Jack A. Rall

Research Description:

Our research goals are to understand the basic mechanisms that control muscle relaxation and to engineer proteins that modulate these mechanisms. The speed of relaxation is determined by the rate of Ca2+ removal from the contractile elements and rate of cross-bridge detachment. Ca2+ removal is controlled by the rate of Ca2+: a) dissociation from the regulatory protein troponin C (TnC), b) uptake by a membrane bound Ca2+ pump and c) binding to the soluble protein parvalbumin. The aim is to understand the quantitative contribution of these factors to the rate of relaxation in different muscle types.

Our hypothesis is that the Ca2+ dissociation rate from TnC is an important determinant of muscle relaxation rate. To test this hypothesis, we: a) generated mutants of TnC that exhibit varying Ca2+ affinities and Ca2+ dissociation rates, b) developed a simplified in vitro system to screen mutant properties and c) tested the effects of TnC mutants on relaxation rate in skinned skeletal muscle fibers. We found that the Ca2+ dissociation rate from TnC complexed with troponin I (TnI), its binding partner in muscle, is similar to the Ca2+ dissociation rate from TnC in myofibril suspensions and is similar to muscle relaxation rate. Furthermore, the muscle relaxation rate can be slowed by a mutant of TnC that slows Ca2+ dissociation from the TnC-TnI complex but cannot be increased by a TnC mutant with faster Ca2+ dissociation rate. These results suggest that the Ca2+ dissociation rate from TnC and the rate of cross-bridge detachment are comparable and contribute equally to the rate of muscle relaxation.

We are now studying the molecular mechanisms of these effects and comparing the regulatory mechanisms in skeletal muscle with those in cardiac muscle.

We employ the tools of molecular biology, biochemistry and physiology. Techniques include protein mutagenesis, stopped flow technology to determine the kinetics of Ca2+ exchange with Ca2+-binding proteins in solution and in myofibrils, pulsed laser light activation of Ca2+- chelating compounds that lead to rapid changes in intracellular Ca2+ that either activate or inactivate muscle fibers and measurements of muscle mechanics.