The regulation of muscle contraction by Ca2+ is essential for normal muscle function. The penultimate event in the process leading to activation of striated muscle is the binding of Ca2+ to Troponin C (TnC), thereby altering the interaction of this protein with the other subunits of the troponin complex, troponin I and troponin T, and leading to contraction. Two isoforms of TnC have been identified, one present in fast skeletal muscle and one found in cardiac and slow skeletal muscle. The greatest sequence divergence between these two proteins is found in the amino terminal helix and in the first Ca2+ binding domain, which is inactive in the cardiac isoform. The goal of this project is to determine the relationship of the structure of fast skeletal muscle TnC to its function in regulating muscle contraction. Much is known about the Ca2+ binding properties, structure, and mechanism of action of TnC. However, there remain many questions regarding the role of the four CA2+ binding sites and the significance of the amino terminal differences between TnC isoforms in the physiological regulation of contraction which, until recently, could not be addressed directly. Two of the CA2+ binding sites have a high affinity for both CA2+ and Mg2+ (sites III and IV) and are thought to play a structural, rather than a regulatory, role. Sites III and IV are thought to be occupied by Mg2+ under normal physiological conditions. The remaining two sites also bind Ca2+, but with a lower affinity and a higher specificity (sites I and II, CA2+-specific sites). A great deal of experimental evidence indicates that the CA2+-specific sites play a critical role in the initiation of muscle contraction upon Ca2+ binding. The expression of a complementary DNA (cDNA) clone for chicken fast skeletal muscle TnC in bacteria will permit us to directly examine the role of each of these Ca2+ binding sites in mediating muscle contraction. Using site-specific mutagenesis techniques, the primary amino acid sequence of TnC will be altered in the Ca2+ binding domains. The physical and functional properties of the mutated proteins will be studied both in vitro and in skinned skeletal muscle fibers. Using this approach to eliminate CA2+ binding to each site both individually and in combination with other sites, while minimizing changes in protein conformation, we will address the following questions: 1) Is Ca2+ binding to both sites I and II necessary for the initiation of muscle contraction?; 2) Are both Ca2+-Mg2+ sites required for the interaction of TnC with other components of the thin filament?; and 3) Do the Ca2+-Mg2+ sites (sites III and IV) play a regulatory function during periods of prolonged contraction when they may be occupied by Ca2+? Similarly, the functional significance of the amino terminal helix will be tested by examining the regulatory properties of a series of fast skeletal muscle TnC deletion mutants. A comparison of our results with complementary studies of chicken cardiac TnC from other laboratories should lead to an understanding of why cardiac TnC is functional with only one Ca2+-specific site.
