The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system
Kv2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires s...
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doaj-14d69f69dfd44e53b916bab7eaac212b2020-11-24T23:17:55ZengElsevierBiotechnology Reports2215-017X2015-09-017C172310.1016/j.btre.2015.04.007The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp systemKyoko Kikuchi0Mika Sugiura1Chizuko Nishizawa-Harada2Tadashi Kimura3Laboratory for Drug Discovery and Glycoscience and Glycotechnology Research Group, Biotechnology Research institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, JapanLaboratory for Drug Discovery and Glycoscience and Glycotechnology Research Group, Biotechnology Research institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, JapanLaboratory for Drug Discovery and Glycoscience and Glycotechnology Research Group, Biotechnology Research institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, JapanLaboratory for Drug Discovery and Glycoscience and Glycotechnology Research Group, Biotechnology Research institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, JapanKv2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires skilful operators and expensive equipments. In this research, we created a simple and sensitive drug screen method using bacterial giant spheroplasts and the automated patch clamp which does not require special skills. We expressed a eukaryotic voltage-gated ion channel Kv2.1 in Escherichia coli using prokaryotic codon, and prepared giant spheroplasts large enough for the patch clamp. Human Kv2.1 currents were successfully recorded from giant spheroplasts with the automated system, and Kv2.1-expressed E. coli spheroplasts could steadily reacted to the dose–response assay with TEA and 4-AP. Collectively, our results indicate for the first time that the bacterial giant spheroplast can be applied for practical pharmaceutical assay using the automated patch clamp.http://www.sciencedirect.com/science/article/pii/S2215017X15000260Ion channelHuman Kv2.1Escherichia coliGiant spheroplastAutomated patch clampTEA (tetraethylammonium)4-AP (4-aminopyridine)Drug screening |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Kyoko Kikuchi Mika Sugiura Chizuko Nishizawa-Harada Tadashi Kimura |
spellingShingle |
Kyoko Kikuchi Mika Sugiura Chizuko Nishizawa-Harada Tadashi Kimura The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system Biotechnology Reports Ion channel Human Kv2.1 Escherichia coli Giant spheroplast Automated patch clamp TEA (tetraethylammonium) 4-AP (4-aminopyridine) Drug screening |
author_facet |
Kyoko Kikuchi Mika Sugiura Chizuko Nishizawa-Harada Tadashi Kimura |
author_sort |
Kyoko Kikuchi |
title |
The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system |
title_short |
The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system |
title_full |
The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system |
title_fullStr |
The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system |
title_full_unstemmed |
The application of the Escherichia coli giant spheroplast for drug screening with automated planar patch clamp system |
title_sort |
application of the escherichia coli giant spheroplast for drug screening with automated planar patch clamp system |
publisher |
Elsevier |
series |
Biotechnology Reports |
issn |
2215-017X |
publishDate |
2015-09-01 |
description |
Kv2.1, the voltage-gated ion channel, is ubiquitously expressed in variety of tissues and dysfunction of this ion channel is responsible for multiple diseases. Electrophysiological properties of ion channels are so far characterized with eukaryotic cells using the manual patch clamp which requires skilful operators and expensive equipments. In this research, we created a simple and sensitive drug screen method using bacterial giant spheroplasts and the automated patch clamp which does not require special skills. We expressed a eukaryotic voltage-gated ion channel Kv2.1 in Escherichia coli using prokaryotic codon, and prepared giant spheroplasts large enough for the patch clamp. Human Kv2.1 currents were successfully recorded from giant spheroplasts with the automated system, and Kv2.1-expressed E. coli spheroplasts could steadily reacted to the dose–response assay with TEA and 4-AP. Collectively, our results indicate for the first time that the bacterial giant spheroplast can be applied for practical pharmaceutical assay using the automated patch clamp. |
topic |
Ion channel Human Kv2.1 Escherichia coli Giant spheroplast Automated patch clamp TEA (tetraethylammonium) 4-AP (4-aminopyridine) Drug screening |
url |
http://www.sciencedirect.com/science/article/pii/S2215017X15000260 |
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