The long-term objective of this application is to understand the molecular mechanism of transport through the mitochondrial inner membrane anion channel (IMAC) and to determine how it is regulated both physiologically and pharmacologically. It is believed that IMAC together with the K+/H+ antiporter mediate net salt efflux in respiring mitochondria, to compensate for the influx of salts, mediated by K+ uniport pathways together with electroneutral anion:H+ symport pathways. Since respiration drives both fluxes, the net flux is dependent on the relative activities of the four transport pathways involved, and volume homeostasis can be achieved by regulating these pathways. Volume homeostasis is necessary to preserve the integrity of the inner membrane which is essential for oxidative phosphorylation. It has been suggested that mitochondrial swelling may be critical in the genesis of irreversible tissue damage in the ischemic heart and other tissues. It has also been proposed that small changes in mitochondrial volume may be responsible for the regulation of a number of metabolic pathways.
The specific aims of the project are [1] To purify and identify IMAC using both chemical labeling and reconstituted activity to follow IMAC through the purification steps. [2] To examine the properties of IMAC reconstituted in proteoliposomes and planar lipid bilayers. [3) To determine the amino acid composition and N-terminal sequence of IMAC. [4] To raise antibodies to IMAC to examine its distribution in various tissues and species. [5] To determine whether the reconstituted protein is able to cotransport K+ and NH4+ with polyanions including PPi and ATP. Standard procedures will be used to reconstitute the protein and previously identified properties of IMAC will be used to identify and purify the protein. These include labeling with [14 C]dicyclohexylcarbodiimide and [14C ]N-ethylmaleimide. Affinity chromatography using mercurial- and Cibacron Blue gels will be exploited for separation, since IMAC binds both these ligands. alpha-ketoglutarate transport will be assayed in proteoliposomes using a coupled enzyme assay and chloride transport will be assayed using the Cl- sensitive fluorescent dye SPQ. Planar lipid bilayers will be used to assay the transport of a variety of anions and to characterize properties of reconstituted IMAC including substrate, selectivity, voltage sensitivity, open and closed times and regulatory mechanisms.
