Inward rectifying potassium (Kir) channels are key regulators of diverse physiological processes and may represent novel drug targets for diseases. In most cases, however, the lack of selective pharmacological probes has hindered progress toward defining their specific cellular functions as well as their drugability and therapeutic potential. Here the investigators propose to develop robust primary and secondary assays to support probe development efforts directed toward the astroglial inward rectifier channel Kir4.1. This channel is expressed predominately in glial cells of the nervous system, inner ear and kidney tubule and may be a drug target for glial-cell cancers, disorders of myelination and hypertension. In Aim 1, the investigators will develop a fluorescence-based thallium (Tl+) flux assay of Kir4.1 channel function for high-throughput screening (HTS) in either 384- or 1536- well plates. The assay will be validated for HTS by performing a screen of approximately 3,000 small molecules for modulators of Kir4.1 activity. In Aim 2, the investigators will develop additional high- throughput Tl+ flux and moderate-throughput electrophysiological assays to support their subsequent probe development campaign. Lay summary: The long-term objective of this work is to develop novel chemical tools which to probe the structure, function and therapeutic potential of a potassium channel expressed in the nervous system and kidney. These studies may lay the foundation for the development of novel drugs to treat cancer, movement disorders and high blood pressure. PUBLIC HEALTH RELEVANCE: Inward rectifying potassium (Kir) channels play key physiological roles in diverse cell types and may represent novel therapeutic targets. However, the lack of selective small-molecule probes has hindered efforts to understand the integrative physiology and pharmacology of most Kir channels. Here we propose to develop robust fluorescence and electrophysiological assays to support high-throughput screening (HTS) and probe development efforts directed toward the astroglial potassium channel Kir4.1.