The Physiology Core is dedicated to the needs of the program project for measurement of CFTR function. CFTR-dependent C1~ channel function will be measured in cells before and after treatment with CFTR genetic replacement pharmaceuticals. It will be used by the clinical project, and all of the basic science subprojects which are involved in vector production or use. The capabilities of this Core include (1) measurement of bioelectric properties using short circuit techniques, (2) net C1~ transport using fluorescent dyes in cultured monolayers, single cells, and with imaging techniques, in mixed cell populations, (3) patch clamp, and (4) bilayer reconstitution. Confluent cell cultures will be subjected to short circuit current analysis in Ussing chambers specifically designed for cultured confluent cells. Measurements of net ~C1 flux will be accomplished using the fluorescent dye, 6-methoxy-N-(3- sulfopropyl) quinolinium (SPQ). Fluorescence measurements on cell monolayers or at the single cell level will be employed when cultures are not confluent. Alternatively, fluorescence ratio imaging will be used to measure C1~ where only sparse or mixed cell populations such as might be obtained from patients, are available. The protocols to be employed are those which have been specifically designed to distinguish CFTR- mediated transepithelial C1~ flux or CFTR-mediated C1~ flux from other epithelial ion transport function. These protocols include ion substitution, ion gradients, and use of specific inhibitors or activators of the CFTR versus other ion transport proteins of the secretory epithelia. Radioisotopic procedures will be employed for evaluation of net ionic flux and barrier properties of cell membranes and cell cultures. A technician will carry out the short circuit current measurements and support the core, and a full time postdoctoral associate will be available for the image analysis and fluorescence studies. The Core will be in the environment of a complete electrophysiology and membrane transport facility, thereby producing an enormous saving in costs and efforts. Thus, patch clamp, bilayer, front face dual emission- dual excitation spectrofluorimeter, a filter based dual excitation spectrofluorimeter, microspectrofluorimeter capabilities, and one Ussing chamber set-up will be made available to the CORE as needed. Only partial funding (20%) for the purchase of an image analysis system is requested, the additional costs to be distributed amongst other sources. Since the personnel working in this CORE will be in the environment of a state-of-the-art physiology laboratory, they will interact with support personnel, postdoctorals, students, visiting scientists and senior faculty who provide expertise in the approaches, technologies and analyses which will be needed.
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