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Human CNNM2 is not a Mg2+ transporter per se

  • Ion channels, receptors and transporters
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Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

A Protocol for this article was published on 02 February 2017

Abstract

CNNM2 is associated with the regulation of serum Mg concentration, and when mutated, with severe familial hypomagnesemia. The function and cellular localization of CNNM2 and its isomorphs (Iso) remain controversial. The objective of this work was to examine the following: (1) the transcription-responsiveness of CNNM2 to Mg starvation, (2) the cellular localization of Iso1 and Iso2, (3) the ability of Iso1 and Iso2 to transport Mg2+, and (4) the complex-forming ability and spectra of potential interactors of Iso1 and Iso2. The five main findings are as follows. (1) Mg-starvation induces CNNM2 overexpression that is markedly higher in JVM-13 cells (lymphoblasts) compared with Jurkat cells (T-lymphocytes). (2) Iso1 and Iso2 localize throughout various subcellular compartments in transgenic HEK293 cells overexpressing Iso1 or Iso2. (3) Iso1 and Iso2 do not transport Mg2+ in an electrogenic or electroneutral mode in transgenic HEK293 cells overexpressing Iso1 or Iso2. (4) Both Iso1 and Iso2 form complexes of a higher molecular order. (5) The spectrum of potential interactors of Iso1 is ten times smaller than that of Iso2. We conclude that sensitivity of CNNM2 expression to extracellular Mg2+ depletion depends on cell type. Iso1 and Iso2 exhibit a dispersed pattern of cellular distribution; thus, they are not exclusively integral to the cytoplasmic membrane. Iso1 and Iso2 are not Mg2+ transporters per se. Both isomorphs form protein complexes, and divergent spectra of potential interactors of Iso1 and Iso2 indicate that each isomorph has a distinctive function. CNNM2 is therefore the first ever identified Mg2+ homeostatic factor without being a Mg2+ transporter per se.

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References

  1. Arjona FJ, de Baaij JHF, Schlingmann KP, Lameris ALL, van Wijk E, Flik G, Regele S, Korenke GC, Neophytou B, Rust S, Reintjes N, Konrad M, Bindels RJ, Hoenderop JG (2014) CNNM2 Mutations Cause Impaired Brain Development and Seizures in Patients with Hypomagnesemia. PLoS Genet 10(4): doi: 10.1371/journal.pgen.1004267

  2. Barbagallo M, Belvedere M, Dominguez LJ (2008) Magnesium homeostasis and aging. Magnes Res 22:235–46. doi:10.1684/mrh.2009.0187

    Google Scholar 

  3. Billia F, Hauck L, Grothe D, Konecny F, Rao V, Kim RH, Mak TW (2013) Parkinson-susceptibility gene DJ-1/PARK7 protects the murine heart from oxidative damage in vivo. Proc Natl Acad Sci U S A 110:6085–90. doi:10.1073/pnas.1303444110

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Bricker DK, Taylor EB, Schell JC, Orsak T, Boutron A, Chen YC, Cox JE, Cardon CM, Van Vranken JG, Dephoure N, Redin C, Boudina S, Gygi SP, Brivet M, Thummel CS, Rutter J (2012) A mitochondrial pyruvate carrier required for pyruvate uptake in yeast, Drosophila, and humans. Science 337:96–100. doi:10.1126/science.1218099

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, Mueller R, Nolan T, Pfaffl MW, Shipley GL, Vandesompele J, Wittwer CT (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–22. doi:10.1373/clinchem.2008.112797

    Article  CAS  PubMed  Google Scholar 

  6. Cooper AA, Gitler AD, Cashikar A, Haynes CM, Hill KJ, Bhullar B, Liu K, Xu K, Strathearn KE, Liu F, Cao S, Caldwell KA, Caldwell GA, Marsischky G, Kolodner RD, Labaer J, Rochet JC, Bonini NM, Lindquist S (2006) Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313:324–8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. de Baaij JH, Stuiver M, Meij IC, Lainez S, Kopplin K, Venselaar H, Müller D, Bindels RJ, Hoenderop JG (2012) Membrane topology and intracellular processing of cyclin M2 (CNNM2). J Biol Chem 287:13644–55. doi:10.1074/jbc.M112.342204

