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)

Number of Access:1,098（2025-08-29 12:28 Counts）

Identifier to cite or link to this item: https://hdl.handle.net/11094/92516

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Title	Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells
Creator	NRID 1000020573231 1000020573231 ORCID 0000-0003-1004-8537 0000-0003-1004-8537 researchmap permalink n-phys n-phys Scopus Author ID 26633163700 26633163700 CreatorName Kubota, Tomoya
	CreatorName Takahashi, Satoe
	CreatorName Yamamoto, Risa
	CreatorName Sato, Ruka
	CreatorName Miyanooto, Aya
	CreatorName Yamamoto, Reina
	CreatorName Yamauchi, Kosuke
	CreatorName Homma, Kazuaki
	NRID 1000020359847 1000020359847 researchmap permalink mtakahas mtakahas Scopus Author ID 15038719500 15038719500 CreatorName Takahashi, Masanori P.
Subject	gating pore current
	hypokalemic periodic paralysis
	proton
	voltage gated sodium channel
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.
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.
Publisher	Company of Biologists Ltd
Source Title	DMM Disease Models and Mechanisms
Volume (Issue)	16(6)
Date of Issued	2023-06-27
Language	English
Handle URL	https://hdl.handle.net/11094/92516
PISSN	17548403
eISSN	17548411
NCID	AA12344126
Relation.isIdenticalTo	DOI (Publisher Version) https://doi.org/10.1242/dmm.049704
Access Rights	open access
CopyRight	This article is licensed under a Creative Commons Attribution 4.0 International License.
oaire:version	Version of Record
Category	学術雑誌論文 Journal Article

Resource Type	journal article
Local Type	Journal Article
DCMI Type	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	English
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	English
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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