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.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Study Section
General Medicine B Study Section (GMB)
Program Officer
Ketchum, Christian J
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Rosalind Franklin University
Schools of Medicine
North Chicago
United States
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Nanazashvili, Mikheil; Sánchez-Rodríguez, Jorge E; Fosque, Ben et al. (2018) LRET Determination of Molecular Distances during pH Gating of the Mammalian Inward Rectifier Kir1.1b. Biophys J 114:88-97
Sackin, Henry; Nanazashvili, Mikheil; Makino, Shin-ichi (2015) Direct injection of cell-free Kir1.1 protein into Xenopus oocytes replicates single-channel currents derived from Kir1.1 mRNA. Channels (Austin) 9:196-9
Frindt, Gustavo; Li, Hui; Sackin, Henry et al. (2013) Inhibition of ROMK channels by low extracellular K+ and oxidative stress. Am J Physiol Renal Physiol 305:F208-15
Yang, Lei; Edvinsson, Johan; Sackin, Henry et al. (2012) Ion selectivity and current saturation in inward-rectifier K+ channels. J Gen Physiol 139:145-57
Sackin, Henry; Nanazashvili, Mikheil; Li, Hui et al. (2012) Residues at the outer mouth of Kir1.1 determine K-dependent gating. Biophys J 102:2742-50
Wang, Hao-Ran; Wu, Meng; Yu, Haibo et al. (2011) Selective inhibition of the K(ir)2 family of inward rectifier potassium channels by a small molecule probe: the discovery, SAR, and pharmacological characterization of ML133. ACS Chem Biol 6:845-56
Sackin, Henry; Nanazashvili, Mikheil; Li, Hui et al. (2011) Modulation of Kir1.1 inactivation by extracellular Ca and Mg. Biophys J 100:1207-15
Sackin, Henry; Nanazashvili, Mikheil; Li, Hui et al. (2010) A conserved arginine near the filter of Kir1.1 controls Rb/K selectivity. Channels (Austin) 4:203-14
Sackin, Henry; Nanazashvili, Mikheil; Li, Hui et al. (2009) An intersubunit salt bridge near the selectivity filter stabilizes the active state of Kir1.1. Biophys J 97:1058-66
Sackin, Henry; Nanazashvili, Mikheil; Li, Hui et al. (2007) External K activation of Kir1.1 depends on the pH gate. Biophys J 93:L14-6

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