STUDIES ON THE MITOCHONDRIAL INNER MEMBRANE ANION UNIPORT

Abstract

This thesis describes four new observations on the mitochondrial pH dependent anion uniporter. Firstly, the effect of osmolality of the supporting medium which shows that anion-conductivity increases with increase in matrix volume. Secondly, partial purification and identification of the thermostable cytoplasmic factor which stimulates mitochondrial anion uniport as inorganic ortho-phosphate and the demonstration that the stimulation is produced by matrix inorganic phosphate. Thirdly, the potent inhibition of the anion uniporter by Zn2+ ions. Fourthly, the finding that this channel is inhibited by several zwitterionic buffers, some of which produce substantial inhibition at concentrations used for buffering. These observations have all been published in refereed international journals. The inhibition by zwitterionic buffers is of practical importance and shows the need to test the effects of these buffers, and avoid those with inhibitory action, when studying transport processes. The inhibition by zinc ions promises to be a useful tool in the investigation of this anion uniporter as it is the most specific inhibitor of the mitochondrial uniporter yet discovered. Zinc should therefore be useful not only as a probe to test the involvement of the anion uniporter in mitochondrial functions but also to help follow the molecular components of the uniporter in attempts to extract and isolate them. The effect of osmolality and matrix volume is of relevance to the problem of the regulation of mitochondrial metabolism with regard to the rate of oxidation and the balance between energy conservation as A TP and thermogenesis by uncoupling. Under some conditions, notably in response to hormones such as vasopressin, adrenalin and glucagon, mitochondrial matrix volume is known to regulate mitochondrial metabolism and the anion uniporter must be involved in volume changes to provide counter ions for K+ movement in and out of the matrix. Phosphate is of particular importance not only as a counter ion but also because of the effect of matrix phosphate on the anion uniporter. Reports from other laboratories have suggested that the cytoplasmic stimulatory factor, which we have shown to be phosphate, increases in response to thyroid hormones. Work in this and other laboratories has shown that Mg2+ ions inhibit the anion uniporter which suggests that the effects of matrix phosphate and matrix volume may be mediated by matrix Mg2+ ion concentration. These observations provide further evidence for the existence of an anion uniporter which connects the mitochondrial matrix to the cytosol. The modulation of the conductivity by matrix phosphate, matrix volume, pH and Ca2+ ions suggest that this anion uniporter functions in the regulation of mitochondrial metabolism by hormones and cytosolic conditions.