Molecules and Cells

Korean Society for Molecular and Cellular Biology (한국분자세포생물학회)
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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)
  • Received : 2021.05.20
  • Accepted : 2021.09.06
  • Published : 2021.10.31
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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

Acknowledgement

This work was supported by grants from the National Research Foundation of Korea (NRF-2018R1A5A2025964), EDISON (EDucation-research Integration through Simulation On the Net) Program (NRF-2016M3C1A6936605) and the Korea Health Technology R&D Project, through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant No. HP20C0199). This work was also supported by a grant from the M.D., Ph.D./Medical Scientist Training Programs through KHIDI to Y.K.J. We thank Prof. Chansik Hong for technical advices and helpful discussions.

References

  1. Al Khamici, H., Hossain, K.R., Cornell, B.A., and Valenzuela, S.M. (2016). Investigating sterol and redox regulation of the ion channel activity of CLIC1 using tethered bilayer membranes. Membranes (Basel) 6, 51. https://doi.org/10.3390/membranes6040051
  2. Bannister, J.P., Young, B.A., Sivaprasadarao, A., and Wray, D. (1999). Conserved extracellular cysteine residues in the inwardly rectifying potassium channel Kir2.3 are required for function but not expression in the membrane. FEBS Lett. 458, 393-399. https://doi.org/10.1016/S0014-5793(99)01096-0
  3. Berman, J.M. and Awayda, M.S. (2013). Redox artifacts in electrophysiological recordings. Am. J. Physiol. Cell Physiol. 304, C604-C613. https://doi.org/10.1152/ajpcell.00318.2012
  4. Calderon-Rivera, A., Andrade, A., Hernandez-Hernandez, O., Gonzalez-Ramirez, R., Sandoval, A., Rivera, M., Gomora, J.C., and Felix, R. (2012). Identification of a disulfide bridge essential for structure and function of the voltage-gated Ca(2+) channel alpha(2)delta-1 auxiliary subunit. Cell Calcium 51, 22-30. https://doi.org/10.1016/j.ceca.2011.10.002
  5. Chen, C., Calhoun, J.D., Zhang, Y., Lopez-Santiago, L., Zhou, N., Davis, T.H., Salzer, J.L., and Isom, L.L. (2012). Identification of the cysteine residue responsible for disulfide linkage of Na+ channel alpha and beta2 subunits. J. Biol. Chem. 287, 39061-39069. https://doi.org/10.1074/jbc.M112.397646
  6. Cho, H.C., Tsushima, R.G., Nguyen, T.T., Guy, H.R., and Backx, P.H. (2000). Two critical cysteine residues implicated in disulfide bond formation and proper folding of Kir2.1. Biochemistry 39, 4649-4657. https://doi.org/10.1021/bi992469g
  7. Choi, W., Clemente, N., Sun, W., Du, J., and Lu, W. (2019). The structures and gating mechanism of human calcium homeostasis modulator 2. Nature 576, 163-167. https://doi.org/10.1038/s41586-019-1781-3
  8. Demura, K., Kusakizako, T., Shihoya, W., Hiraizumi, M., Nomura, K., Shimada, H., Yamashita, K., Nishizawa, T., Taruno, A., and Nureki, O. (2020). Cryo-EM structures of calcium homeostasis modulator channels in diverse oligomeric assemblies. Sci. Adv. 6, eaba8105. https://doi.org/10.1126/sciadv.aba8105
  9. Deutsch, C. (2003). The birth of a channel. Neuron 40, 265-276. https://doi.org/10.1016/S0896-6273(03)00506-3
  10. Dreses-Werringloer, U., Lambert, J.C., Vingtdeux, V., Zhao, H., Vais, H., Siebert, A., Jain, A., Koppel, J., Rovelet-Lecrux, A., Hannequin, D., et al. (2008). A polymorphism in CALHM1 influences Ca2+ homeostasis, Abeta levels, and Alzheimer's disease risk. Cell 133, 1149-1161. https://doi.org/10.1016/j.cell.2008.05.048
  11. Drozdzyk, K., Sawicka, M., Bahamonde-Santos, M.I., Jonas, Z., Deneka, D., Albrecht, C., and Dutzler, R. (2020). Cryo-EM structures and functional properties of CALHM channels of the human placenta. Elife 9, e55853. https://doi.org/10.7554/elife.55853
