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

Figures

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  Fig. 1. CALHM1 protein sequence and structure. (A) Schematic topology of CALHM1 showing four transmembrane domains (TMD), two extracellular loops (EL), short N-terminal helix (NTH), and four C-terminal helices (CH1-4). The four conserved cysteine residues are presented as red dots; C42 and C44 in EL1, C127, and C161 in EL2. (B) Amino acid sequences of human CALHM1-3, mouse calhm1, and killifish calhm1 were aligned using the Clustal Omega algorithm. The four cysteine residues are highlighted within grey boxes. (C) The octameric structure of killifish calhm1 is depicted, and the locations of conserved cysteine residues are highlighted in red color (PDB accession code: 6LMT). (D) A three-dimensional drawing of CALHM1 monomer; colored ribbon-type representation of domains is similar to that in (A). The locations of eight non-conserved cysteine residues are highlighted in orange color. (E) Magnified view of the two disulfide bonds: C42-C127 and C44-C161 (red color).

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  Fig. 2. Electrical activity of CALHM1 and loss of function in Cys mutants. (A) Representative traces of WT CALHM1 current activated by step-like depolarizations ranging from –40 to 80 mV (5 s) every 20 s under standard condition (2 mM [Ca2+]e and 27°C), nominal Ca2+-free, and at 37°C. (B and C) I-V curves and G-V curves of WT obtained under the three conditions (n = 12 and 10-12 for I-V curves and G-V curves, respectively). (D) No current was observed in cells expressing C42S. (E) I-V curves of C42S obtained under the three conditions (n = 6-8). (F) Summary of current densities of wild-type CALHM1 (n = 64) and all the cysteine mutants under standard condition (n = 4-6).

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  Fig. 3. Surface expression of CALHM1 and cysteine mutants. (A) Representative confocal images of CHO cells expressing the wild-type CALHM1 (uppermost row) and four cysteine-to-serine mutants are shown. Four images are shown per row for each cell, with the blue DAPI fluorescent signal shown in the first column, the red WGA signal of plasma membrane in the second column, the green CALHM1-associated signal in the third column, and the merged signal shown in the fourth column. Despite the considerable portion of wild-type CALHM1 proteins expressed in the cytosolic space, distinct membrane expression of CALHM1 was confirmed in yellow-colored regions. (B) Line scanning of fluorescent images was processed using ImageJ software. (C) Surface expression was confirmed using surface biotinylation followed by immunoblotting. The total and surface represented whole cell lysate and surface fraction, respectively.

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  Fig. 4. Multimeric formation of wild-type CALHM1 and cysteine mutants. (A) 10 µg of freshly prepared cell lysate from wild-type CALHM1-transfected CHO cells was solubilized in SDS sample buffer without (lane 1) or with DTT (0.1, 1, and 10 mM, lanes 2-4), followed by separation on SDS-PAGE and immunoblotting using GFP-antibodies. The predicted molecular weight for monomeric, dimeric, and tetrameric forms of CALHM1 are marked with arrows on the right side. (B) Immunoblotting of four cysteine-to-serine mutants was performed. The oligomerization of the ion channel was reduced in two cysteine mutants (C44S and C161S) but not in the other two mutants (C42S and C127S). (C) Relative immunoreactive band intensities of monomer and tetramer in WT without or with DTT. (D) The expressions pattern of monometric- or tetrametric form were altered in C42S and C127S comparing with WT, while not in C44S and C161S according to the concentrations of DTT.

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  Fig. 5. No interaction of cysteine mutants and WT CALHM1. (A) Wild-type CALHM1 cDNA was injected alone or with C42S mutant cDNA. The ratios of WT CALHM1 and C42S cDNA were 10:0, 10:1, 10:2, 10:5, and 10:10 (n = 5-9). The current densities did not differ between the groups (open bars). In addition, a combination of WT CALHM1 and C42S cDNA was transfected into CHO cells with varying amounts (9:1, 1:1, 1:9), which of the total amount was 300 ng (n = 11-15). I CALHM1 decreased based on the amount of wild-type vector (**P < 0.01, ***P < 0.001, grey bars). n.s., not significant. (B) Co-immunoprecipitation blot represents that C42S CALHM1 do not interacts with WT CALHM1. CALHM1 interacts with itself, but could not assemble with C42S CALHM1 through hetero-multimerization. (C) Representative traces of the currents recorded in CHO cells expressing CALHM3 alone and co-expression of CALHM1/CALHM3 or C42S/CALHM3 under standard condition (2 mM [Ca2+]e and 27°C). (D) Summary of the membrane current densities in CHO cells co-expressing CALHM3, WT CALHM1 and C42S cDNA. The current densities of triple co-expression of CALHM1, C42S, and CALHM3 did not differ from that of CALHM1, CALHM3, and GFP (n = 5). In…

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  Fig. 6. Different effects of the acute and sustained treatment with reducing agent on ICALHM1 and protein expression. (A and B) Acute treatment with the reducing agents, DTT (2 mM, upper panel) and TCEP (2 mM, lower panel), did not change I CALHM1 under standard condition (2 mM [Ca2+]e and 27°C). Representative trace (A) and summary of the current density (B) are shown (n = 8 and n = 13 for DTT and TCEP, respectively). n.s., not significant. (C) I CALHM1 gradually decreased following sustained treatment with cell-impermeable reducing agent, TCEP (n = 13 and n = 15 for control and TCEP, respectively). (D and E) CALHM1 membrane expression decreased markedly from 2 h after TCEP treatment (n = 5, closed bars). Total amount of CALHM1 expression levels also decreased when TCEP was treated, but the expression decreased gradually from 2 to 6 h (n = 5, open bars, one-way ANOVA; **P < 0.01, ***P < 0.001).