Pflügers Archiv - European Journal of Physiology

, Volume 466, Issue 10, pp 1941–1952 | Cite as

P2X4 receptor regulation of transient receptor potential melastatin type 6 (TRPM6) Mg2+ channels

  • Jeroen H. F. de Baaij
  • Maxime G. Blanchard
  • Marla Lavrijsen
  • Jens Leipziger
  • René J. M. Bindels
  • Joost G. J. Hoenderop
Ion channels, receptors and transporters


The transient receptor potential melastatin type 6 (TRPM6) ion channel regulates the body Mg2+ homeostasis by mediating transcellular Mg2+ absorption in kidney and intestine. Here, the P2X4 receptor was established as a novel regulator of TRPM6 activity. Using RT-qPCR on a mouse tissue panel, P2x4 and P2x6 were shown to be expressed in the epithelium of the colon and of the kidney, two major sites of Mg2+ reabsorption. While P2x4 was highly expressed in the colon, both P2x4 and P2x6 mRNA were prominently expressed in the distal convoluted tubule segment of the kidney, a segment with high Trpm6 expression. Using whole-cell patch clamp, an inhibitory role of P2X4 on TRPM6 activity was determined. Expression of P2X6, which does not form functional channels in mammalian cells, did not affect the function of TRPM6. The inhibition was dependent on the activity of P2X4, since a P2X4 mutant with altered ATP sensitivity was not able to inhibit TRPM6. Additionally, P2X4 was unable to inhibit TRPM7, a close homologue of TRPM6, suggesting that the inhibition is specific for TRPM6. To identify the intracellular signaling molecules that mediate the P2X4-dependent inhibition of TRPM6, the cells were treated with inhibitors of protein kinase c, protein kinase a, and phosphoinositide 3-kinase. However, none of these inhibitors prevented the inhibition of TRPM6 by P2X4. In conclusion, we propose that P2X4 receptor mediated purinergic signaling is a new regulatory mechanism of TRPM6 Mg2+ channels.


Magnesium Colon Purinergic receptor P2X4 TRPM6 



This work was supported by grants from the Netherlands Organization for Scientific Research (ZonMw 9120.8026, NWO ALW 818.02.001), a NWO Vici Grant for J.G.J. Hoenderop (016.130.668) and EURenOmics funding from the European Union seventh Framework Program (FP7/2007–2013, agreement no 305608). The authors want to state their gratitude to AnneMiete van der Kemp, Lonneke Duijkers, Fariza Bouallala, and Pauline de Bruijn for their excellent technical support. In particular, we would like to thank Dr. Sjoerd Verkaart for valuable discussions.