    Article  PubMed  PubMed Central  Google Scholar 

  8. del Toro D, Alberch J, Lázaro-Diéguez F, Martín-Ibáñez R, Xifró X, Egea G, Canals JM (2009) Mutant Huntingtin impairs post-golgi trafficking to lysosomes by delocalizing optineurin Rab8 complex from the golgi apparatus. Mol Biol Cell 20:1478–92. doi:10.1091/mbc.E08-07-0726

    Article  PubMed  PubMed Central  Google Scholar 

  9. Fleig A, Schweigel-Röntgen M, Kolisek M (2013) Solute Carrier Family SLC41, what do we really know about it? Wiley Interdiscip Rev Membr Transp Signal 2(6). doi: 10.1002/wmts.95

  10. Goytain A, Quamme GA (2005) Functional characterization of ACDP2 (ancient conserved domain protein), a divalent metal transporter. Physiol Genomics 22:82–9

    Article  Google Scholar 

  11. Goytain A, Hines RM, Quamme GA (2008) Huntingtin-interacting proteins, HIP14 and HIP14L, mediate dual functions, palmitoyl acyltransferase and Mg2+ transport. J Biol Chem 283:33365–74. doi:10.1074/jbc.M801469200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hahn FM, Xuan JW, Chambers AF, Poulter CD (1996) Human isopentenyl diphosphate: dimethylallyl diphosphate isomerase: overproduction, purification, and characterization. Arch Biochem Biophys 332:30–4

    Article  CAS  PubMed  Google Scholar 

  13. Heinrich A, Nees F, Lourdusamy A et al (2013) From gene to brain to behavior: schizophrenia-associated variation in AMBRA1 alters impulsivity-related traits. Eur J Neurosci 38:2941–5. doi:10.1111/ejn.12201

    PubMed  Google Scholar 

  14. Hermosura MC, Monteilh-Zoller MK, Scharenberg AM, Penner R, Fleig A (2002) Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNuM. J Physiol 539:445–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Iotti S, Malucelli E (2008) In vivo assessment of Mg2+ in human brain and skeletal muscle by 31P-MRS. Magnes Res 21:157–62

    CAS  PubMed  Google Scholar 

  16. Kolisek M, Zsurka G, Samaj J, Weghuber J, Schweyen RJ, Schweigel M (2003) Mrs2p is an essential component of the major electrophoretic Mg2+ influx system in mitochondria. EMBO J 22:1235–44

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Kolisek M, Launay P, Beck A, Sponder G, Serafini N, Brenkus M, Froschauer EM, Martens H, Fleig A, Schweigel M (2008) SLC41A1 is a novel mammalian Mg2+ carrier. J Biol Chem 283:16235–47. doi:10.1074/jbc.M707276200

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kolisek M, Nestler A, Vormann J, Schweigel-Röntgen M (2012) Human gene SLC41A1 encodes for the Na+/Mg2+ exchanger. Am J Physiol Cell Physiol 302:C318–26. doi:10.1152/ajpcell.00289.2011

    Article  CAS  PubMed  Google Scholar 

  19. Kolisek M, Galaviz-Hernández C, Vázquez-Alaniz F, Sponder G, Javaid S, Kurth K, Nestler A, Rodríguez-Moran M, Verlohren S, Guerrero-Romero F, Aschenbach JR, Vormann J (2013) SLC41A1 is the only magnesium responsive gene significantly overexpressed in placentas of preeclamptic women. Hypertens Pregnancy 32:378–89. doi:10.3109/10641955.2013.810237

