This renewal application describes ongoing studies on characterization of key proteins regulating Ca release from junctional sarcoplasmic reticulum (SR) in cardiac muscle. Four proteins will be studied, which are abundantly expressed in heart, co-localize to the junctional SR membrane, and form a complex. The four proteins investigated are junctin, triadin 1, calsequestrin, and the ryanodine receptor. The interactions stabilizing the complex of these four proteins at the junctional SR membrane will be resolved and the role of the complex in regulating Ca release elucidated. Junctin and triadin 1 are homologous, integral membrane proteins which bind to each other, to calsequestrin, and to the ryanodine receptor. They are responsible for anchoring calsequestrin to the ryanodine receptor at the lumenal face of the SR membrane. The sites of junctin and triadin 1 binding to each other, to calsequestrin, and to the ryanodine receptor will be localized, and the effects of junctin and triadin 1 on the channel activity of the ryanodine receptor will be defined, alone and in combination with calsequestrin. Junctin appears to promote junction formation with sarcolemmal membranes, and an attempt will be made to identify putative sarcolemmal docking protein(s) which may bind to the cytoplasmic domain of junctin. Calsequestrin is the major Ca-binding protein located in the lumen of the junctional SR of cardiac muscle, which stores the Ca required for Ca release. The sites of calsequestrin binding to junctin and to triadin 1 will be localized. Junctin and triadin 1 effects on Ca binding to calsequestrin will be characterized, and the effects of calsequestrin on the channel activity of the ryanodine receptor will be determined. Calsequestrin will be crystallized, and its 3-D structure determined in the presence and absence of Ca, complexed with and without the calsequestrin-binding domains of junctin and triadin 1. To assess the function(s) of junctin, triadin 1, and calsequestrin in intact myocytes and in live animals, the wild-type and mutated proteins will be overexpressed in transgenic mouse hearts. Effects of overexpression of the proteins on cardiac membrane biochemistry, ultrastructure, and intact cellular physiology will be investigated. Completion of these studies will increase our understanding of the role(s) of junctin, triadin 1, and calsequestrin in regulating the channel activity of the ryanodine receptor, in maintaining the molecular architecture at the junctional SR membrane, and in controlling the strength of the heartbeat. New animal models useful for investigation of cardiac hypertrophy and heart failure will result.
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