  1. 1.
    Bourzac JF, L’Eriger K, Larrivee JF, Arguin G, Bilodeau MS, Stankova J, Gendron FP (2013) Glucose transporter 2 expression is down regulated following P2X7 activation in enterocytes. J Cell Physiol 228(1):120–129. doi: 10.1002/jcp.24111 PubMedCrossRefGoogle Scholar
  2. 2.
    Clark K, Middelbeek J, Morrice NA, Figdor CG, Lasonder E, van Leeuwen FN (2008) Massive autophosphorylation of the Ser/Thr-rich domain controls protein kinase activity of TRPM6 and TRPM7. PLoS One 3(3):e1876. doi: 10.1371/journal.pone.0001876 PubMedCrossRefPubMedCentralGoogle Scholar
  3. 3.
    Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS (2011) Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev 63(3):641–683. doi: 10.1124/pr.110.003129 PubMedCrossRefPubMedCentralGoogle Scholar
  4. 4.
    Collo G, North RA, Kawashima E, Merlo-Pich E, Neidhart S, Surprenant A, Buell G (1996) Cloning OF P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels. J Neurosci 16(8):2495–2507PubMedGoogle Scholar
  5. 5.
    Cooke HJ, Wunderlich J, Christofi FL (2003) “The force be with you”: ATP in gut mechanosensory transduction. News Physiol Sci 18:43–49PubMedGoogle Scholar
  6. 6.
    Coutinho-Silva R, Stahl L, Cheung KK, de Campos NE, de Oliveira SC, Ojcius DM, Burnstock G (2005) P2X and P2Y purinergic receptors on human intestinal epithelial carcinoma cells: effects of extracellular nucleotides on apoptosis and cell proliferation. Am J Physiol Gastrointest Liver Physiol 288(5):G1024–1035. doi: 10.1152/ajpgi.00211.2004 PubMedCrossRefGoogle Scholar
  7. 7.
    Dai LJ, Kang HS, Kerstan D, Ritchie G, Quamme GA (2001) ATP inhibits Mg(2+) uptake in MDCT cells via P2X purinoceptors. Am J Physiol Renal Physiol 281(5):F833–840PubMedGoogle Scholar
  8. 8.
    de Baaij JH, Groot Koerkamp MJ, Lavrijsen M, van Zeeland F, Meijer H, Holstege FC, Bindels RJ, Hoenderop JG (2013) Elucidation of the distal convoluted tubule transcriptome identifies new candidate genes involved in renal magnesium handling. Am J Physiol Renal Physiol. doi: 10.1152/ajprenal.00322.2013 PubMedGoogle Scholar
  9. 9.
    de Baaij JHF, Hoenderop JGJ, Bindels RJM (2012) Regulation of magnesium balance: lessons learned from human genetic disease. Clinical Kidney Journal 5(Suppl 1):i15–i24CrossRefGoogle Scholar
  10. 10.
    Egan TM, Khakh BS (2004) Contribution of calcium ions to P2X channel responses. J Neurosci 24(13):3413–3420. doi: 10.1523/jneurosci.5429-03.2004 PubMedCrossRefGoogle Scholar
  11. 11.
    Garcia-Alcocer G, Padilla K, Rodriguez A, Miledi R, Berumen LC (2012) Distribution of the purinegic receptors P2X(4) and P2X(6) during rat gut development. Neurosci Lett 509(2):92–95. doi: 10.1016/j.neulet.2011.12.044 PubMedCrossRefGoogle Scholar
  12. 12.
    Glass R, Loesch A, Bodin P, Burnstock G (2002) P2X4 and P2X6 receptors associate with VE-cadherin in human endothelial cells. Cell Mol Life Sci 59(5):870–881PubMedCrossRefGoogle Scholar
  13. 13.
    Groenestege WM, Hoenderop JG, van den Heuvel L, Knoers N, Bindels RJ (2006) The epithelial Mg2+ channel transient receptor potential melastatin 6 is regulated by dietary Mg2+ content and estrogens. J Am Soc Nephrol 17(4):1035–1043. doi: 10.1681/ASN.2005070700 PubMedCrossRefGoogle Scholar
  14. 14.
    Groenestege WM, Thebault S, van der Wijst J, van den Berg D, Janssen R, Tejpar S, van den Heuvel LP, van Cutsem E, Hoenderop JG, Knoers NV, Bindels RJ (2007) Impaired basolateral sorting of pro-EGF causes isolated recessive renal hypomagnesemia. J Clin Invest 117(8):2260–2267. doi: 10.1172/JCI31680 PubMedCrossRefPubMedCentralGoogle Scholar
  15. 15.
    Groschel-Stewart U, Bardini M, Robson T, Burnstock G (1999) P2X receptors in the rat duodenal villus. Cell Tissue Res 297(1):111–117PubMedCrossRefGoogle Scholar
  16. 16.