    Article  CAS  PubMed  Google Scholar 

  20. Kolisek M, Sponder G, Mastrototaro L, Smorodchenko A, Launay P, Vormann J, Schweigel-Röntgen M (2013) Substitution p.A350V in Na+/Mg2+ exchanger SLC41A1, potentially associated with Parkinson’s disease, is a gain-of-function mutation. PLoS One 8(8). doi: 10.1371/journal.pone.0071096

  21. Kolisek M, Montezano AC, Sponder G, Anagnostopoulou A, Vormann J, Touyz RM, Aschenbach JR (2015) PARK7/DJ-1 dysregulation by oxidative stress leads to magnesium deficiency: implications in degenerative and chronic diseases. Clin Sci (Lond) 129:1143–50. doi:10.1042/CS20150355

    Article  CAS  Google Scholar 

  22. Lee HJ, Lee K, Im H (2012) α-Synuclein modulates neurite outgrowth by interacting with SPTBN1. Biochem Biophys Res Commun 424:497–502. doi:10.1016/j.bbrc.2012.06.143

    Article  CAS  PubMed  Google Scholar 

  23. Meyer TE, Verwoert GC, Hwang SJ, et al (2010) Genetic Factors for Osteoporosis Consortium; Meta Analysis of Glucose and Insulin Related Traits Consortium. Genome-wide association studies of serum magnesium, potassium, and sodium concentrations identify six Loci influencing serum magnesium levels. PLoS Genet 6(8). doi: 10.1371/journal.pgen.1001045.

  24. Monteilh-Zoller MK, Hermosura MC, Nadler MJ, Scharenberg AM, Penner R, Fleig A (2003) TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions. J Gen Physiol 121:49–60

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Montell C (2003) Mg2+ homeostasis: the Mg2+nificent TRPM chanzymes. Curr Biol 13:R799–801

    Article  CAS  PubMed  Google Scholar 

  26. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, Kurosaki T, Kinet JP, Penner R, Scharenberg AM, Fleig A (2001) LTRPC7 is a Mg.ATP-regulated divalent cation channel required for cell viability. Nature 411:590–5

    Article  CAS  PubMed  Google Scholar 

  27. Nestler A, Sponder G, Rutschmann K, Mastrototaro L, Weise C, Vormann J, Schweigel-Röntgen M, Kolisek M (2013) Nature of SLC41A1 complexes: report on the split-ubiquitin yeast two hybrid assay. Magnes Res 26:56–66. doi:10.1684/mrh.2013.0339

    CAS  PubMed  Google Scholar 

  28. Nishizawa Y, Morii H, Durlach J (2007) New perspectives in magnesium research (nutrition and health). Springer-Verlag Ltd., London

    Book  Google Scholar 

  29. Ohi K, Hashimoto R, Yamamori H, Yasuda Y, Fujimoto M, Umeda-Yano S, Fukunaga M, Watanabe Y, Iwase M, Kazui H, Takeda M (2013) The impact of the genome-wide supported variant in the cyclin M2 gene on gray matter morphology in schizophrenia. Behav Brain Funct 9:40. doi:10.1186/1744-9081-9-40

    Article  PubMed  PubMed Central  Google Scholar 

  30. Okatsu K, Oka T, Iguchi M, Imamura K, Kosako H, Tani N, Kimura M, Go E, Koyano F, Funayama M, Shiba-Fukushima K, Sato S, Shimizu H, Fukunaga Y, Taniguchi H, Komatsu M, Hattori N, Mihara K, Tanaka K, Matsuda N (2012) PINK1 autophosphorylation upon membrane potential dissipation is essential for Parkin recruitment to damaged mitochondria. Nat Commun 3:1016. doi:10.1038/ncomms2016

    Article  PubMed  PubMed Central  Google Scholar 

  31. Quamme GA (2010) Molecular identification of ancient and modern mammalian magnesium transporters. Am J Physiol Cell Physiol 298:C407–29. doi:10.1152/ajpcell.00124.2009

    Article  CAS  PubMed  Google Scholar 

  32. Rao VS, Srinivas K, Sujini GN, Sunand Kumar GN (2014) Protein-protein interaction detection: methods and analysis. Int J Proteomics 2014:147648. doi:10.1155/2014/147648

