MODULATION OF CA FLUXES IN HEART SARCOPLASMIC RETICULUM

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The goal of this proposal is to characterize heart SR in terms of its role in the physiology and function of the heart. Recently, we have developed a highly purified and stable preparation of sarcoplasmic reticulum (SR) from heart. The availability of such a preparation encourages us to carry out a detailed molecular biology approach on heart SR analogous to that currently in progress in our laboratory with skeletal muscle SR. Specifically, we aim to define the nature of modulation of the Ca++ uptake process in heart by cAMP and Ca++ -calmodulin dependent protein kinases. A second focus is on defining molecular aspects of the Ca++ release mechanism and its modulation. Both approaches will make us of resolution and reconstitution methodology. In this way we plan to: 1) test directly the influence of phospholamban and its phosphorylation and dephosphorylation on calcium pumping in heart SR; 2) test the influence of phospholamban from heart on the calcium pump from skeletal muscle SR; 3) use target inactivation analysis on heart SR to measure the size of calcium pump protein and phospholamban in different states of the pump cycle and after modulation of phospholamban (phosphorylation/dephosphorylation of phospholamban); 4) isolate fractions referable to terminal and lateral cisternae of heart SR and characterize them with regard to Ca++ uptake and release and their modulation; 5) attempt to isolate and characterize the channel(s) involved in Ca++ release, 6) study the effect of Ca++ -calmodulin and cAMP dependent phosphorylation on Ca++ release. A third focus is to define other functions which are carried out by heart SR. The significance of these studies is that they provide basic knowledge of the regulation of heart muscle contraction and relaxation which in part are mediated by heart SR.