Cardiac cells contain two cation-transporting adenosine triphosphatases (ATPases) which play essential roles in cardiac function. The Ca2+-ATPase is the predominant protein component of the sarcoplasmic reticulum (SR). It initiates and maintains cardiac relaxation by transferring calcium from the myofilaments into the sarcoplasmic reticulum. The sarcolemmal (Na+ + K+)-ATPase is important in generating the resting membrane potential and conserving ionic gradients. Normal cardiac function requires the localization of the Ca2+-ATPase and the (Na+ + K+)-ATPase to the appropriate membrane system. The specific mechanisms for this """"""""targeting"""""""" of newly synthesized proteins has been an active area of investigation. Our major goal for this proposal is to characterize """"""""targeting"""""""" domains intrinsic to the ion-pump which are responsible for the specificity of targeting. To analyse these """"""""targeting"""""""" domains, we will generate a family of chimeric ATPase genes, formed by interchanging regions from the wild-type Ca 2+-ATPase and (Na+ + K+)-ATPase genes. The mutant genes will be transferred to cultured myogenic cells and the intracellular disposition of the expressed proteins will be determined by a variety of im- munolabelling techniques. The folding patterns of the chimeric proteins and their ability to assemble into heterodimers will also be determined. These mutant proteins may also support partial reactions of the ion-pumping sequence thereby providing further insight into structure-function relationships. Once the Ca2+-ATPase is targeted into cardiac-sarcoplasmic reticulum, its activity is regulated by the protein phospholamban. During phase II of the award we will examine the molecular basis for the stimulation by phospholamban of the cardiac Ca2+-ATPase using recombinant DNA technology.
| Robinson, S W; Cho, P W; Levitsky, H I et al. (1996) Arterial delivery of genetically labelled skeletal myoblasts to the murine heart: long-term survival and phenotypic modification of implanted myoblasts. Cell Transplant 5:77-91 |