  12. Duan, J., Li, J., Chen, G.L., Ge, Y., Liu, J., Xie, K., Peng, X., Zhou, W., Zhong, J., Zhang, Y., et al. (2019). Cryo-EM structure of TRPC5 at 2.8-A resolution reveals unique and conserved structural elements essential for channel function. Sci. Adv. 5, eaaw7935. https://doi.org/10.1126/sciadv.aaw7935
  13. Duan, J., Li, J., Zeng, B., Chen, G.L., Peng, X., Zhang, Y., Wang, J., Clapham, D.E., Li, Z., and Zhang, J. (2018). Structure of the mouse TRPC4 ion channel. Nat. Commun. 9, 3102. https://doi.org/10.1038/s41467-018-05247-9
  14. Foskett, J.K. (2020). Structures of CALHM channels revealed. Nat. Struct. Mol. Biol. 27, 227-228. https://doi.org/10.1038/s41594-020-0391-y
  15. Fujiwara, Y., Takeshita, K., Nakagawa, A., and Okamura, Y. (2013). Structural characteristics of the redox-sensing coiled coil in the voltage-gated H+ channel. J. Biol. Chem. 288, 17968-17975. https://doi.org/10.1074/jbc.M113.459024
  16. Gamper, N., Stockand, J.D., and Shapiro, M.S. (2005). The use of Chinese hamster ovary (CHO) cells in the study of ion channels. J. Pharmacol. Toxicol. Methods 51, 177-185. https://doi.org/10.1016/j.vascn.2004.08.008
  17. Gibson, D.G., Young, L., Chuang, R.Y., Venter, J.C., Hutchison, C.A., 3rd, and Smith, H.O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343-345. https://doi.org/10.1038/nmeth.1318
  18. Hong, C., Kwak, M., Myeong, J., Ha, K., Wie, J., Jeon, J.H., and So, I. (2015). Extracellular disulfide bridges stabilize TRPC5 dimerization, trafficking, and activity. Pflugers Arch. 467, 703-712. https://doi.org/10.1007/s00424-014-1540-0
  19. Hwang, E.M., Kim, E., Yarishkin, O., Woo, D.H., Han, K.S., Park, N., Bae, Y., Woo, J., Kim, D., Park, M., et al. (2014). A disulphide-linked heterodimer of TWIK-1 and TREK-1 mediates passive conductance in astrocytes. Nat. Commun. 5, 3227. https://doi.org/10.1038/ncomms4227
  20. Isacoff, E.Y., Jan, L.Y., and Minor, D.L., Jr. (2013). Conduits of life's spark: a perspective on ion channel research since the birth of neuron. Neuron 80, 658-674. https://doi.org/10.1016/j.neuron.2013.10.040
  21. Jeon, Y.K., Choi, S.W., Kwon, J.W., Woo, J., Choi, S.W., Kim, S.J., and Kim, S.J. (2021). Thermosensitivity of the voltage-dependent activation of calcium homeostasis modulator 1 (calhm1) ion channel. Biochem. Biophys. Res. Commun. 534, 590-596. https://doi.org/10.1016/j.bbrc.2020.11.035
  22. Kashio, M., Wei-Qi, G., Ohsaki, Y., Kido, M.A., and Taruno, A. (2019). CALHM1/CALHM3 channel is intrinsically sorted to the basolateral membrane of epithelial cells including taste cells. Sci. Rep. 9, 2681. https://doi.org/10.1038/s41598-019-39593-5
  23. Ma, Z., Siebert, A.P., Cheung, K.H., Lee, R.J., Johnson, B., Cohen, A.S., Vingtdeux, V., Marambaud, P., and Foskett, J.K. (2012). Calcium homeostasis modulator 1 (CALHM1) is the pore-forming subunit of an ion channel that mediates extracellular Ca2+ regulation of neuronal excitability. Proc. Natl. Acad. Sci. U. S. A. 109, E1963-E1971.
  24. Ma, Z., Taruno, A., Ohmoto, M., Jyotaki, M., Lim, J.C., Miyazaki, H., Niisato, N., Marunaka, Y., Lee, R.J., Hoff, H., et al. (2018). CALHM3 is essential for rapid ion channel-mediated purinergic neurotransmission of GPCR-mediated tastes. Neuron 98, 547-561.e10. https://doi.org/10.1016/j.neuron.2018.03.043
  25. Narayan, M. (2012). Disulfide bonds: protein folding and subcellular protein trafficking. FEBS J. 279, 2272-2282. https://doi.org/10.1111/j.1742-4658.2012.08636.x
  26. Okui, M., Murakami, T., Sun, H., Ikeshita, C., Kanamura, N., and Taruno, A. (2021). Posttranslational regulation of CALHM1/3 channel: N-linked glycosylation and S-palmitoylation. FASEB J. 35, e21527.
  27. Ramesh, A., Peleh, V., Martinez-Caballero, S., Wollweber, F., Sommer, F., van der Laan, M., Schroda, M., Alexander, R.T., Campo, M.L., and Herrmann, J.M. (2016). A disulfide bond in the TIM23 complex is crucial for voltage gating and mitochondrial protein import. J. Cell Biol. 214, 417-431. https://doi.org/10.1083/jcb.201602074
