Kv channels play a crucial role in determining the resting membrane potential, shaping action potential repolarization and setting spike frequency. It is becoming increasingly clear that the subcellular localization of these channels is an important component of cellular excitability. However, very little is known;about the localization of specific ion channels within cardiac myocytes. Kv2.1 is one of the delayed rectifier K+ channels expressed in both atria and ventricle, where it plays an important role in the late phase of repolarization of the cardiac action potential and helps set the QT interval. Kv2.1 is unusual among the voltage-gated K+ channels in that its function is modulated by hypoxia/ischemia, redox, mitochondrial Ca2+ and muscarinic agonists. It is intriguing that channel localization is also altered by these stimuli, suggesting a tight relationship between channel function and localization. In the brain, the altered function of Kv2.1 following ischemic insult appears to be neuroprotective. It is therefore likely that Kv2.1 plays a similar role in the heart. The hypothesis of this proposal is that the localization of Kv2.1 to subcellular microdomains places the channel in proximity to the singaling pathways that modulate its function in response to cellular stimuli, permitting dynamic regulation of cardiac excitability on a beat-to-beat basis. Because it is unknown where in a cardiac myocyte Kv2.1 is expressed, the goal of the mentored phase of this proposal is to establish cardiac myocytes from both atria and ventricle as a model system for studying the trafficking of Kv2.1. AdenoviraI-mediated expression of tagged channels will be used to study the localization and dynamics of Kv2.1 in living myocytes, thus building a foundation for the independent phase research. During the second phase, the emphasis will be on investigating the role of Kv2.1 in the cardiac cellular response to ischemia and intracellular Ca2+. We propose that the localization of Kv2.1, and consequently channel function will be altered by these stimuli. Idiopathic arrhythmias and cardiac ischemia are serious threats to human health whose underlying causes are not well understood. Therefore, defects in ion channel localization may be an as-yet unrecognized cause of human cardiac disease, emphasizing the importance of understanding how these proteins are trafficked and localized in the heart.