Calcium Buffering in Presynaptic Nerve Terminals I. Evidence for Involvement of a Nonmitochondrial A TP-Dependent Sequestration Mechanism M O R D E C A I P. B L A U S T E I N , R O N A L D W. R A T Z L A F F , C. K E N D R I C K , and E R I K S. S C H W E I T Z E R
NANCY
From the Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110. Dr. Kendrick's present address is Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
A B $ T R AC T A latent A T P - d e p e n d e n t Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. T h e data indicate that a fraction of the Ca uptake (measured with ~Ca) is associated with the intraterminal mitochondria; it is blocked by r u t h e n i u m red, by FCCP, and by azide + dinitrophenol + oligomycin. T h e r e is, however, a residual A T P - d e p e n d e n t Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondriai) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. T h e proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the A T P - d e p e n d e n t Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca 2+ concentration (buffered with EGTA) is
NANCY
From the Department of Physiology and Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110. Dr. Kendrick's present address is Department of Biochemistry, University of Wisconsin, Madison, Wisconsin 53706.
A B $ T R AC T A latent A T P - d e p e n d e n t Ca storage system is enriched in preparations of pinched-off presynaptic nerve terminals (synaptosomes), and is exposed when the terminals are disrupted by osmotic shock or saponin treatment. T h e data indicate that a fraction of the Ca uptake (measured with ~Ca) is associated with the intraterminal mitochondria; it is blocked by r u t h e n i u m red, by FCCP, and by azide + dinitrophenol + oligomycin. T h e r e is, however, a residual A T P - d e p e n d e n t Ca uptake that is insensitive to the aforementioned poisons; this (nonmitochondriai) Ca uptake is blocked by tetracaine, mersalyl and A-23187. Moreover, A-23187 rapidly releases previously accumulated Ca from these (nonmitochondrial) storage sites, whereas the Ca chelator, EGTA, does not. T h e proteolytic enzyme, trypsin, spares the mitochondria but inactivates the nonmitochondrial Ca uptake mechanism. Chemical measurements of total Ca indicate that the A T P - d e p e n d e n t Ca uptake at the nonmitochondrial sites involves the net transfer of Ca from medium to tissue fragments. This system can sequester Ca when the ambient-ionized Ca 2+ concentration (buffered with EGTA) is