    Article  PubMed  PubMed Central  Google Scholar 

  33. Romanuik TL, Wang G, Holt RA, Jones SJ, Marra MA, Sadar MD (2009) Identification of novel androgen-responsive genes by sequencing of LongSAGE libraries. BMC Genomics 10:476. doi:10.1186/1471-2164-10-476

    Article  PubMed  PubMed Central  Google Scholar 

  34. Rose EJ, Hargreaves A, Morris D, Fahey C, Tropea D, Cummings E, Caltagirone E, Bossu P, Chiapponi C, Piras F, Spalletta G, Gill M, Corvin A, Donohoe G (2014) Effects of a novel schizophrenia risk variant rs7914558 at CNNM2 on brain structure and attributional style. Br J Psychiatry 204:115–21. doi:10.1192/bjp.bp.113.131359

    Article  PubMed  Google Scholar 

  35. Sargenti A, Farruggia G, Malucelli E, Cappadone C, Merolle L, Marraccini C, Andreani G, Prodi L, Zaccheroni N, Sgarzi M, Trombini C, Lombardo M, Iotti S (2014) A novel fluorescent chemosensor allows the assessment of intracellular total magnesium in small samples. Analyst 139:1201–7. doi:10.1039/c3an01737k

    Article  CAS  PubMed  Google Scholar 

  36. Schägger H, von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal Biochem 199:223–31

    Article  PubMed  Google Scholar 

  37. Schefe JH, Lehmann KE, Buschmann IR, Unger T, Funke-Kaiser H (2006) Quantitative real-time RT-PCR data analysis: current concepts and the novel “gene expression’s CT difference” formula. J Mol Med (Berl) 84:901–10

    Article  CAS  Google Scholar 

  38. Schmitz C, Perraud AL, Johnson CO, Inabe K, Smith MK, Penner R, Kurosaki T, Fleig A, Scharenberg AM (2003) Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell 114:191–200

    Article  CAS  PubMed  Google Scholar 

  39. Sihag RK, Cataldo AM (1996) Brain beta-spectrin is a component of senile plaques in Alzheimer’s disease. Brain Res 743:249–57

    Article  CAS  PubMed  Google Scholar 

  40. Sponder G, Svidova S, Schweigel M, Vormann J, Kolisek M (2010) Splice-variant 1 of the ancient domain protein 2 (ACDP2) complements the magnesium-deficient growth phenotype of Salmonella enterica sv. typhimurium strain MM281. Magnes Res 23:105–14. doi:10.1684/mrh.2010.0206

    CAS  PubMed  Google Scholar 

  41. Stuiver M, Lainez S, Will C, Terryn S, Günzel D, Debaix H, Sommer K, Kopplin K, Thumfart J, Kampik NB, Querfeld U, Willnow TE, Nemec V, Wagner CA, Hoenderop JG, Devuyst O, Knoers NV, Bindels RJ, Meij IC, Müller D (2011) CNNM2, encoding a basolateral protein required for renal Mg2+ handling, is mutated in dominant hypomagnesemia. Am J Hum Genet 88:333–43. doi:10.1016/j.ajhg.2011.02.005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Takahashi K, Taira T, Niki T, Seino C, Iguchi-Ariga SM, Ariga H (2001) DJ-1 positively regulates the androgen receptor by impairing the binding of PIASx alpha to the receptor. J Biol Chem 276:37556–63

    Article  CAS  PubMed  Google Scholar 

  43. Takeuchi F, Isono M, Katsuya T, Yamamoto K, Yokota M, Sugiyama T, Nabika T, Fujioka A, Ohnaka K, Asano H, Yamori Y, Yamaguchi S, Kobayashi S, Takayanagi R, Ogihara T, Kato N (2010) Blood pressure and hypertension are associated with 7 loci in the Japanese population. Circulation 121:2302–9. doi:10.1161/CIRCULATIONAHA.109.904664

