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Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation

Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investiga...

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Autores principales: Kwon, Jae Won, Jeon, Young Keul, Kim, Jinsung, Kim, Sang Jeong, Kim, Sung Joon
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Korean Society for Molecular and Cellular Biology 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560582/
https://www.ncbi.nlm.nih.gov/pubmed/34711692
http://dx.doi.org/10.14348/molcells.2021.0131
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author Kwon, Jae Won
Jeon, Young Keul
Kim, Jinsung
Kim, Sang Jeong
Kim, Sung Joon
author_facet Kwon, Jae Won
Jeon, Young Keul
Kim, Jinsung
Kim, Sang Jeong
Kim, Sung Joon
author_sort Kwon, Jae Won
collection PubMed
description Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I (CALHM1)). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I (CALHM1). Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I (CALHM1) for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I (CALHM1) and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane.
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spelling pubmed-85605822021-11-12 Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation Kwon, Jae Won Jeon, Young Keul Kim, Jinsung Kim, Sang Jeong Kim, Sung Joon Mol Cells Research Article Calcium homeostasis modulator 1 (CALHM1) is a membrane protein with four transmembrane helices that form an octameric ion channel with voltage-dependent activation. There are four conserved cysteine (Cys) residues in the extracellular domain that form two intramolecular disulfide bonds. We investigated the roles of C42-C127 and C44-C161 in human CALHM1 channel biogenesis and the ionic current (I (CALHM1)). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as I (CALHM1). Immunoblotting analysis revealed dithiothreitol-sensitive multimeric CALHM1, which was markedly reduced in C44S and C161S, but preserved in C42S and C127S. The mixed expression of C42S and wild-type did not show a dominant-negative effect. While the heteromeric assembly of CALHM1 and CALHM3 formed active ion channels, the co-expression of C42S and CALHM3 did not produce functional channels. Despite the critical structural role of the extracellular cysteine residues, a treatment with the membrane-impermeable reducing agent tris(2-carboxyethyl) phosphine (TCEP, 2 mM) did not affect I (CALHM1) for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both I (CALHM1) and the surface expression of CALHM1 in a time-dependent manner. We propose that the intramolecular disulfide bonds are essential for folding, oligomerization, trafficking and maintenance of CALHM1 in the plasma membrane, but dispensable for the voltage-dependent activation once expressed on the plasma membrane. Korean Society for Molecular and Cellular Biology 2021-10-31 2021-10-29 /pmc/articles/PMC8560582/ /pubmed/34711692 http://dx.doi.org/10.14348/molcells.2021.0131 Text en © The Korean Society for Molecular and Cellular Biology. All rights reserved. https://creativecommons.org/licenses/by-nc-sa/3.0/This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/3.0/ (https://creativecommons.org/licenses/by-nc-sa/3.0/)
spellingShingle Research Article
Kwon, Jae Won
Jeon, Young Keul
Kim, Jinsung
Kim, Sang Jeong
Kim, Sung Joon
Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation
title Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation
title_full Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation
title_fullStr Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation
title_full_unstemmed Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation
title_short Intramolecular Disulfide Bonds for Biogenesis of CALHM1 Ion Channel Are Dispensable for Voltage-Dependent Activation
title_sort intramolecular disulfide bonds for biogenesis of calhm1 ion channel are dispensable for voltage-dependent activation
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8560582/
https://www.ncbi.nlm.nih.gov/pubmed/34711692
http://dx.doi.org/10.14348/molcells.2021.0131
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