Modulators of CL-dependent Transport Processes (RMI)

Vanderbilt University, Nashville TN

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Abstract

DESCRIPTION (provided by applicant): Coupled movement of Na+, K+, and Cl-through cell membranes occurs primarily through Na-K 2CI and K-CI co-transporters, which belong to the Slc12 family of membrane transporters. Until the identification of the multiple genes that constitute this family, the physiology of cation-chloride co-transporters was dominated by the critical role that some of these co-transporters play in water and ion homeostasis in the kidney. In fact, the co-transporters are best known through their inhibitors: the loop and thiazide diuretics which are widely used in clinical medicine to volume deplete patients. The cloning of the human and mouse genomes identified 9-10 members of the Slc12 family of solute carriers. Among them, 7 are functionally well characterized: there are 1 Na-CI co-transporters, 2 Na-K-2CI co-transporters, and 4 K-CI co-transporters. These transporters participate in a wide variety of function which range from fluid secretion/absorption in multiple epithelia, modulation of synaptic transmission, cell volume control and regulation, cell proliferation. Mutations in cation-chloride co-transporters are responsible for diseases such as salt wasting disorders and peripheral nerve degeneration. They are also possibly involved in hypertension, age-related loss of hearing, neurological and psychiatric disorders. Whereas loop diuretics such as furosemide and bumetanide inhibit most cation-chloride co-transporters, these drugs are rather unspecific and not very potent. Thus, there is a critical need for the development of new compounds targeting cation-chloride co-transporter function. The development of novel fluorescent-based reagents, sensitive to anions and cations, now permits the development of better methods for High Throughput Screening (HTS). We propose to 1) Develop a fluorescence-based assay to monitor the activity of cation-chloride co-transporters. This will be achieved through heterologous expression of NKCC1 and KCC2 together with anion-sensitive fluorescent protein in HEK-293 cells. Based on studies showing that K+ channels can carry thallium, we will also examine the feasibility of use of a thallium-sensitive indicator dyes. 2) Provide a validation the assay for High Throughput Screening. This will be achieved through a detailed analysis of effects of a known inhibitor (e.g. bumetanide) on fluorescent signals generated by the movement of the surrogate anion or cation through the cell membrane and a through a test screening of a library consisting of 10,000 compounds. The development of a High Throughput fluorescent method to measure cation-chloride co-transporter function will allow us to transit to actual HT screens to identify novel compounds targeting the function of cation-chloride co-transporters.