Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells

Kubota, Tomoya; Takahashi, Satoe; Yamamoto, Risa; Sato, Ruka; Miyanooto, Aya; Yamamoto, Reina; Yamauchi, Kosuke; Homma, Kazuaki; Takahashi, Masanori P.

doi:https://doi.org/10.1242/dmm.049704

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells

Kubota, Tomoya; Takahashi, Satoe; Yamamoto, Risa et al.

DMM Disease Models and Mechanisms, 2023, 16(6)

アクセス数:1,052件（2025-07-17 21:19 集計）

固定URL: https://hdl.handle.net/11094/92516

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タイトル	Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells
著者	NRID 1000020573231 1000020573231 ORCID 0000-0003-1004-8537 0000-0003-1004-8537 researchmap permalink n-phys n-phys Scopus Author ID 26633163700 26633163700 著者名 Kubota, Tomoya
	著者名 Takahashi, Satoe
	著者名 Yamamoto, Risa
	著者名 Sato, Ruka
	著者名 Miyanooto, Aya
	著者名 Yamamoto, Reina
	著者名 Yamauchi, Kosuke
	著者名 Homma, Kazuaki
	NRID 1000020359847 1000020359847 researchmap permalink mtakahas mtakahas Scopus Author ID 15038719500 15038719500 著者名 Takahashi, Masanori P.
キーワード等	gating pore current
	hypokalemic periodic paralysis
	proton
	voltage gated sodium channel
内容	Kubota T., Takahashi S., Yamamoto R., et al. Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells. DMM Disease Models and Mechanisms 16, A18 (2023); https://doi.org/10.1242/dmm.049704.
抄録	Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium channel (mKir2.1) and HypoPP2-associated Nav1.4 variants. Whole cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane potential to comparable levels to myofibers, and that some Nav1.4 variants induced notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants using a ratiometric pH indicator, SNARF-4F. Our optical method provides a potential in vitro platform for high-throughput drug screen, not only for HypoPP but also for other channelopathies caused by VSD mutations.
出版者	Company of Biologists Ltd
収録物名	DMM Disease Models and Mechanisms
巻(号)	16(6)
刊行年月	2023-06-27
言語	英語
ハンドルURL	https://hdl.handle.net/11094/92516
PISSN	17548403
eISSN	17548411
NCID	AA12344126
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1242/dmm.049704
アクセス権	open access
権利情報	This article is licensed under a Creative Commons Attribution 4.0 International License.
出版タイプ	Version of Record
カテゴリ	学術雑誌論文 Journal Article

資源タイプ	学術雑誌論文
ローカル資源タイプ	学術雑誌論文
DCMI資源タイプ	text
DCTERMS.bibliographicCitation	DMM Disease Models and Mechanisms. 2023, 16(6)
DC.title	Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells
DC.creator	Kubota, Tomoya
	Takahashi, Satoe
	Yamamoto, Risa
	Sato, Ruka
	Miyanooto, Aya
	Yamamoto, Reina
	Yamauchi, Kosuke
	Homma, Kazuaki
	Takahashi, Masanori P.
DC.publisher	Company of Biologists Ltd
DC.language' scheme='DCTERMS.RFC1766	英語
DC.type' scheme='DCTERMS.DCMIType	text
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2023-06-27
DC.identifier	https://doi.org/10.1242/dmm.049704
DC.subject	gating pore current
	hypokalemic periodic paralysis
	proton
	voltage gated sodium channel
DC.description	Kubota T., Takahashi S., Yamamoto R., et al. Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells. DMM Disease Models and Mechanisms 16, A18 (2023); https://doi.org/10.1242/dmm.049704.
DCTERMS.abstract	Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium channel (mKir2.1) and HypoPP2-associated Nav1.4 variants. Whole cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane potential to comparable levels to myofibers, and that some Nav1.4 variants induced notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants using a ratiometric pH indicator, SNARF-4F. Our optical method provides a potential in vitro platform for high-throughput drug screen, not only for HypoPP but also for other channelopathies caused by VSD mutations.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	92516
citation_title	Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells
citation_author	Kubota, Tomoya
	Takahashi, Satoe
	Yamamoto, Risa
	Sato, Ruka
	Miyanooto, Aya
	Yamamoto, Reina
	Yamauchi, Kosuke
	Homma, Kazuaki
	Takahashi, Masanori P.
citation_publisher	Company of Biologists Ltd
citation_language	英語
citation_date	2023-06-27
citation_journal_title	DMM Disease Models and Mechanisms
citation_volume	16
citation_issue	6
citation_doi	https://doi.org/10.1242/dmm.049704
citation_public_url	https://hdl.handle.net/11094/92516

Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells

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