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http://dx.doi.org/10.14348/molcells.2021.0131

Intramolecular Disulfide Bonds for Biogenesis of Calcium Homeostasis Modulator 1 Ion Channel Are Dispensable for Voltage-Dependent Activation  

Kwon, Jae Won (Department of Physiology, Seoul National University College of Medicine)
Jeon, Young Keul (Department of Physiology, Seoul National University College of Medicine)
Kim, Jinsung (Department of Physiology, Seoul National University College of Medicine)
Kim, Sang Jeong (Department of Physiology, Seoul National University College of Medicine)
Kim, Sung Joon (Department of Physiology, Seoul National University College of Medicine)
Publication Information
Molecules and Cells / v.44, no.10, 2021 , pp. 758-769 More about this Journal
Abstract
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 (ICALHM1). Replacing Cys with Ser or Ala abolished the membrane trafficking as well as ICALHM1. 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 ICALHM1 for up to 30 min. Interestingly, incubation with TCEP (2 mM) for 2-6 h reduced both ICALHM1 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.
Keywords
calcium homeostasis modulator 1; disulfide bond; membrane trafficking; oligomerization; reducing agent;
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