    Hausmann R, Bodnar M, Woltersdorf R, Wang H, Fuchs M, Messemer N, Qin Y, Gunther J, Riedel T, Grohmann M, Nieber K, Schmalzing G, Rubini P, Illes P (2012) ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors. J Biol Chem 287(17):13930–13943. doi: 10.1074/jbc.M112.345207 PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Hess MW, Hoenderop JG, Bindels RJ, Drenth JP (2012) Systematic review: hypomagnesaemia induced by proton pump inhibition. Aliment Pharmacol Ther 36(5):405–413. doi: 10.1111/j.1365-2036.2012.05201.x PubMedCrossRefGoogle Scholar
  18. 18.
    Kawate T, Michel JC, Birdsong WT, Gouaux E (2009) Crystal structure of the ATP-gated P2X(4) ion channel in the closed state. Nature 460(7255):592–598. doi: 10.1038/nature08198 PubMedCrossRefPubMedCentralGoogle Scholar
  19. 19.
    Khakh BS, Zhou X, Sydes J, Galligan JJ, Lester HA (2000) State-dependent cross-inhibition between transmitter-gated cation channels. Nature 406(6794):405–410. doi: 10.1038/35019066 PubMedCrossRefGoogle Scholar
  20. 20.
    Lameris AL, Hess MW, van Kruijsbergen I, Hoenderop JG, Bindels RJ (2013) Omeprazole enhances the colonic expression of the Mg transporter TRPM6. Pflugers Arch. doi: 10.1007/s00424-013-1306-0 PubMedGoogle Scholar
  21. 21.
    Lameris AL, Monnens LA, Bindels RJ, Hoenderop JG (2012) Drug-induced alterations in Mg2+ homoeostasis. Clin Sci (Lond) 123(1):1–14. doi: 10.1042/CS20120045 CrossRefGoogle Scholar
  22. 22.
    Le KT, Babinski K, Seguela P (1998) Central P2X4 and P2X6 channel subunits coassemble into a novel heteromeric ATP receptor. J Neurosci 18(18):7152–7159PubMedGoogle Scholar
  23. 23.
    Leipziger J (2003) Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284(3):F419–432. doi: 10.1152/ajprenal.00075.2002 PubMedGoogle Scholar
  24. 24.
    Leipziger J (2011) Luminal nucleotides are tonic inhibitors of renal tubular transport. Curr Opin Nephrol Hypertens 20(5):518–522. doi: 10.1097/MNH.0b013e3283487393 PubMedCrossRefGoogle Scholar
  25. 25.
    Limaye CS, Londhey VA, Nadkart MY, Borges NE (2011) Hypomagnesemia in critically ill medical patients. J Assoc Physicians India 59:19–22PubMedGoogle Scholar
  26. 26.
    Marques RD, de Bruijn PI, Sorensen MV, Bleich M, Praetorius HA, Leipziger J (2012) Basolateral P2X receptors mediate inhibition of NaCl transport in mouse medullary thick ascending limb (mTAL). Am J Physiol Renal Physiol 302(4):F487–494. doi: 10.1152/ajprenal.00570.2011 PubMedCrossRefGoogle Scholar
  27. 27.
    Matos JE, Sorensen MV, Geyti CS, Robaye B, Boeynaems JM, Leipziger J (2007) Distal colonic Na(+) absorption inhibited by luminal P2Y(2) receptors. Pflugers Arch 454(6):977–987. doi: 10.1007/s00424-007-0248-9 PubMedCrossRefGoogle Scholar
  28. 28.
    Meyer AH, Katona I, Blatow M, Rozov A, Monyer H (2002) In vivo labeling of parvalbumin-positive interneurons and analysis of electrical coupling in identified neurons. J Neurosci 22(16):7055–7064PubMedGoogle Scholar
  29. 29.
    Nair AV, Hocher B, Verkaart S, van Zeeland F, Pfab T, Slowinski T, Chen YP, Schlingmann KP, Schaller A, Gallati S, Bindels RJ, Konrad M, Hoenderop JG (2012) Loss of insulin-induced activation of TRPM6 magnesium channels results in impaired glucose tolerance during pregnancy. Proc Natl Acad Sci U S A 109(28):11324–11329. doi: 10.1073/pnas.1113811109 PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Nijenhuis T, Renkema KY, Hoenderop JG, Bindels RJ (2006) Acid–base status determines the renal expression of Ca2+ and Mg2+ transport proteins. J Am Soc Nephrol 17(3):617–626. doi: 10.1681/asn.2005070732 PubMedCrossRefGoogle Scholar
  31. 31.
    North RA, Surprenant A (2000) Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 40:563–580. doi: 10.1146/annurev.pharmtox.40.1.563 PubMedCrossRefGoogle Scholar
  32. 32.
    Novak I (2011) Purinergic signalling in epithelial ion transport: regulation of secretion and absorption. Acta Physiol (Oxf) 202(3):501–522. doi: 10.1111/j.1748-1716.2010.02225.x CrossRefGoogle Scholar
  33. 33.
    Orriss IR, Key ML, Brandao-Burch A, Patel JJ, Burnstock G, Arnett TR (2012) The regulation of osteoblast function and bone mineralisation by extracellular nucleotides: The role of p2x receptors. Bone 51(3):389–400. doi: 10.1016/j.bone.2012.06.013 PubMedCrossRefGoogle Scholar
