The long term objective of this project is to develop a better understanding of K+ channel function by using peptide toxins to probe channel structure. K+ channels play an important role in many cellular processes. For example, they underlie electrical impulses in the nervous system, they control the pace of the heart, and they couple the blood glucose concentration to insulin secretion. Therefore, efforts to understand K+ channel structure and function relate directly to human health and disease. Due to the difficulty in applying standard protein structure methods to ion channels we are investigating the molecular basis of K+ channel function through a combination of electrophysiological and molecular biological methods. K+ channels with specifically designed mutations will be expressed and studied in Xenopus oocytes using two- electrode voltage clamp and patch recording methods. (1) We will probe the pore entryway of a Shaker voltage dependent K+ channel using a scorpion toxin of known structure to deduce features of the channel structure. This is a very important region on the channel as it is analogous to the active site of an enzyme. (2) We will also study the mechanism and site of action of a second inhibitor peptide toxin (that we have recently purified) to investigate the structure a new region on a different voltage-dependent K+ channel cloned from mammalian brain. (3) Finally, we will purify new K+ channel inhibitors from natural sources. There are many K+ channels for which we have no high-affinity peptide ligands. In particular, we have no peptide ligands for the entire class of inward rectifier channels which includes the G-protein regulated K+ channels (that modulate heart rate) and the ATP regulated K+ channels (that control blood sugar). Identification of active agents against these channels will open new approaches to understand their function.
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