Temperature dependence of the mitochondrial inner membrane anion channel. The relationship between temperature and inhibition by protons.

In this paper, we investigate the temperature and pH dependence of the mitochondrial inner membrane anion channel (IMAC) that is believed to be involved in mitochondrial volume homeostasis. At pH 7. 4, the flux of malonate is highly temperature-dependent with rates increasing from 1 nmol/min mg at 5 degrees C to 1900 nmol/min mg at 45 degrees C. The Arrhenius plot is nonlinear with the activation energy increasing from 21 kJ/mol (Q10 = 1.3) to 193 kJ/mol (Q10 = 13) as the temperature is decreased. This temperature dependence is unusual and not seen with solutes that are transported through the bilayer such as NH4OAc, malonamide, and KSCN (plus valinomycin) or even for cytochrome c oxidase-dependent uptake of potassium (plus valinomycin). The temperature dependence of IMAC is closely related to the inhibition of IMAC by protons. Thus, we find that the pIC50 for protons decreases from 9.3 (Hill coefficient = 1.0) at 5 degrees C to 7.1 (Hill coefficient = 2.5) at 45 degrees C. This behavior is explained on the basis of a new kinetic model for IMAC in which the net open probability is not only modulated by the binding of three protons but also by temperature via effects on the open probability of the unprotonated channel and the pK of one of the inhibitory protonation sites.

The mitochondrial inner membrane anion channel is inhibited by DIDS.

The mitochondrial inner membrane anion channel (IMAC) is a channel, identified by flux studies in intact mitochondria, which has a broad anion selectivity and is maintained closed or inactive by matrix Mg2+ and H+. We now present evidence that this channel, like many other chloride/anion channels, is reversibly blocked/inhibited by stilbene-2,2'-disulfonates. Inhibition of malonate transport approaches 100% with IC50 values of 26, 44, and 88 mu M for DIDS, H2-DIDS, and SITS respectively and Hill coefficients < or = 1. In contrast, inhibition of Cl- transport is incomplete, reaching a maximum of about 30% at pH 7.4 and 65% at pH 8.4 with an IC50 which is severalfold higher than that for malonate. The IC50 for malonate transport is decreased about 50% by pretreatment of the mitochondria with N-ethylmaleimide. Raising the assay pH from 7.4 to 8.4 increases the IC50 by about 50%, but under conditions where only the matrix pH is made alkaline the IC50 is decreased slightly. These properties and competition studies suggest that DIDS inhibits by binding to the same site as Cibacron blue 3GA. In contrast, DIDS does not appear to compete with the fluorescein derivative Erythrosin B for inhibition. These findings not only provide further evidence that IMAC may be more closely related to other "Cl-" channels than previously thought, but also suggest that other Cl- channels may be sensitive to some of the many regulators of IMAC which have been identified.