Studies on the regulation of muscle contraction by troponin

Abstract Two major areas of our current research interests will be discussed. The first deals with our attempts to understand the Ca 2+ -binding properties of Tn in situ and the second involves our recent antibody studies which are aimed at elucidating the structure of the Tn complex as well as the function of the various subunits in Ca 2+ regulation. Two different methods, one direct and one indirect, have been used to measure Ca 2+ binding to troponin in thin filaments. Both methods demonstrate that actin lowers the affinity of the Ca 2+ specific sites on Tn for Ca 2+ by approximately an order of magnitude while the affinity of the Ca 2+ Mg 2+ sites remains unchanged. Furthermore, the Ca 2+ dependence of myofibrillar ATPase activation corresponded exactly with Ca 2+ binding to the Ca 2+ -specific sites as measured by the fluorescence change of TnC DANZ specifically incorporated into myofibrils (H.G. Zot and J.D. Potter, J. Biol. Chem. , 257, 7678 (1982)). These results lead us to two major conclusions. The first is that if actin alters the structure of Tn(as indicated by a change in Ca 2+ affinity of the Ca 2+ -specific sites) then Ca 2+ binding to the Ca 2+ -specific sites must alter the structure of actin. Since previous studies have shown that the Ca 2+ -specific sites are the sites involved in regulating actomyosin interaction, this postulated change in actin structure brought about by Ca 2+ binding to these sites, may be the key event in muscle activation. The second conclusion is that the exchange of Ca 2+ with the Ca 2+ -specific sites in muscle is probably faster than previously estimated from studies on isolated Tn where the affinity of the Ca 2+ -specific sites is an order of magnitude higher. This is important since these sites must exchange Ca 2+ rapidly in order to be able to respond to the rapid [Ca 2+ ] transients which occur in muscle. A monoclonal antibody (IgG- ϰ 2 γ 2 ) to STnI has been produced using the somatic cell fusion of spleen cells from CB 6 F 1 /J mice immunized with STnI with cells of the myeloma cell line SP2/O-Ag-14. The reaction of this antibody (C135) towards STnI has been determined using 1. the ELISA technique 2. a triple sandwich immunodot technique 3. by immunodot on nitrocellulose after gel electrophoresis of skeletal myofibrils. C135 also interacts with cardiac TnI (CTnI) suggesting that both STnI and CTnI share a common antigenic determinant. The effect of C135 on the Ca 2+ dependence of the myofibrillar ATPase activity was also investigated. At low Ca 2+ concentrations, where the ATPase was inhibited, the antibody had no effect. However, in the presence of Ca 2+ , where the myofibrils were fully activated, the antibody greatly inhibited the ATPase activity. These results suggest that the binding of the antibody to STnI in the myofibrils ‘locks’ the conformation of this troponin submit in an inhibitory state, similar to the one in which STnI is still bound to actin in the absence of Ca 2+ . Work is in progress to determine the binding site of C135 on fragments of STnI generated by CNBr cleavage. These two areas will be discussed in terms of our current overall view of the mechanism of Ca 2+ regulation of muscle contraction.