In the cardiovascular system, inwardly-rectifying potassium (Kir) channels are a critical component in the cardiac action potential, regulating both the shape and duration of the electrical events that underlie each heartbeat. A unique feature of this family of proteins is the range of cellular factors that regulate their activity, including inorganic cations, products of cellular metabolism, and changes in cellular acidity, and even properties of the cell membrane. Adding further diversity, the activity of Kir channels is regulated dynamically, from moment to moment, by voltage-dependent block by intracellular cations, and more tonically regulated by ligands that influence channel gating. Recent studies have begun to link genetic mutations that disrupt blockade or gating of specific Kir channels to various cardiac arrhythmias, vascular defects, and more severe disorders including Andersen's syndrome, Bartter's syndrome, and DEND (Diabetes with Epilepsy and Neuromuscular Defects), demonstrating a broad spectrum of physiological roles of this class of channels in many organ systems. The long-term goal of the proposed research is to understand the general and specific mechanisms underlying regulation of Kir channels by (1) their permeating ions, (2) soluble cellular factors that alter channel activity, and (3) interactions with other proteins. To tackle these questions, detailed experiments measuring the electrical activity of Kir channels will be combined with computer modeling, and powerful new biochemical techniques to introduce useful artificial/unnatural amino acids at specific locations in proteins. A detailed understanding of the fundamental mechanisms of Kir channel regulation will provide a basis for ultimately developing interventions to regulate disorders of electrical excitability in the heart, vasculature, and beyond.