Keith D. Garlid

Evidence for the existence of an inner membrane anion channel in mitochondria

Mitochondria normally exhibit very low electrophoretic permeabilities to physiologically important anions such as chloride, bicarbonate, phosphate, succinate, citrate, etc. Nevertheless, considerable evidence has accumulated which suggests that heart and liver mitochondria contain a specific anion-conducting channel. In this review, a postulated inner membrane anion channel is discussed in the context of other known pathways for anion transport in mitochondria. This anion channel exhibits the following properties. It is anion-selective and inhibited physiologically by protons and magnesium ions. It is inhibited reversibly by quinine and irreversibly by dicyclohexylcarbodiimide. We propose that the inner membrane anion channel is formed by inner membrane proteins and that this pathway is normally latent due to regulation by matrix Mg2+. The physiological role of the anion channel is unknown; however, this pathway is well designed to enable mitochondria to restore their normal volume following pathological swelling. In addition, the inner membrane anion channel provides a potential futile cycle for regulated non-shivering thermogenesis and may be important in controlled energy dissipation.

On the nature of ion leaks in energy-transducing membranes

Diffusion is the implicit null hypothesis for ion transport across biological membranes. A proper model of ionic diffusion across the permeability barrier is needed to distinguish among leaks, channels and carriers and to determine whether changes in flux reflect changes in permeability (regulation) or merely changes in the driving force. These issues arise in all biomembranes, but they are particularly confounding in energy-transducing membranes on account of their characteristically high electrical gradients. This paper examines the nature of the barrier to ion leaks, using the classical Eyring rate theory. We introduce new practical procedures for estimating permeability coefficients from ion flux data. We also reach some general conclusions regarding ion leaks across energy-transducing membranes. (1) The dependence of ion flux on the electrical membrane potential is invariably non-linear (non-ohmic). (2) Non-ohmic behavior does not imply variable permeability. (3) Ohmic behavior is exceptional and its occurrence should alert us to the possibility of an underlying carrier or channel. (4) Leak pathways are very likely localized to protein-lipid interfaces and will exhibit quasi-specific properties such as saturation and competition. (5) The inherent non-ohmicity of leaks and the requirement for efficient energy transduction impose constraints upon the magnitude of allowable Gibbs free-energy changes in biological systems. (6) Nature adapts to these constraints by devising mechanisms for step-wise splitting of the partial reactions of energy transduction.

Unmasking the mitochondrial KH exchanger: Swelling-induced K+-loss

Abstract Matrix swelling induces a rapid, transient, energy-independent potassium efflux in rat liver mitochondria. Swelling-induced K+-loss is electroneutral; therefore it does not reflect electrophoretic diffusion secondary to increased membrane permeability. Matrix swelling unmasks an endogenous K H transport mechanism in the mitochondrial membrane, providing a valuable experimental approach to the study of K+ transport in mitochondria.

Mitochondrial Volume Control

Mitochondria lead a precarious existence within the cell, requiring dynamic regulation of matrix volume in order to survive, even under normal physiological conditions (Garlid, 1980). As a mechanism for maintaining the structural and functional integrity of mitochondria, volume control involves nothing less than the preservation of the machinery of oxidative phosphorylation.

Unmasking the mitochondrial KH exchanger: Tetraethylammonium-induced K+-loss

Mitochondria respiring in media containing 80 mM tetraethylammonium ions lose all of their endogenous K+ within 7 minutes. K+-loss is associated with uptake of tetraethylammonium ions. K+ efflux under these conditions is energy-dependent and electroneutral. It is concluded that tetraethylammonium uptake unmasks the endogenous KH exchanger. Considered in relation to the chemiosmotic theory, these results support the existence of a “carrier-brake” mechanism which modulates KH exchange to maintain volume homeostasis in vivo.

New insights into mechanisms of anion uniport through the uncoupling protein of brown adipose tissue mitochondria

GDP-sensitive Cl- uniport is a widely studied property of the uncoupling protein of brown adipose tissue mitochondria; nevertheless, little is known about its mechanism and there is even controversy over whether this protein transports Cl-. Using a fluorescent probe assay, we have demonstrated non-ohmic, electrophoretic, GDP-sensitive Cl- uniport into proteoliposomes reconstituted with purified uncoupler protein. We have also identified a large number of new anionic substrates for this porter that also inhibit Cl- uniport competitively. Anion transport, its inhibition by GDP and anion inhibition of Cl- uniport are all strongly dependent on anion hydrophobicity. These surprising results are consequential for hypotheses of common transport mechanisms in the gene family of mitochondrial anion porters.

Sodium/Proton Antiporters in the Mitochondrial Inner Membrane

The two mitochondrial Na+/H+ antiporters differ in several important respects, and the most physiologically significant of these may be their differences in regulation. The Mg2+-dependent Na+/H+ antiporter controls mitochondrial volume in a dangerous, high-K+ environment. To play this vital role, this porter must always lie poised far from K+/H+ equilibrium; i.e., it must be under dynamic regulation, as proposed in the Mg2+ carrier-brake hypothesis (7). Being regulated, it is not necessary for this antiporter to be cation-selective, since all electroneutral cation movements will be followed by redistributions of anions and water. On the other hand, there is no indication at present that the Mg2+-independent Na+/H+ antiporter is regulated. This transporter is therefore required to exhibit high discrimination against K+ in order to prevent the collapse of matrix volume dueto uncontrolled loss of K+ salts and water (4). Do the properties of the mitochondrial Na+/H+ antiporters help us in any way to understand the plasmalemmal Na+/H+ antiporters? I believe they do, if we allow that there are a limited number of ways in which nature constructs such porters. The difference in cation selectivities very likely reflects a fundamental structural difference between the two mitochondrial antiporters, and this difference appears to be mirrored in two types of plasmalemmal Na+/H+ antiporters. Thus, the Mg2+-independent Na+/H+ antiporter resembles the renal tubular Na+/H+ antiporter in its discrimination against K+ and its competitive inhibition by Li+. On the other hand, the Mg2+-dependent Na+/H+ antiporter resembles a cardiac sarcolemmal Na+/H+ antiporter which transports all alkali cations, including Na+ and K+, and which is inhibited by DCCD and amphiphilic amines (S. Kakar, A. Askari and K. Garlid, in preparation). The existence of the latter class of antiporter in plasmalemma may seem unlikely at first glance, since it would tend to catalyze Na+/K+ exchange and dissipate the effects of the Na+,K+-ATPase. Nevertheless, a sound design principle would be followed if the cell, like mitochondria, were to regulate volume by governing a passive back-flow process rather than an active transport process. In conclusion, it seems premature to conclude that plasma membranes contain only one type of Na+/H+ antiporter. Nor does it seem likely that there is an unlimited variety of such transporters. I propose as a working hypothesis that antiporters from both mitochondria and plasmalemma may be separated into two classes: Na+-selective and non-Na+-selective.(ABSTRACT TRUNCATED AT 400 WORDS)

Lithium Ion is a Competitive Inhibitor of the Na+ Selective Na+/H+ Antiporter from Mitochondria

The existence of an inner membrane Na+/H+ antiporter in mitochondria was predicted by Peter Mitchell (1961) and later demonstrated experimentally by Mitchell and Moyle (1967; 1969). These and subsequent studies by Douglas and Cockrell (1974) and Brierley (1976) led to the conclusions that mitochondria possess one Na+/H+ antiporter and that this porter also catalyzes K+/H+ exchange at a much lower rate. Indeed K+/H+ exchange was so low that some workers (Rosen and Futai, 1980) concluded that mitochondria do not possess such activity.