The inward rectifier family (Kir) of channels play an important role in K transport in a variety of tissues including the heart, macrophage and kidney. The IRK (Kir2) subfamily of K channels are characterized by strong inward rectification, with little current in the outward direction. In contrast, the ROMK (Kir1) subfamily of K channels exhibit a much weaker inward rectification, with significant outward current. ROMK is predominantly expressed in the kidney where it mediates K secretion into the lumen of the cortical collecting tubule (CCT) and recycles K for the triple cotransporter at the luminal membrane of the thick ascending limb of Henle (TALH). Members of both the Kir1 and Kir2 subfamilies have been cloned and sequenced. Evidence suggests that both these channels consist of 4 subunits, each with 2 transmembrane spanning segments. Recent crystallographic information (Doyle et. al. Science 280: 69-77, 1998) about a bacterial K channel (KcsA) has revealed it to be a tetrameric channel with each of the 4 subunits possessing 2 membrane spanning segments, similar to the topology of inward rectifiers. This discovery provides a unique opportunity to employ electrophysiological methods to understand how specific structural elements of inward rectifiers control K permeation and gating through IRK and ROMK, using the KcsA channel as a model. In the proposed experiments, I will utilize the techniques of patch-clamp recording, 2- electrode voltage clamp, site directed mutagenesis, and chimeric constructs to focus on 4 aspects of permeation through inward rectifier channels: (1) ion binding within the channel, (2) control by pore-lining residues, (3) regulation by external K, and (4) control of permeation by the C-terminus. Results of these experiments, together with the known crystal structure of KcsA, should provide basic information about: (1) how K traverses inward rectifier channels, (2) which regions of the channel control permeation, and (3) how these regions interact with each other. This would be relevant not only to K handling by the kidney, but also to K transport by the entire (Kir) family of ion channels.
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