Functional variation of troponin, a muscle regulatory protein, during evolution of chordate animals

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In vertebrate striated muscles, troponin regulates muscle contraction in a Ca^<++>-dependent manner and acts as an inhibitor of actin-myosin interaction during contraction. In ascidian (Halocynthia roretzi) smooth muscle, however, troponin regulates actin-myosin contraction as an activator in the presence of calcium ions and not an inhibitor in the absence of calcium ions as in vertebrates. In this study, functional variation of troponin in striated and smooth muscles of protochordates was examined. The animals we used mainly are Amphioxus, Branchiostoma belcheri, that has striated muscle abundantly in the entire body and ascidian Ciona intestinalis that has smooth muscle in adult and striated muscle in larva but uses a common TnI, an inhibitory subunit of troponin, in both adult and larval ages. For the functional analysis, we used recombinant troponin subunits, TnT, TnI, and TnC that were generated in an E. coli expression system. The troponin components were used in an actin-myosin ATPase activity assay to determine the role of troponin in the actin-myosin interaction during muscle contraction. Our results are summarized as follows. 1) The troponin complex of TnT, TnI, and TnC in amphioxus striated muscle functions similar to the troponin in vertebrate striated muscle and different from the troponin in smooth muscle of ascidian, H. roretzi. 2) The troponin complex in ascidian (Ciona intestinalis) which uses a common TnI in both adult smooth and larval striated muscles functions similar to the troponin in the smooth muscle of ascidian, H. roretzi and different from vertebrate striated muscle troponin. These results show evolutionary variations in the role of troponin in protochordates.