The proposed project continues the original specific goal of understanding K permeation and gating through the renal, inward rectifying, K channel: ROMK (Kir1.1). However, the project now encompasses a more general theme of understanding the general mechanics of K channel gating. This is particularly timely in view of recent crystallographic studies on bacterial KcsA and Ca-activated BK channels suggesting that K channel gating is associated with specific structural changes. In this model, the K selectivity filter not only selects among cations but also functions as an outer gate for the channel. A second, inner gate, in series with the outer gate, is believed to consist of a hinged section of the inner transmembrane helix. Particular sensors (voltage, ligand, pH) that are coupled to this inner gate would define the functional characteristics of a particular channel. Electrophysiological experiments would be conducted to test this two-gate hypothesis, using ROMK and its mutants and chimeras, expressed in Zenopus oocytes. Oocytes would be studied with: the two electrode voltage clamp (TEVC), the cut-open oocyte technique and patch-clamp recording of single channels. This proposal is a unique opportunity to combine available crystallographic information with a functional electrophysiological study to elucidate what may be a general paradigm for K channel gating, as well as a specific mechanism for regulating renal K secretion.
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