The proposed research will extend the previous grant of identical title (GM23990) and will incorporate personnel and techniques developed in a previous grant entitled """"""""Membrane Binding of Ca2 ion and K ion Ionophores"""""""" (HL 16117). The proposed research will test the ability of all major fractions of skeletal and cardiac sarcoplasmic reticulum (SR) to produce and maintain a Ca2 ion gradient under a number of physiologically-relevant conditions, including pH, phosphate potential, external Ca2 ion and monovalent cation concentration. The basic approach consists of studying the kinetics of the Ca2 ion-ATPase pump (initial rate, t 1/2 and maximal free internal Ca2 ion concentration at steady-state), in isolation from other SR functions (cation and anion permeability and gated Ca2 ion release). The passive cation and anion permeability mechanisms will then be studied and integrated into a total mechanism of SR function. The basic technique consists of the use of fluorescent indicators of the free Ca2 ion concentration in the SR lumen together in stopped-flow rapid mixing experiments. The technique is capable of continuous monitoring and of revolving transport events with half-times which of 20 msec or longer, will be augmented by on-line date acquisition and computer modelling. The publications of the applicant provide strong evidence that the pump operates by a Ca2 ion/2K ion exchange mechanism proposed by Kanazawa et al. in l971. The proposal will extend this work by determining the rate and equilibrium constants of 13 identified steps in the cyclic mechanism. This will provide further characterization of the pump's ionophore (Ca2 ion binding site), of conformational changes involved in its translocation, and of the K ion/Ca2 ion competition reactions on the inside surface. Conformationally-correlated fluorescence changes in covalently-linked probes will also be studied and the possibility of allosteric interaction between pump molecules will be evaluated. The resulting information will be used in the analysis of the effects of lipid composition, drugs (chlorpromazine and dantrolene) on SR behavior and in the comparison of skeletal and cardiac SR.
