The major goals of this proposal are to evaluate the factors which control the rate and selectivity of cation transport catalyzed by A23187, its derivatives, ionomycin and X-14547A. Equilibrium and kinetic studies of metal ion-ionophore complexation will be conducted in methanol-water solvent mixtures and in biphasic water-phospholipid vesicle systems. Spectroscopic and potentiometric techniques will be employed to determine complex stability constants. Stopped-flow and temperature-jump relaxation methods will be utilized to study complexation kinetics. Transport sequences will be determined employing solvent extraction systems, vesicle bilayer membranes, mitochondria and spermatoza. The rate limiting transport steps will be identified from spectral observation of the carrier molecule under pseudo steady-state transport conditions. The nature and dynamics of ionophore-membrane interactions will be defined by complimentary spectroscopic and physical methods. This work will combine fluorescence lifetime, polarization and quenching techniques with UV absorption, circular dichroism and surface chemicals approaches. Alterations of transport selectivity arising from systematic variation of membrane properties and the chemical basis of charge selective transport will be studied. The physicochemical characterization of novel divalent cation ionophores and A23187 derivatives will be undertaken and a potential Li+ selective ionophore will be synthesized. The results of the proposed studies are required to interpret the biological actions of ionophores, particularly with regard to cellular control by Ca2+. The findings will foster the use of ionophores as pharmacological agents in such areas as cardiac function and the control of hypertension. New carriers with useful and well characterized properties will become available to the scientific community. The transport mechanism and membrane interaction work will contribute to our understanding of the movement of lipophilic compounds across membranes.
