(92) TRANSFECTED AEQUORIN IN THE MEASUREMENT OF CA2+ TRANSPORT IN YEAST MITOCHONDRIA

P.C. Bradshaw, D.W. Jung, M. Litsky and D.R. Pfeiffer
Department of Medical Biochemistry, Ohio State University, Columbus, Ohio 43210

We have transformed a plasmid encoding a mitochondrial-targeted apoaequorin into the yeast Saccharomyces cerevisiae to study Ca2+ transport in isolated mitochondria and the expression of transporters in cells. This was necessary since isolated mitochondria could not be loaded with fluorescent probes using the acetoxymethyl ester loading procedure. The expressed apoaequorin appears to be sequestered in the mitochondrial matrix since in the presence of low levels of external [Ca2+] (0.4 uM) the rapid discharge of aequorin luminescence is dependent on additon of the Ca2+ ionophore ETH129 and respiration. The endogenous matrix [Ca2+] concentration was approximately 125 nM. When isolated nonrespiring mitochondria were suspended in a series of [Ca2+] buffers (0 to 1.5 uM in 0.3 M KCl medium with 10 mM phosphate) matrix [Ca2+] can increase or decrease from 125 nM, and after several minutes approaches the external [Ca2+] value. The rate of change in matrix [Ca2+] vs. external [Ca2+] fits a sigmoid curve with a half-maximal effect at 0.8 uM external [Ca2+]. Energization of mitochondria by respiration (ethanol) or ATP decreases the rate of increase of matrix [Ca2+], possibility due to matrix phosphate accumulation. In the absence of phosphate, respiration or ATP opens the permeability transition pore allowing Ca2+ entry and rapid discharge of aequorin luminescence. The change in matrix [Ca2+] was slowed by KCl, and Mg2+, increased by uncoupler, but no effect was observed with ruthenium red, NaCl, carboxyatractyloside, oligomycin, diltiazem, quinine or nigericin. The results indicate that although yeast mitochondria do not have Ca2+ uniporter activity, a mechanism for flux exists that allows equilibration of [Ca2+] gradients across the inner mitochondrial membrane. Supported by a grant from the American Heart Association.