REGULATION OF THE SODIUM/MYO-INOSITOL COTRANSPORTER

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Myo-inositol is an abundant sugar present in all cells. It has two very important functions: 1) It is a precursor of phosphatidylinositol, an important component of the plasma membrane of all cells, and 2) it is a compatible osmolyte, i.e.-in contrast to sodium and potassium, its intracellular concentration can increase 10 fold without disturbing cell function. Although the concentration of myo-inositol in plasma is 0.05mM, its concentration in most cells is lO-2OmM. It enters cells against such a steep concentration gradient on a sodium/myo-inositol cotransporter that is driven by the electrochemical gradient for sodium. Although intracellular deficiency of myoinositol has been implicated in the neuropathy and nephropathy of diabetes mellitus, virtually nothing is known about the cotransporter or its regulation. This project is designed to gain an understanding of the function and regulation of the sodium/myo-inositol cotransporter by cloning and sequencing its cDNA from cultured kidney cells, that like the renal medulla, raise their myo-inositol levels to >80mM in response to hypertonicity. At that time, there is increased cotransporter activity, some evidence for increased mRNA for the cotransporter. The cDNA will be sequenced to reveal the primary structure of the cotransporter and facilitate the production of antibodies against it. The antibodies will be used to quantify the number of cotransporters in renal cells in order to determine whether the cells accumulate more myo-inositol because there are more cotransporters, and if there are more, whether that is due to increased synthesis and/or decreased degradation of the cotransporter. The cDNA will be used to construct an RNA probe to measure cell mRNA levels for the cotransporter to determine whether there is in fact an increase in cotransporter mRNA and whether synthesis and/or degradation of the mRNA is regulated. If mRNA synthesis is increased, the gene for the cotransporter will be cloned so that the mechanism of regulation hypertonicity and by other factors can be studied. The cDNA will be used to develop probes to clone the cDNA for sodium/ myo-inositol cotransporters located in the apical plasma membrane of kidney and small intestines where it functions to transport myo-inositol from lumen to blood. The sequence data should yield information about the signal that results in the cotransporter being located in the apical plasma membrane of some epithelia and in the basolateral plasma membrane of other epithelia.