The regulation of extramitochondrial free calcium ion concentration by rat liver mitochondria

Abstract

The mechanism by which rat liver mitochondria regulate the extramitochondrial concentration of free Ca2+ was investigated. At 30.degree. C and pH 7.0, mitochondria can maintain a steady-state .**GRAPHIC**. (the negative logarithm of the free extramitochondrial Ca2+ concentration) of 6.1 (0.8 .mu.M). This represents a true steady state, as slight displacements in .**GRAPHIC**. away from 6.1 result in net Ca2+ uptake or efflux in order to restore .**GRAPHIC**. to its original value. In the absence of added permeant weak acid, the steady-state .**GRAPHIC**. is virtually independent of the Ca2+ accumulated in the matrix until 60 nmol of Ca2+/mg of protein is taken up. The steady-state .**GRAPHIC**. is also independent of the membrane potential, as long as the latter parameter is above a critical value. When the membrane potential is below this value, .**GRAPHIC**. is variable and appears governed by thermodynamic equilibration of Ca2+ across a Ca2+ uniport. Permeant weak acids increase and N-ethylmaleimide decreases, the capacity of mitochondria to buffer .**GRAPHIC**. in the region of 6 (1 .mu.M-free Ca2+) while accumulating Ca2+. Permeant acids delay the build up of the transmembrane pH gradient as Ca2+ is accumulated. Consequently, the fall in membrane potential is delayed to values insufficient to maintain a .**GRAPHIC**. of 6. The steady-state .**GRAPHIC**. is affected by temperature, incubation pH and Mg2+. The activity of the Ca2+ uniport, rather than that of the respiratory chain, is rate-limiting when .**GRAPHIC**. is greater than 5.3 (free Ca2+ less than 5 .mu.M). When the Ca2+ electrochemical gradient is in excess, the activity of the uniport decreases by 2-fold for every 0.12 increase in .**GRAPHIC**. (fall in free Ca2+). At .**GRAPHIC**. 6.1, the activity of the Ca2+ uniport is kinetically limited to 5 nmol of Ca2+/min per mg of protein, even when the Ca2+ electrochemical gradient is large. A steady-state cycling of Ca2+ through independent influx and efflux pathways provides a model which is kinetically and thermodynamically consistent with the present observations, and which predicts an extremely precise regulation of .**GRAPHIC**. by liver mitochondria in vivo.