Improved method for heterologous expression of ion channels in<i>Xenopus</i>Oocyte: A PCR shortcut to Oocyte expression

Kim, Han‐Seop; Lee, Chang-Ho; Moon, Eunpyo; Min, Churl K.

doi:10.1080/12265071.1999.9647483

Cited by 1 publication

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References 9 publications

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“…The use of capped RNA is favored for programming certain translation systems (e.g., Xenopus oocytes) because uncapped RNA are rapidly degraded after microinjection into the cells (Quick and Lester, 1994;Kim et al, 1999). To achieve this, DNA templates were enzymatically linearized prior to in vitro transcription, and cRNA was produced in vitro in the presence of Ribo m7G cap analog (Promega).…”

Section: Electrophysiological Measurement Of Putative Bacterial K-atpmentioning

confidence: 99%

Identification and characterization of a novel bacterial ATP-sensitive K+ channel

et al. 2010

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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.

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Section: Electrophysiological Measurement Of Putative Bacterial K-atpmentioning

confidence: 99%

Identification and characterization of a novel bacterial ATP-sensitive K+ channel

et al. 2010

Self Cite

View full text Add to dashboard Cite

show abstract

Improved method for heterologous expression of ion channels inXenopusOocyte: A PCR shortcut to Oocyte expression

Cited by 1 publication

References 9 publications

Identification and characterization of a novel bacterial ATP-sensitive K+ channel

Identification and characterization of a novel bacterial ATP-sensitive K+ channel

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