    Article  PubMed  Google Scholar 

  44. van Humbeeck C, Cornelissen T, Vandenberghe W (2011) Ambra1: a Parkin-binding protein involved in mitophagy. Autophagy 7:1555–6

    Article  PubMed  PubMed Central  Google Scholar 

  45. Wang CY, Shi JD, Yang P, Kumar PG, Li QZ, Run QG, Su YC, Scott HS, Kao KJ, She JX (2003) Molecular cloning and characterization of a novel gene family of four ancient conserved domain proteins (ACDP). Gene 306:37–44

    Article  CAS  PubMed  Google Scholar 

  46. Wolf FI, Trapani V (2011) MagT1: a highly specific magnesium channel with important roles beyond cellular magnesium homeostasis. Magnes Res 24:S86–91. doi:10.1684/mrh.2011.0288

    CAS  PubMed  Google Scholar 

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Acknowledgments

Our gratitude is due to Martin Marak (Freie Universität Berlin) for competent technical support of the project, to Dr. Svenja Plöger-Meissner (Freie Universität Berlin) for help with the confocal microscopy, to Dr. Mandana Rezwan (Dualsystems Biotech AG) for cooperation with the construction of the cell lines, and to Dr. Katrin Rutschmann (Dualsystems Biotech AG) for cooperation with the SU-YTHa. Our thanks are also extended to Dr. Theresa Jones for linguistic corrections.

This work was supported by research grants from the German Research Foundation (DFG), KO-3586/3-1 and KO-3586/3-2 to MK and by research grant from Protina Pharmazeutische GmbH to JV and MK.

Contributions

MK designed the study; GS, LMa, KK, LMe, ZZ, NA, AS, KW, AF, RP, and MK performed the experiments and analyzed the data; GS, AF, RP, SI, JRA, and JV contributed to the study design; MK wrote the manuscript. All authors read, edited, and approved the manuscript.

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Author notes

  1. Katharina Kurth

    Present address: Landesuntersuchungsanstalt Sachsen, Jägerstraße 8/10, Dresden, Germany

  2. Lucia Merolle

    Present address: Elettra—Sincrotrone Trieste S.C.p.A., Strada Statale 14 AREA Science Park, Basovizza, Italy

  3. Alina Smorodchenko

    Present address: Institute of Vegetative Anatomy, Charité - Universitätsmedizin Berlin, Campus Charité Mitte, Philippstrasse 12, Berlin, Germany

Authors and Affiliations

  1. Institute of Veterinary-Physiology, Free University of Berlin, Oertzenweg 19b, 14163, Berlin, Germany

    Gerhard Sponder, Lucia Mastrototaro, Katharina Kurth, Nasrin Abdulhanan, Alina Smorodchenko, Katharina Wolf, Jörg R. Aschenbach & Martin Kolisek

  2. Department of Pharmacy and Biotechnology, University of Bologna, Via Massarenti 9, Bologna, Italy

    Lucia Merolle & Stefano Iotti

  3. The Queen’s Medical Center, Center for Biomedical Research, 1356 Lusitana Street, UH Tower 8, Honolulu, HI, USA

    Zheng Zhang, Andrea Fleig & Reinhold Penner

  4. Institute for Prevention and Nutrition (IPEV), Adalperostrasse 37, Munich, Ismaning, Germany

    Jürgen Vormann

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  1. Gerhard Sponder
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Correspondence to Martin Kolisek.

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JV is a scientific consultant of Protina Pharmazeutische GmbH. Other authors have no conflict of interests to disclose.

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Gerhard Sponder, Lucia Mastrototaro, Katharina Kurth, Lucia Merolle and Zheng Zhang contributed equally to this work.

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Sponder, G., Mastrototaro, L., Kurth, K. et al. Human CNNM2 is not a Mg2+ transporter per se. Pflugers Arch - Eur J Physiol 468, 1223–1240 (2016). https://doi.org/10.1007/s00424-016-1816-7

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