivo.
