| Title |
Identification and Characterization of a Novel Bacterial ATP-Sensitive K+ Channel |
| Author |
Seung Bum Choi1, Jong-Uk Kim2, Hyun Joo3, and Churl K. Min1* |
| Address |
1Department of Biological Sciences, Ajou University, Suwon 443-749, Republic of Korea, 2Department of Molecular Science and Technology, Ajou University, Suwon 443-749, Republic of Korea, 3Department of Molecular Physiology and Biophysics, College of Medicine, Inje University, Busan 614-735, Republic of Korea |
| Bibliography |
Journal of Microbiology, 48(3),325-330, 2010,
|
| DOI |
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| Key Words |
bacterial ATP-sensitive K+ channel, C. violaceum, in silico homology analysis, Xenopus oocyte expression, TEVC |
| Abstract |
Five bacterial species that are most likely to have putative prokaryotic inward rectifier K+ (Kir) channels were selected by in silico sequence homology and membrane topology analyses with respect to the number of transmembrane domains (TMs) and the presence of K+ selectivity filter and/or ATP binding sites in reference to rabbit heart inward rectifier K+ channel (Kir6.2). A dot blot assay with genomic DNAs when probed with whole rabbit Kir6.2 cDNA further supported the in silico analysis by exhibiting a stronger hybridization in species with putative Kir’s compared to one without a Kir. Among them, Chromobacterium violaceum gave rise to a putative Kir channel gene, which was PCR-cloned into the bacterial expression vector pET30b(+), and its expression was induced in Escherichia coli and confirmed by gel purification and immunoblotting. On the other hand, this putative bacterial Kir channel was functionally expressed inXenopus oocytes and its channel activity was measured electrophysiologically by using two electrode voltage
clamping (TEVC). Results revealed a K+ current with characteristics similar to those of the ATP-sensitive K+ (K-ATP) channel. Collectively, cloning and functional characterization of bacterial ion channels could be greatly facilitated by combining the in silico analysis and heterologous expression in Xenopus oocytes. |
