The proposed project relies on previous structural knowledge of avian Kir2.2 and bacterial Kir channels to probe the molecular details of ROMK (Kir1.1) channel gating. This continues the original specific goal of understanding potassium (K) permeation and gating through the renal, inward rectifying, K channel ROMK (Kir1.1);which plays an important role in control of systemic K and water balance. Results of this study would not only be relevant for renal diseases like antenatal Bartter's syndrome but might also be important for hypertension if a partial reduction of ROMK function lowers blood pressure without significantly disrupting serum electrolytes. Starting with crystallographic models of the avian Kir2.2 and prokaryotic KirBac closed-states, the proposed experiments would examine conformational changes associated with Kir1.1 channel gating (opening &closing), using direct measurement of state-dependent molecular distances with lanthanide resonance energy transfer (LRET) optical techniques in an in vitro proteoliposome system.
AIM 1 describes steady-state LRET determinations of Kir1.1b dimensions using novel single-Cys dimeric constructs, labeled with a single donor and a single acceptor. In this aim we would also conduct electrophysiological measurements to validate these single-Cys dimers as models for ROMK gating.
AIM 2 proposes state-dependent LRET measurements to evaluate alternative hypotheses for initiation of channel opening by the C-terminal domain;
and AIM 3 examines alternative motions of the primary hydrophobic gate at the ROMK bundle-crossing of inner transmembrane helices. This would help resolve the Kir open-state conformation, which has been controversial. Finally, in AIM 4 we would evaluate the hypothesis that PIP2 binding, required for ROMK opening, sets a pre-open condition by shortening the linker between the C-terminal domain and the interfacial slide helix. The proposed expts rely on a variety of innovations: (1) cell- free, in vitro, eukaryotic protein expression, (2) evaluation of cell-free Kr protein by direct injection into Xenopus oocytes, followed by whole-cell and excised patch recording (3) unambiguous LRET state-dependent molecular distance measurements using single-Cys ROMK dimers.
The proposed project would characterize the underlying molecular basis for ion channel gating (opening &closing) in the renal inward rectifier (ROMK) family of potassium (K) channels. Results of this study would not only be relevant for renal diseases like Bartter's syndrome but would also have profound impli- cations for G-protein regulated inward rectifier channels in the heart and nervous system, as well as K channels in pancreatic beta cells that are implicated in diabetes and hypoglycemia. This could ultimately be used for targeted drug design to correct a variety of congenital ion channelopathies affecting the kidney, heart, and pancreas.
|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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