PROJECT SUMMARY/ABSTRACT Heterotetrameric inward rectifier potassium (Kir) channels composed of Kir4.1 (KCNJ10) and Kir5.1 (KCNJ16) play key roles in regulation of sodium, potassium, and water balance by the distal convoluted tubule (DCT) of the renal tubule. Loss-of- function mutations in KCNJ10 lead to EAST syndrome, which is characterized by neurological dysfunction (e.g. epilepsy, ataxia, and sensorineural deafness) and renal salt wasting. While genetic approaches have been invaluable for understanding the physiology of renal Kir4.1/5.1, the pharmacological tools for probing the therapeutic potential of these channels have been limited to non-specific inhibitors. As part of a NIDDK-funded high-throughput screening program aimed at developing potent and specific Kir4.1/5.1 small- molecule modulators, we have recently discovered the first known potentiators/activators of Kir4.1/5.1. The major focus of my F32 fellowship will be to optimize the potency and selectivity of one of these potentiators, termed VU206, to understand its molecular mechanism of action and test its ability to rescue the function of select EAST mutant channels. In Aim 1, medicinal chemistry will be employed to create iterative analog libraries of VU206 to evaluate the relationship between changes on the functional groups of the chemical scaffold and potency and selectivity toward Kir4.1/5.1. An established quantitative thallium flux assay will be used to screen VU206 and analog libraries against Kir4.1/5.1 and ten different Kir channels. Potentiators with the greatest potency and selectivity will be confirmed using “gold standard” manual patch clamp electrophysiology. Finally, VU206 and/or optimized potentiators will be tested against native rat distal convoluted tubule Kir4.1/5.1 channels as a step towards developing an in vivo-active channel potentiator. In Aim 2, we will determine the molecular mechanism of action of VU206. I will use single channel analysis to test the hypothesis that VU206 potentiates Kir4.1/5.1 activity via increased open-state probability (Po), as opposed to increased unitary conductance. Effects of VU206 on Po would be consistent with opening of the helix-bundle crossing (HBC) gate, a well-established mechanism for regulating the channel by changes in intracellular pH. Next, we will systematically evaluate the sensitivity of EAST gating mutations, R65P, A167V, and R175Q, to changes in intracellular pH and VU206 to determine whether channel dysfunction occurs via a conserved molecular mechanism. Successful completion of these Aims will lead to the development of first-in-class potentiators of Kir4.1/5.1, provide important new insights into how Kir4.1/5.1 channels can be opened pharmacologically, and contribute to a growing “toolkit” for evaluating the integrative physiology and therapeutic potential of Kir4.1/5.1 channels in the regulation of renal tubule function. Importantly, this fellowship experience will prepare me for a successful career as an i...