Identification and characterization of a novel bacterial ATP-sensitive K+ channel.
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 in Xenopus 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.
Choi
Seung Bum
SB
Department of Biological Sciences, Ajou University, Suwon, 443-749, Republic of Korea.
Kim
Jong-Uk
JU
Joo
Hyun
H
Min
Churl K
CK
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D002861:Chromobacterium / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D005798:Genes, Bacterial; D066298:In Vitro Techniques; D054086:KATP Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D024661:Potassium Channels, Inwardly Rectifying / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014982:Xenopus laevis
D001426:Bacterial Proteins; D017931:DNA Primers; D054086:KATP Channels; D024661:Potassium Channels, Inwardly Rectifying; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate