Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes

Kawai, Takafumi; Mizutani, Natsuki; Okamura, Yasushi

doi:https://doi.org/10.1016/j.bbamem.2024.184396

Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes

Kawai, Takafumi; Mizutani, Natsuki; Okamura, Yasushi

Biochimica et Biophysica Acta (BBA) - Biomembranes, 2024, 1867(1), 184396

アクセス数:499件（2025-10-24 11:46 集計）

固定URL: https://hdl.handle.net/11094/98373

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タイトル	Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes
著者	NRID 1000070614915 1000070614915 researchmap permalink t_kawa t_kawa Scopus Author ID 55465095900 55465095900 著者名 Kawai, Takafumi
	ORCID 0009-0005-9575-9961 0009-0005-9575-9961 researchmap permalink zdl134skhf zdl134skhf 著者名 Mizutani, Natsuki
	NRID 1000080201987 1000080201987 researchmap permalink yasushiokamura yasushiokamura Scopus Author ID 55322083200 55322083200 ORCID 0000-0001-5386-7968 0000-0001-5386-7968 著者名 Okamura, Yasushi
キーワード等	PLCζ
	Ion channel
	Membrane potential
	Calcium
	Xenopus oocyte
内容	Kawai Takafumi, Mizutani Natsuki, Okamura Yasushi. Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1867, 184396 (2024); https://doi.org/10.1016/j.bbamem.2024.184396.
抄録	Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP₂ levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP₂ levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP₂. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca²⁺ influx driven by the increased driving force of Ca²⁺. High Ca²⁺ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca²⁺-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP₂-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca²⁺-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP₂ levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP₂ levels, (2) PIP₂ levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP₂ levels were reduced by Ca²⁺ administration. Therefore, PLCζ effectively supports understanding how PIP₂ regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP₂ and the PLC pathway in Xenopus oocytes.
出版者	Elsevier BV
収録物名	Biochimica et Biophysica Acta (BBA) - Biomembranes
巻(号)	1867(1)
ページ	184396
刊行年月	2024-11-05
言語	英語
ハンドルURL	https://hdl.handle.net/11094/98373
PISSN	00052736
関連情報 (isIdenticalTo)	DOI (出版社版) https://doi.org/10.1016/j.bbamem.2024.184396
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
DCTERMS.bibliographicCitation	Biochimica et Biophysica Acta (BBA) - Biomembranes. 2024, 1867(1), p. 184396
DC.title	Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes
DC.creator	Kawai, Takafumi
	Mizutani, Natsuki
	Okamura, Yasushi
DC.publisher	Elsevier BV
DC.language' scheme='DCTERMS.RFC1766	英語
DCTERMS.issued' scheme='DCTERMS.W3CDTF	2024-11-05
DC.identifier	https://doi.org/10.1016/j.bbamem.2024.184396
DC.subject	PLCζ
	Ion channel
	Membrane potential
	Calcium
	Xenopus oocyte
DC.description	Kawai Takafumi, Mizutani Natsuki, Okamura Yasushi. Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1867, 184396 (2024); https://doi.org/10.1016/j.bbamem.2024.184396.
DCTERMS.abstract	Phosphatidylinositol 4,5-bisphosphate (PIP₂) is a key membrane lipid regulating various ion channel activities. Currently, several molecular tools are used to modulate PIP₂ levels, each of which has distinct advantages and drawbacks. In this study, we proposed a novel methodology using heterologous Xenopus oocytes to precisely manipulate PIP₂ levels using phospholipase C (PLC)-ζ, which hydrolyzes PIP₂. Xenopus oocytes injected with PLCζ exhibited notable hyperpolarization-induced Ca²⁺ influx driven by the increased driving force of Ca²⁺. High Ca²⁺ sensitivity of PLCζ facilitated hyperpolarization-induced PLC activity in Xenopus oocytes that was voltage- and Ca²⁺-dependent. This study demonstrated the regulatory capacity of PLCζ in modulating PIP₂-sensitive ion channels, such as the KCNQ2/3 and GIRK channels, in a voltage- and Ca²⁺-dependent manner. Moreover, activation pathway of PLCζ only requires a two-electrode voltage clamp setup, making it a convenient molecular tool to manipulate PIP₂ levels in combination with a voltage-sensing phosphatase (VSP). PLCζ has distinct characteristics and advantages compared to VSP: (1) Hyperpolarization, but not depolarization, reduced the PIP₂ levels, (2) PIP₂ levels were decreased without any increase in phosphatidylinositol 4-monophosphate (PIP) levels, and (3) PIP₂ levels were reduced by Ca²⁺ administration. Therefore, PLCζ effectively supports understanding how PIP₂ regulates ion channels, alongside VSP. Overall, this study highlights the unique characteristics of PLCζ and its distinct advantages in analyzing ion channel regulation by PIP₂ and the PLC pathway in Xenopus oocytes.
DC.rights	This article is licensed under a Creative Commons Attribution 4.0 International License.
DC.format	98373
citation_title	Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes
citation_author	Kawai, Takafumi
	Mizutani, Natsuki
	Okamura, Yasushi
citation_publisher	Elsevier BV
citation_language	英語
citation_date	2024-11-05
citation_journal_title	Biochimica et Biophysica Acta (BBA) - Biomembranes
citation_volume	1867
citation_issue	1
citation_firstpage	184396
citation_doi	https://doi.org/10.1016/j.bbamem.2024.184396
citation_public_url	https://hdl.handle.net/11094/98373

Voltage- and Ca²⁺-inducible PLC activity for analyzing PI(4,5)P₂ sensitivity of ion channels in Xenopus oocytes

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