Identification of a Novel Bacterial K+ Channel

Identification of a Novel Bacterial K+ Channel In an attempt to explore unknown K+ channels in mammalian cells, especially ATP-sensitive K+ (KATP) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian KATP channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K+ channel current that was inwardly rectified and had a reversal potential close to K+ equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific KATP channel openers or blockers did not alter the K+ current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of KATP channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K+ currents were inhibited by protopine (a nonspecific K+ channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K+ channel in mammalian cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Identification of a Novel Bacterial K+ Channel

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Publisher
Springer-Verlag
Copyright
Copyright © 2011 by Springer Science+Business Media, LLC
Subject
Life Sciences; Human Physiology; Biochemistry, general
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s00232-011-9386-2
Publisher site
See Article on Publisher Site

Abstract

In an attempt to explore unknown K+ channels in mammalian cells, especially ATP-sensitive K+ (KATP) channels, we compared the sequence homology of Kir6.1 and Kir6.2, two pore-forming subunits of mammalian KATP channel genes, with bacterial genes that code for selective proteins with confirmed or putative ion transport properties. BLAST analysis revealed that a prokaryotic gene (ydfJ) expressed in Escherichia coli K12 strain shared 8.6% homology with Kir6.1 and 8.3% with Kir6.2 genes. Subsequently, we cloned and sequenced ydfJ gene from E. coli K12 and heterologously expressed it in mammalian HEK-293 cells. The whole-cell patch-clamp technique was used to record ion channel currents generated by ydfJ-encoded protein. Heterologous expression of ydfJ gene in HEK-293 cells yielded a novel K+ channel current that was inwardly rectified and had a reversal potential close to K+ equilibrium potential. The expressed ydfJ channel was blocked reversibly by low concentration of barium in a dose-dependent fashion. Specific KATP channel openers or blockers did not alter the K+ current generated by ydfJ expression alone or ydfJ coexpressed with rvSUR1 or rvSUR2B subunits of KATP channel complex. Furthermore, this coexpressed ydfJ/rvSUR1 channels were not inhibited by ATP dialysis. On the other hand, ydfJ K+ currents were inhibited by protopine (a nonspecific K+ channel blocker) but not by dofetilide (a HERG channel blocker). In summary, heterologously expressed prokaryotic ydfJ gene formed a novel functional K+ channel in mammalian cells.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Jul 9, 2011

References

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