title: Computational and functional analyses of a small-molecule binding site in ROMK.
abstract: The renal outer medullary potassium channel (ROMK, or Kir1.1, encoded by KCNJ1) critically regulates renal tubule electrolyte and water transport and hence blood volume and pressure. The discovery of loss-of-function mutations in KCNJ1 underlying renal salt and water wasting and lower blood pressure has sparked interest in developing new classes of antihypertensive diuretics targeting ROMK. The recent development of nanomolar-affinity small-molecule inhibitors of ROMK creates opportunities for exploring the chemical and physical basis of ligand-channel interactions required for selective ROMK inhibition. We previously reported that the bis-nitro-phenyl ROMK inhibitor VU591 exhibits voltage-dependent knock-off at hyperpolarizing potentials, suggesting that the binding site is located within the ion-conduction pore. In this study, comparative molecular modeling and in silico ligand docking were used to interrogate the full-length ROMK pore for energetically favorable VU591 binding sites. Cluster analysis of 2498 low-energy poses resulting from 9900 Monte Carlo docking trajectories on each of 10 conformationally distinct ROMK comparative homology models identified two putative binding sites in the transmembrane pore that were subsequently tested for a role in VU591-dependent inhibition using site-directed mutagenesis and patch-clamp electrophysiology. Introduction of mutations into the lower site had no effect on the sensitivity of the channel to VU591. In contrast, mutations of Val(168) or Asn(171) in the upper site, which are unique to ROMK within the Kir channel family, led to a dramatic reduction in VU591 sensitivity. This study highlights the utility of computational modeling for defining ligand-ROMK interactions and proposes a mechanism for inhibition of ROMK.
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Swale
authors.affiliation: Center for Structural Biology, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Sheehan
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee.
authors.forename: Sreedatta
authors.initials: S
authors.lastname: Banerjee
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee.
authors.forename: Afeef S
authors.initials: AS
authors.lastname: Husni
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee.
authors.forename: Thuy T
authors.initials: TT
authors.lastname: Nguyen
authors.affiliation: Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee; Center for Structural Biology, Vanderbilt University Medical Center, Nashville, Tennessee.
authors.forename: Jens
authors.initials: J
authors.lastname: Meiler
authors.affiliation: Department of Anesthesiology, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee; Institute of Chemical Biology, Vanderbilt University Medical Center, Nashville, Tennessee; Institute for Global Health, Vanderbilt University Medical Center, Nashville, Tennessee. Electronic address: jerod.s.denton@vanderbilt.edu.
authors.forename: Jerod S
authors.initials: JS
authors.lastname: Denton
mesh_terms: D000595:Amino Acid Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000494:pharmacology*; D001665:Binding Sites; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D026902:Potassium Channel Blockers / Q000494:pharmacology*; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: C000604076:6-nitro-2-((6-nitro-1H-benzimidazol-2-yl)methoxymethyl)-1H-benzimidazole; D001562:Benzimidazoles; D026902:Potassium Channel Blockers; D024661:Potassium Channels, Inwardly Rectifying