SMALL ANKYRINS IN EXCITABLE CELLS

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The sarcoplasmic reticulum (SR) is embedded in the myoplasm, where it surrounds the contractile structures of each myofibril. Without a mechanism to stabilize the membrane and to link it at regular intervals to the contractile apparatus, the SR might be damaged during the contractile cycle, causing the uncontrolled release of its stores of Ca2+ and profound affects on muscle structure and function. In healthy muscle, of course, this does not occur, due in part to the presence of regular links between the SR and the Z disks of myofibrils. Here we propose to characterize a protein that may form these connections. We have discovered small, alternatively spliced forms of the structural protein, ankyrin, in striated muscle cells. The small ankyrins (abbreviated sAnk1) are enriched in the SR specifically at sites flanking the M and Z disks of nearby myofibrils. Ankyrin in mammalian erythrocytes stabilizes the cell membrane by linking it to the spectrin-based cytoskeleton. We hypothesize that sAnk1 stabilizes the SR by attaching it to the cytoskeleton of striated muscle cells -- the contractile apparatus. We propose 4 specific aims to test this idea and its developmental and functional consequences. (i) sAnk1 has an unique, highly hydrophobic amino terminal sequence that is likely to anchor it to membranes and target it specifically to the SR. We will perform biochemical and cellular transfection experiments to determine how sAnk1 associates preferentially with the SR. (ii) sAnk1 may concentrate in the SR near M and Z disks as a result of specific binding to proteins present in those structures. We will use the yeast two-hybrid screen to identify these ligands, and immunolabeling at the confocal and ultrastructural levels to localize them in the sarcomere. (iii) If sAnk1 links the SR to the contractile apparatus, it and its ligands should play an important role in the organization and differentiation of the SR in developing muscle. We will use biochemical, immunological and structural approaches to determine how sAnk1 and its ligands are expressed and organized during development. (iv) If sAnk1 is important for sarcoplasmic organization, inhibiting its activity should disrupt the relationship of the SR with the contractile apparatus and alter Ca2+ homeostasis. We will use transgenic techniques to manipulate the levels of sAnk1 and its ligands in muscle cells, and physiological and ultrastructural techniques to assess the effects of these manipulations on the organization and function of the SR. Our studies should therefore help to elucidate the mechanisms responsible for the formation and organization of the SR in immature and adult striated muscle. These mechanisms may have an important role in excitation-contraction coupling, and in maintaining the structure and function of the SR in immature, adult, and aging skeletal muscle.