The proposed study will examine the mechanisms underlying inward rectification in cloned potassium channels, utilizing a combination of molecular biological and electrophysiological techniques. In previous experiments, novel inward rectifying potassium (Kir) channels were cloned and characterized, and polyamines (spermine, spermidine and putrescine) were identified as soluble factors responsible for intrinsic rectification. Based on background and preliminary data, the hypothesis is developed that polyamines cause intrinsic rectification by voltage-dependent block of the Kir channel pore. In order to extend preliminary data and examine the above hypothesis, four experimental series are proposed to address the following questions: (1) What are the polyamine structural requirements for Kir channel blockade? (2) What pore structures are involved in polyamine block of potassium channels? (3) What does the Kir pore look like to permeating ions and water? (4) Can a theoretical basis be developed for understanding ion channel block by polyamines? The results of the proposed experiments, answering the above questions, will provide detailed insight into the fundamental mechanism of inward rectification, a critical determinant of the functional diversity of potassium channels. Inward rectification is essential for regulation of cell excitability and potassium homeostasis in cardiac, brain and other tissues. The work will therefore provide information that may ultimately underlie the development of rational therapies for the treatment of cardiac arrhythmias, epilepsy and other disorders of cell excitability.
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