Conformational changes in the human Cx43/GJA1 gap junction channel visualized using cryo-EM

Hyuk Joon Lee; Hyung Jin Cha; Hyeongseop Jeong; Seu Na Lee; Chang Won Lee; Minsoo Kim; Jejoong Yoo; Jae Sung Woo

doi:10.1038/s41467-023-36593-y

Conformational changes in the human Cx43/GJA1 gap junction channel visualized using cryo-EM

Hyuk Joon Lee, Hyung Jin Cha, Hyeongseop Jeong, Seu Na Lee, Chang Won Lee, Minsoo Kim, Jejoong Yoo, Jae Sung Woo

Department of Life Sciences

Research output: Contribution to journal › Article › peer-review

5 Citations (Scopus)

Abstract

Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43—gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)—that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.

Original language	English
Article number	931
Journal	Nature communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-36593-y
Publication status	Published - 2023 Dec

Bibliographical note

Funding Information:
The authors thank Bumhan Ryu at IBS and Jin-Seok Choi at KARA for their assistance in data collection. We also thank Amer Alam at the University of Minnesota for critical reading of the manuscript, and Jeesoo Kim and Jong-Seo Kim at the Seoul National University for mass spectrometry experiments and data analyses. This work was supported by the Suh Kyungbae Foundation (SUHF-18010097 to J.-S.W.), the National Research Foundation (NRF) grants funded by the Ministry of Science and ICT (NRF-2018R1C1B6004447 to J.-S.W. and NRF-2020R1A2C1101424 to J.Y.), and by the National Supercomputing Center (KSC-2020-CRE-0080 to J.Y.).

Publisher Copyright:
© 2023, The Author(s).

ASJC Scopus subject areas

Physics and Astronomy(all)
Chemistry(all)
Biochemistry, Genetics and Molecular Biology(all)

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title = "Conformational changes in the human Cx43/GJA1 gap junction channel visualized using cryo-EM",

abstract = "Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43—gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)—that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.",

author = "Lee, {Hyuk Joon} and Cha, {Hyung Jin} and Hyeongseop Jeong and Lee, {Seu Na} and Lee, {Chang Won} and Minsoo Kim and Jejoong Yoo and Woo, {Jae Sung}",

note = "Funding Information: The authors thank Bumhan Ryu at IBS and Jin-Seok Choi at KARA for their assistance in data collection. We also thank Amer Alam at the University of Minnesota for critical reading of the manuscript, and Jeesoo Kim and Jong-Seo Kim at the Seoul National University for mass spectrometry experiments and data analyses. This work was supported by the Suh Kyungbae Foundation (SUHF-18010097 to J.-S.W.), the National Research Foundation (NRF) grants funded by the Ministry of Science and ICT (NRF-2018R1C1B6004447 to J.-S.W. and NRF-2020R1A2C1101424 to J.Y.), and by the National Supercomputing Center (KSC-2020-CRE-0080 to J.Y.). Publisher Copyright: {\textcopyright} 2023, The Author(s).",

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AU - Lee, Seu Na

AU - Lee, Chang Won

AU - Kim, Minsoo

AU - Yoo, Jejoong

AU - Woo, Jae Sung

N1 - Funding Information: The authors thank Bumhan Ryu at IBS and Jin-Seok Choi at KARA for their assistance in data collection. We also thank Amer Alam at the University of Minnesota for critical reading of the manuscript, and Jeesoo Kim and Jong-Seo Kim at the Seoul National University for mass spectrometry experiments and data analyses. This work was supported by the Suh Kyungbae Foundation (SUHF-18010097 to J.-S.W.), the National Research Foundation (NRF) grants funded by the Ministry of Science and ICT (NRF-2018R1C1B6004447 to J.-S.W. and NRF-2020R1A2C1101424 to J.Y.), and by the National Supercomputing Center (KSC-2020-CRE-0080 to J.Y.). Publisher Copyright: © 2023, The Author(s).

PY - 2023/12

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N2 - Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43—gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)—that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.

AB - Connexin family proteins assemble into hexameric hemichannels in the cell membrane. The hemichannels dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). We report the cryo-electron microscopy structures of Cx43 GJICh, revealing the dynamic equilibrium state of various channel conformations in detergents and lipid nanodiscs. We identify three different N-terminal helix conformations of Cx43—gate-covering (GCN), pore-lining (PLN), and flexible intermediate (FIN)—that are randomly distributed in purified GJICh particles. The conformational equilibrium shifts to GCN by cholesteryl hemisuccinates and to PLN by C-terminal truncations and at varying pH. While GJIChs that mainly comprise GCN protomers are occluded by lipids, those containing conformationally heterogeneous protomers show markedly different pore sizes. We observe an α-to-π-helix transition in the first transmembrane helix, which creates a side opening to the membrane in the FIN and PLN conformations. This study provides basic structural information to understand the mechanisms of action and regulation of Cx43 GJICh.

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ER -

Conformational changes in the human Cx43/GJA1 gap junction channel visualized using cryo-EM

Abstract

Bibliographical note

ASJC Scopus subject areas

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