Membrane transport of Cl has an important role in cellular regulatory, absorptive and secretory processes. The techniques presently in use for measurement of Cl transport, including patch-clamp, microelectrodes and 36C1 tracer methods, have restricted sensitivities, time resolution and applicability. Based on the utility of fluorescence probes in examining physiological questions (fluoresceins to study pH, fura-2 to study Ca), we have been developing new fluorescence methods to study Cl transport in membrane vesicles, cells and intact epithelia. We have found that the fluorescence of several quinolinium derivatives is Cl-sensitive and specific and responds rapidly to changes in (C1). The probes can be loaded non-invasively into vesicles and cells to provide a continuous record of (C1).
The aims of this grant request are: to optimize the chemical structure of the Cl indicator for use in measurement of cell (C1), to characterize the best Cl indicators to provide a prescription for their use in physiological measurements, and to use Cl indicators to characterize mechanisms of proximal tubule Cl transport. Using well-established methods in synthetic organic chemistry, the structure of the Cl indicator will be modified to maximize sensitivity, to optimize cell loading and trapping, and to produce the most favorable fluorescent properties including a wavelength shift in the emission spectrum with changes in (C1). Probes will be characterized in terms of their Cl sensitivity, specificity, response time, cell loading, toxicity and intracellular calibration. Mechanisms of Cl transport will be examined in isolated brush border and basolateral membrane vesicles from the renal proximal tubule, in isolate proximal tubule cells and in the intact proximal tubule. Cuvette fluorimetry and fluorescence microscopy will be used. The availability of a good fluorescent Cl indicator should have wide applications in transport physiology and facilitate the understanding of Cl transport mechanisms and Cl regulation in both epithelial and non-epithelial membranes.
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