The principal goal of this project is to extend our understanding of the mechanisms of enzymes of particular relevance to the nervous system. A study of sodium, potassium pump (Na,K-ATPase) and calcium pump (SERCA ATPase) mechanisms, in collaboration with J. Froehlich (U. Md.), tests a hypothesis that certain of our transient kinetic observations reflect oligomeric interactions of multiple pump molecules during their catalytic cycle. A dependent hypothesis is that such oligomeric interactions contribute to the efficiency of cation transport. Our current evidence suggests that these interactions mediate """"""""out-of-phase"""""""" free energy transfers between two or more catalytic subunits, and that these interactions may be a general property of P-type cation pumps examined in their native state. Recently we have examined the effects of the small intrinsic membrane protein protein, phospholamban(PLB), on the transient kinetics and oligomeric state of the SERCA1 and SERCA2a calcium pumps. PLB with both SERCA1 and SERCA2a, but is normally co-expressed only with SERCA2a, the cardiac muscle isoform. Activation of SERCA2a by beta-1-agonists is known to increase the efficiency of Ca2+ transport and cardiac sarcoplasmic reticulum (CSR) Ca2+ stores. This involves PKA-dependent phosphorylation of PLB. The effect of PLB interaction is to decrease the Vmax and the apparent Ca2+ affinity of these pumps, whereas phosphorylation of phospholamban reverses both of these effects. The mechanism responsible for the increased pump efficiency resulting from activation of SERCA2a by beta-1 agonists has not been determined. However, saturation transfer EPR measurements of spin-labeled SERCA2a indicate that PLB-binding changes the SERCA2a from an oligomer to one that is functionally monomeric. Pre-steady state kinetic investigations of Ca2+-ATPase by Froehlich et al suggest that that the skeletal muscle isoform, SERCA1, is an oligomer in the absence of PLB. To account for the faster turnover and higher Vmax in SERCA1 relative to SERCA2a, oligomeric pump interactions were hypothesized to contribute to a rapid phase of E2P hydrolysis in SERCA1 which is absent in SERCA2a. We have tested whether the absence of PLB regulation of SERCA2a converts it to a functionally oligomeric state resembling SERCA1. SERCA2a was expressed with (+) or without (-) PLB in High Five insect cells using recombinant baculovirus (provided by the Mahaney lab. Quenched-flow mixing was used to measure the kinetics of EP formation at different ATP concentrations; dephosphorylation was measured by chasing the steady state EP with ADP or EGTA. In rapid mixing experiments, SERCA2a + PLB demonstrated kinetic properties resembling those of a monomer like native cardiac SR Ca2+-ATPase. In contrast, SERCA2a sans PLB showed characteristics similar to those seen in skeletal SR Ca2+-ATPase and suggest that SERCA2a sans PLB, like SERCA1, functions as an oligomer. In parallel experiments the Mahaney lab has found that spin-labeled SERCA2a co-expressed with PLB has a smaller rotational correlation time (63 ms) than either SERCA2a sans PLB (78 ms) or SERCA2a + phosphorylated-PLB (97 ms. The rotational correlation times suggest a more compact structure for SERCA2a + PLB (monomer or compact dimer?) than for SERCA2a sans PLB or SERCA2a + phosphorylated-PLB (less compact dimer?). We conclude that either genetic deletion of PLB or its phosphorylation by PKA converts SERCA2a from a monomer (or uncoupled oligomer) to a coupled oligomer, which exhibits improved Ca2+ pumping efficiency. The manuscript describing this work was recently published (Mahaney et al, 2005). We also collaborate with the Neuronal Cytoskeletal Section of this laboratory to examine the kinetics of cdk5 kinase activation and inhibition. In the course of these studies it has become evident that the interaction of this kinase with its different activator proteins and fragments thereof produce complex effects on its biological activity. Among these, a truncated fragment of the activator protein becames a selective inhibitor for the p25 activator. Because the p25 activator produces hyperphosphorylation of tau in neurons (Zheng et al, 2005), we are currently engaged in studies to define the mechanisms of the inhibition and of the selectivity for the p25-cdk5 complex.