  28. Ren, Y., Wen, T., Xi, Z., Li, S., Lu, J., Zhang, X., Yang, X., and Shen, Y. (2020). Cryo-EM structure of the calcium homeostasis modulator 1 channel. Sci. Adv. 6, eaba8161. https://doi.org/10.1126/sciadv.aba8161
  29. Roh, J.W., Hwang, G.E., Kim, W.K., and Nam, J.H. (2021). Ca2+ sensitivity of anoctamin 6/TMEM16F is regulated by the putative Ca2+-binding reservoir at the N-terminal domain. Mol. Cells 44, 88-100. https://doi.org/10.14348/molcells.2021.2203
  30. Schulteis, C.T., John, S.A., Huang, Y., Tang, C.Y., and Papazian, D.M. (1995). Conserved cysteine residues in the shaker K+ channel are not linked by a disulfide bond. Biochemistry 34, 1725-1733. https://doi.org/10.1021/bi00005a029
  31. Schulteis, C.T., Nagaya, N., and Papazian, D.M. (1996). Intersubunit interaction between amino- and carboxyl-terminal cysteine residues in tetrameric shaker K+ channels. Biochemistry 35, 12133-12140. https://doi.org/10.1021/bi961083s
  32. Syrjanen, J.L., Michalski, K., Chou, T.H., Grant, T., Rao, S., Simorowski, N., Tucker, S.J., Grigorieff, N., and Furukawa, H. (2020). Structure and assembly of calcium homeostasis modulator proteins. Nat. Struct. Mol. Biol. 27, 150-159. https://doi.org/10.1038/s41594-019-0369-9
  33. Tanis, J.E., Ma, Z., and Foskett, J.K. (2017). The NH2 terminus regulates voltage-dependent gating of CALHM ion channels. Am. J. Physiol. Cell Physiol. 313, C173-C186. https://doi.org/10.1152/ajpcell.00318.2016
  34. Taruno, A., Vingtdeux, V., Ohmoto, M., Ma, Z., Dvoryanchikov, G., Li, A., Adrien, L., Zhao, H., Leung, S., Abernethy, M., et al. (2013). CALHM1 ion channel mediates purinergic neurotransmission of sweet, bitter and umami tastes. Nature 495, 223-226. https://doi.org/10.1038/nature11906
  35. Thompson, J.D., Higgins, D.G., and Gibson, T.J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  36. Vingtdeux, V., Chang, E.H., Frattini, S.A., Zhao, H., Chandakkar, P., Adrien, L., Strohl, J.J., Gibson, E.L., Ohmoto, M., Matsumoto, I., et al. (2016). CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice. Sci. Rep. 6, 24250. https://doi.org/10.1038/srep24250
  37. Wang, L., Cvetkov, T.L., Chance, M.R., and Moiseenkova-Bell, V.Y. (2012). Identification of in vivo disulfide conformation of TRPA1 ion channel. J. Biol. Chem. 287, 6169-6176. https://doi.org/10.1074/jbc.M111.329748
  38. Waterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., et al. (2018). SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46(W1), W296-W303. https://doi.org/10.1093/nar/gky427
  39. Xu, S.Z., Sukumar, P., Zeng, F., Li, J., Jairaman, A., English, A., Naylor, J., Ciurtin, C., Majeed, Y., Milligan, C.J., et al. (2008). TRPC channel activation by extracellular thioredoxin. Nature 451, 69-72. https://doi.org/10.1038/nature06414
  40. Yang, W., Wang, Y., Guo, J., He, L., Zhou, Y., Zheng, H., Liu, Z., Zhu, P., and Zhang, X.C. (2020). Cryo-electron microscopy structure of CLHM1 ion channel from Caenorhabditis elegans. Protein Sci. 29, 1803-1815. https://doi.org/10.1002/pro.3904
  41. Yereddi, N.R., Cusdin, F.S., Namadurai, S., Packman, L.C., Monie, T.P., Slavny, P., Clare, J.J., Powell, A.J., and Jackson, A.P. (2013). The immunoglobulin domain of the sodium channel beta3 subunit contains a surface-localized disulfide bond that is required for homophilic binding. FASEB J. 27, 568-580. https://doi.org/10.1096/fj.12-209445
  42. Zha, X.M., Wang, R., Collier, D.M., Snyder, P.M., Wemmie, J.A., and Welsh, M.J. (2009). Oxidant regulated inter-subunit disulfide bond formation between ASIC1a subunits. Proc. Natl. Acad. Sci. U. S. A. 106, 3573-3578. https://doi.org/10.1073/pnas.0813402106
  43. Zuniga, L. and Zuniga, R. (2016). Understanding the cap structure in K2P channels. Front. Physiol. 7, 228. https://doi.org/10.3389/fphys.2016.00228