The molecular basis of chloroethylclonidine block of inward rectifier (Kir2.1 and Kir4.1) K channels
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Background Inwardly rectifying potassium (Kir) channels are expressed in many cell types and contribute to a wide range of physiological processes. Kir channels dysfunction cause several diseases in brain, ear, heart, muscle, kidney and pancreas, and developmental abnormalities. Therefore, a better understanding of Kir channels pharmacology is desirable. In this study we characterized the electrophysiological and molecular basis of the inhibition produced by the α-adrenergic agonist/antagonist chloroethylclonidine of the currents generated by wild type and mutant Kir2.1 and Kir4.1 channels heterologously expressed in HEK293 cells. Methods Macroscopic currents were recorded using the patch clamp technique in the inside out configuration. Results We found that chloroethylclonidine inhibits the Kir2.1 and Kir4.1 channels in a voltage-dependent manner by interacting with pore facing residues in the cytoplasmic and transmembrane domains, respectively. Site-directed mutagenesis experiments demonstrate that chloroethylclonidine interact with Kir2.1 channels in the cytoplasmic pore involving the E224, E299, D255 and D259 residues, whereas in Kir4.1channels T128 and E158 residues located in the transmembrane pore are important for the chloroethylclonidine effect. Conclusions Overall, our results suggest that differences in the cavity of Kir channels are determinants in its interactions with chloroethylclonidine. © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Sp. z o.o. All rights reserved.
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Chloroethylclonidine; Kir2.1 channels; Kir4.1 channels; Patch clamp amino acid; chloroethylclonidine; inwardly rectifying potassium channel; inwardly rectifying potassium channel subunit Kir2.1; inwardly rectifying potassium channel subunit kir4.1; spermine; unclassified drug; chloroethylclonidine; clonidine; inwardly rectifying potassium channel; Kcnj10 (channel); KCNJ2 protein, human; membrane protein; potassium; Article; controlled study; drug mechanism; electrophysiology; human; human cell; hyperpolarization; IC50; inside out patch clamp; membrane permeability; patch clamp technique; protein expression; protein interaction; site directed mutagenesis; steady state; wild type; analogs and derivatives; cell line; cell membrane; cytoplasm; drug effects; HEK293 cell line; metabolism; procedures; Cell Line; Cell Membrane; Clonidine; Cytoplasm; HEK293 Cells; Humans; Membrane Proteins; Mutagenesis, Site-Directed; Potassium; Potassium Channels, Inwardly Rectifying
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