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.

The mitochondrial inner membrane anion channel (IMAC) carries a wide variety of anions and is postulated to be involved in mitochondrial volume homeostasis in conjunction with the K ؉ /H ؉ antiporter, thus allowing the respiratory chain proton pumps to drive salt efflux. How it is regulated is uncertain; however, it is inhibited by matrix Mg 2؉ and matrix protons. Although, in energized mitochondria, the anion flux through IMAC is expected to be in the outward direction, IMAC is most easily assayed in de-energized mitochondria by monitoring the rate of passive mitochondrial swelling that occurs after the addition of the potassium ionophore valinomycin to mitochondria suspended in potassium salts of the test anion (1). By using this assay, it has been shown that potential physiological regulators of IMAC include matrix Mg 2ϩ and matrix protons (5, 6). A number of nonphysiological inhibitors have also been identified. These include amphiphilic amines such as propranolol, the irreversible inhibitor N,NЈ-dicyclohexylcarbodiimide, and tributyltin, which is probably the most potent inhibitor of IMAC identified to date (see Ref. 1 for review of properties).More recently, we demonstrated that IMAC is extremely sensitive to temperature, in a manner that suggests the open probability is temperature-dependent (7). For example, using malonate as the substrate anion, the flux increased about 1000-fold when the temperature was raised from 5 to 40°C. A large part of this stimulation seems to result from a decrease in the pIC 50 of one of the inhibitory protonation sites. The most significant aspect of these findings is that they suggest the activity of IMAC might be significantly higher at physiological pH values and temperatures than what has been predicted from previous studies that were carried out at 25°C. The actual activity under physiological conditions cannot be predicted, however, because the effects of temperature were determined in Mg 2ϩ -depleted mitochondria, and the effect of temperature on the inhibition by matrix Mg 2ϩ is unknown. On the basis of published values for the IC 50 for Mg 2ϩ determined at 25°C (6), Jung and Brierley (8) have pointed out that activation of IMAC is unlikely at physiological concentrations of Mg 2ϩ and, for similar reasons, O'Rourke (3) has stated that the physiological role of IMAC is unclear. Thus, the goal of the present work was to determine the effect of temperature on the inhibition of IMAC by Mg 2ϩ and thus shed light on its physiological significance. * This work was supported in part by the American Heart Association, Ohio Affiliate, Columbus, Ohio. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.§ To whom correspondence should be addressed. Tel.: 419-383-4125; Fax: 419-383-2871; E-mail: abeavis@mco.edu.1 The abbreviations used are: IMAC, inner membrane anion channel; MOPS, 3-(N-morpholino)propanesu...

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/min⅐mg at 5°C to 1900 nmol/ min⅐mg at 45°C. The Arrhenius plot is nonlinear with the activation energy increasing from 21 kJ/mol (Q 10 ‫؍‬ 1.3) to 193 kJ/mol (Q 10 ‫؍‬ 13) as the temperature is decreased. This temperature dependence is unusual and not seen with solutes that are transported through the bilayer such as NH 4 OAc, 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 pIC 50 for protons decreases from 9.3 (Hill coefficient ‫؍‬ 1.0) at 5°C to 7.1 (Hill coefficient ‫؍‬ 2.5) at 45°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 (IMAC) 1 mediates the transport of a wide variety of different anions and is believed to be involved in mitochondrial volume homeostasis. Its properties have been characterized at 25°C by flux studies in intact mitochondria, and many inhibitors have been identified (see Ref. 1 for a review). Recently, it has been shown that IMAC is blocked by DIDS (2) which is an inhibitor of many anion channels (3). The most physiologically important inhibitors of IMAC are protons and magnesium which inhibit from the matrix (inner) side of the inner membrane. At pH 7.4, the pIC 50 for Mg 2ϩ is about 40 M (4), and the pIC 50 for protons is about 7.8 (5); consequently, in freshly isolated mitochondria, which contain about 0.5 mM free Mg 2ϩ (6, 7), IMAC is almost inactive. Thus, IMAC is usually assayed in Mg 2ϩ -depleted mitochondria or under conditions that alkalinize the matrix (1). It has also been shown that N-ethylmaleimide and mercurials stimulate IMAC by increasing the IC 50 for protons and the IC 50 for Mg 2ϩ (8); moreover, the IC 50 for Mg 2ϩ is also found to increase with pH (4). These findings have led to the suggestion that other physiological factors may modulate the IC 50 values for these inhibitors (8). In the present paper, we show that temperature is one of these important factors.One or more anion channels have been observed in the inner mitochondrial membrane using the electrophysiological techniques of patch-clamping (9 -11) and reconstitution into planar lipid bilayers (12, 13). Whether any of these channels represent IMAC is still uncertain; however, many of the properties of IMAC in intact mitochondria can be explained by a simple channel model in which H ϩ and Mg 2ϩ decrease the open probability of IMAC by binding...