title: Lack of negatively charged residues at the external mouth of Kir2.2 channels enable the voltage-dependent block by external Mg2+.
abstract: Kir channels display voltage-dependent block by cytosolic cations such as Mg2+ and polyamines that causes inward rectification. In fact, cations can regulate K channel activity from both the extracellular and intracellular sides. Previous studies have provided insight into the up-regulation of Kir channel activity by extracellular K+ concentration. In contrast, extracellular Mg2+ has been found to reduce the amplitude of the single-channel current at milimolar concentrations. However, little is known about the molecular mechanism of Kir channel blockade by external Mg2+ and the relationship between the Mg2+ blockade and activity potentiation by permeant K+ ions. In this study, we applied an interactive approach between theory and experiment. Electrophysiological recordings on Kir2.2 and its mutants were performed by heterologous expression in Xenopus laevis oocytes. Our results confirmed that extracellular Mg2+ could reduce heterologously expressed WT Kir2.2 currents in a voltage dependent manner. The kinetics of inhibition and recovery of Mg2+ exhibit a 3∼4s time constant. Molecular dynamics simulation results revealed a Mg2+ binding site located at the extracellular mouth of Kir2.2 that showed voltage-dependent Mg2+ binding. The mutants, G119D, Q126E and H128D, increased the number of permeant K+ ions and reduced the voltage-dependent blockade of Kir2.2 by extracellular Mg2+.
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Junwei
authors.initials: J
authors.lastname: Li
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Xie
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Yu
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Suhua
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Zhan
authors.affiliation: Key Laboratory of Neural and Vascular Biology, Ministry of Education, Key Laboratory of Pharmacology and Toxicology for New Drug, Hebei Province, Department of Pharmacology, Hebei Medical University, Shijiazhuang, Hebei Province, China.
authors.forename: Hailin
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Physiology and Biophysics, School of Medicine, Virginia Commonwealth University, Richmond, VA, United States of America.
authors.forename: Diomedes E
authors.initials: DE
authors.lastname: Logothetis
authors.affiliation: Institute of Biophysics, Hebei University of Technology, Tianjin, China.
authors.forename: Hailong
authors.initials: H
authors.lastname: An
mesh_terms: D000818:Animals; D001665:Binding Sites; D002645:Chickens; D018360:Crystallography, X-Ray; D055724:Electrophysiological Phenomena; D005786:Gene Expression Regulation / Q000187:drug effects; D015640:Ion Channel Gating / Q000187:drug effects; D007477:Ions; D008274:Magnesium / Q000737:chemistry*; D008564:Membrane Potentials / Q000187:drug effects; D056004:Molecular Dynamics Simulation; D009154:Mutation; D009865:Oocytes / Q000166:cytology*; D011188:Potassium / Q000737:chemistry; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D007477:Ions; C489461:Kir2.1 channel; C489463:Kir2.2 channel; D024661:Potassium Channels, Inwardly Rectifying; D008274:Magnesium; D011188:Potassium