REGULATION OF INTESTINAL ION CHANNELS

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Traveller's diarrhea is often caused by Escherichia coli which releases a heat-stable toxin (STa) increasing cyclic GMP (cGMP). STa generates cGMP from GTP by activation of a membrane bound guanylate cyclase. This proposal is designed to identify which chloride channels are involved in fluid and salt balance and to investigate their regulation by STa and cGMP. The cultured colonic cell line, T84, a model for transepithelial chloride secretion, exhibits STa-stimulated short circuit current, a measure of transepithelial transport. STa regulation of chloride channels in cultured T84 cells, will be explored in parallel with studies on ileal cells. Using T84 cells, the first aim is to investigate the biophysical properties of STa-induced chloride currents using the whole cell patch clamp technique and to determine using excised patches if STa-activated chloride channels are modulated by cGMP-dependent protein kinase. The second aim is to determine the interrelationship between STa-activated chloride currents and those activated by other chloride secretory agonists like vasoactive intestinal peptide and carbachol. The third aim is to increase our understanding of chloride channels in the ileum by investigating their properties and control mechanisms. Both whole-cell currents and single channel fluctuations will be measured. The whole-cell current mode has the advantage that individual signals can be tested in isolation from other concomitant signals. Single channel recordings determine whether the purported secretory channels are located on the apical membrane. The long term goal of this work is to isolate channels which participate in the control of salt and fluid movements in the intestine and to document their regulatory control in health and in diarrheal states.