Ca 2 ' Regulation of Mechanical Properties of Striated Muscle Mechanistic Studies Using Extraction and Replacement of Regulatory Proteins Richard

Selective extraction of protein subunits from permeabilized preparations of skeletal muscle and myocardium has become an important tool in investigations of the mechanisms underlying the regulation of tension and shortening velocity in these muscles. In some studies, extraction or exchange has been used as a means of introducing genetically engineered or alternate isoforms of a protein into a muscle cell. Several groups have replaced endogenous troponin C (TnC) in heart or skeletal muscle with molecules that have been altered to modify or disrupt Ca'+ binding at the highor low-affinity sites or to modify interactions among domains thought to be important in regulation of contraction by Ca`+.'-8 These studies are producing exciting and potentially important results concerning molecular mechanisms of action of Ca2+ to activate contraction. Other investigators have used replacement protocols to introduce TnC labeled with fluorophores that report Ca2+ binding to TnC and cross-bridge binding to actin.9-13 These probes have particular promise in studies of cooperative activation of thin filaments by Ca'+ and cross-bridge binding, as well as in developing quantitative models of Ca2+ binding and activation of contraction under physiological conditions. Extraction and exchange techniques have also been used to introduce into fibers spectroscopic probes bound to myosin light chain 2 (LC,) in attempts to detect changes in cross-bridge orientation in transitions from rest to rigor or from rest to active contraction.14,15 Besides its use as a means of introducing proteins into muscle fibers, extraction by itself has been an important probe of molecular mechanisms of contraction and its regulation by Ca'+. Under physiological conditions, Ca2+ is thought to bind to low-affinity site(s) on TnC, a subunit of the thin filament,16 which through a series of events allows interaction of the contractile proteins myosin and actin (reviewed in References 17-19). For this reason, a great deal of attention has been focused on characterization of the structure of TnC, its divalent cation binding characteristics, and its interactions with