In this proposal we will study membrane ion transport processes, with particular emphasis on mechanisms involving the chloride anion. We will study not only basic, mechanistic issues related to such ion transport processes, but also issues that relate these ion transport processes to human health and disease. Using the internally dialyzed squid giant axon, which gives direct access to both the inner and outer faces of the axolemma, several important mechanistic issues related to the operation of the Na-K-Cl cotransporter will be addressed. Radioisotopic fluxes and voltage clamping will be used to address these issues. We will test (a) whether high [Cl-]i acts to change the apparent affinity of external and internal binding sites for the cotransported ions; (b) whether the effect of elevated [Cl-]i is due to inhibition of a protein kinase. An examination of possible effects of intracellular K+ on Na-K-Cl cotransporter function will be made. This ion occupies a key spot in the putative order of binding and release. Therefore, a careful examination of its effects will shed considerable light on the actual mechanism of ion binding and release by this cotransporter. Activation by cell shrinkage of this cotransporter will be studied. Voltage-clamping and isotopic fluxes will be combined to examine possible voltage sensitivity of cotransport fluxes. A new direction for our lab is to examine the role of ion transport mechanisms in the development of human fibroblast cell swelling (cytomegaly) in response to infection with the human cytomegalovirus. In particular, we will address the possible role of Na/H exchange and Cl/HCO3 in the development of this pathology. Infection appears to cause the endogenous Na/H exchanger in MRC-5 fibroblasts to become sensitive to cell shrinkage. We will follow up a preliminary finding that suggests a fall of [Cl-]i plays a role in this effect. The effects of the viral infection on the MRC-5 cell's ability to volume regulate will be studied. These studies involve the use of fluorescent probes for ion concentrations and cell volume as well as the use of radioisotopes.
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