The inhibition of mitochondrial calcium transport by lanthanides and Ruthenium Red

Abstract

An EGTA (ethanedioxybis(ethylamine)tetra-acetic acid)-quench technique was developed for measuring initial rates of ⁴⁵Ca²⁺ transport by rat liver mitochondria. This method was used in conjunction with studies of Ca²⁺-stimulated respiration to examine the mechanisms of inhibition of Ca²⁺ transport by the lanthanides and Ruthenium Red. Ruthenium Red inhibits Ca²⁺ transport non-competitively with K_i 3×10^-8m; there are 0.08nmol of carrier-specific binding sites/mg of protein. The inhibition by La³⁺ is competitive (K_i=2×10^-8m); the concentration of lanthanide-sensitive sites is less than 0.001nmol/mg of protein. A further difference between their modes of action is that lanthanide inhibition diminishes with time whereas that by Ruthenium Red does not. Binding studies showed that both classes of inhibitor bind to a relatively large number of external sites (probably identical with the ‘low-affinity’ Ca²⁺-binding sites). La³⁺ competes with Ruthenium Red for most of these sites, but a small fraction of the bound Ruthenium Red (less than 2nmol/mg of protein) is not displaced by La³⁺. The results are discussed briefly in relation to possible models for a Ca²⁺ carrier.