  34. 34.
    Quamme GA (2008) Recent developments in intestinal magnesium absorption. Curr Opin Gastroenterol 24(2):230–235. doi: 10.1097/MOG.0b013e3282f37b59 PubMedCrossRefGoogle Scholar
  35. 35.
    Schlingmann KP, Weber S, Peters M, Niemann Nejsum L, Vitzthum H, Klingel K, Kratz M, Haddad E, Ristoff E, Dinour D, Syrrou M, Nielsen S, Sassen M, Waldegger S, Seyberth HW, Konrad M (2002) Hypomagnesemia with secondary hypocalcemia is caused by mutations in TRPM6, a new member of the TRPM gene family. Nat Genet 31(2):166–170. doi: 10.1038/ng889 PubMedCrossRefGoogle Scholar
  36. 36.
    Stanchev D, Blosa M, Milius D, Gerevich Z, Rubini P, Schmalzing G, Eschrich K, Schaefer M, Wirkner K, Illes P (2009) Cross-inhibition between native and recombinant TRPV1 and P2X(3) receptors. Pain 143(1–2):26–36. doi: 10.1016/j.pain.2009.01.006 PubMedCrossRefGoogle Scholar
  37. 37.
    Stokes L, Scurrah K, Ellis JA, Cromer BA, Skarratt KK, Gu BJ, Harrap SB, Wiley JS (2011) A loss-of-function polymorphism in the human P2X4 receptor is associated with increased pulse pressure. Hypertension 58(6):1086–1092. doi: 10.1161/hypertensionaha.111.176180 PubMedCrossRefGoogle Scholar
  38. 38.
    Tanaka J, Murate M, Wang CZ, Seino S, Iwanaga T (1996) Cellular distribution of the P2X4 ATP receptor mRNA in the brain and non-neuronal organs of rats. Arch Histol Cytol 59(5):485–490PubMedCrossRefGoogle Scholar
  39. 39.
    Taylor AL, Schwiebert LM, Smith JJ, King C, Jones JR, Sorscher EJ, Schwiebert EM (1999) Epithelial P2X purinergic receptor channel expression and function. J Clin Invest 104(7):875–884. doi: 10.1172/JCI7270 PubMedCrossRefPubMedCentralGoogle Scholar
  40. 40.
    Thebault S, Alexander RT, Tiel Groenestege WM, Hoenderop JG, Bindels RJ (2009) EGF increases TRPM6 activity and surface expression. J Am Soc Nephrol 20(1):78–85. doi: 10.1681/ASN.2008030327 PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Thebault S, Cao G, Venselaar H, Xi Q, Bindels RJ, Hoenderop JG (2008) Role of the alpha-kinase domain in transient receptor potential melastatin 6 channel and regulation by intracellular ATP. J Biol Chem 283(29):19999–20007. doi: 10.1074/jbc.M800167200 PubMedCrossRefGoogle Scholar
  42. 42.
    Topala CN, Groenestege WT, Thebault S, van den Berg D, Nilius B, Hoenderop JG, Bindels RJ (2007) Molecular determinants of permeation through the cation channel TRPM6. Cell Calcium 41(6):513–523. doi: 10.1016/j.ceca.2006.10.003 PubMedCrossRefGoogle Scholar
  43. 43.
    Torres GE, Egan TM, Voigt MM (1999) Hetero-oligomeric assembly of P2X receptor subunits. Specificities exist with regard to possible partners. J Biol Chem 274(10):6653–6659PubMedCrossRefGoogle Scholar
  44. 44.
    Turner CM, Vonend O, Chan C, Burnstock G, Unwin RJ (2003) The pattern of distribution of selected ATP-sensitive P2 receptor subtypes in normal rat kidney: an immunohistological study. Cells Tissues Organs 175(2):105–117PubMedCrossRefGoogle Scholar
  45. 45.
    Voets T, Nilius B, Hoefs S, van der Kemp AW, Droogmans G, Bindels RJ, Hoenderop JG (2004) TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem 279(1):19–25. doi: 10.1074/jbc.M311201200 PubMedCrossRefGoogle Scholar
  46. 46.
    Walder RY, Landau D, Meyer P, Shalev H, Tsolia M, Borochowitz Z, Boettger MB, Beck GE, Englehardt RK, Carmi R, Sheffield VC (2002) Mutation of TRPM6 causes familial hypomagnesemia with secondary hypocalcemia. Nat Genet 31(2):171–174. doi: 10.1038/ng901 PubMedCrossRefGoogle Scholar
  47. 47.
    Wang J, Huo K, Ma L, Tang L, Li D, Huang X, Yuan Y, Li C, Wang W, Guan W, Chen H, Jin C, Wei J, Zhang W, Yang Y, Liu Q, Zhou Y, Zhang C, Wu Z, Xu W, Zhang Y, Liu T, Yu D, Zhang Y, Chen L, Zhu D, Zhong X, Kang L, Gan X, Yu X, Ma Q, Yan J, Zhou L, Liu Z, Zhu Y, Zhou T, He F, Yang X (2011) Toward an understanding of the protein interaction network of the human liver. Mol Syst Biol 7:536. doi: 10.1038/msb.2011.67 PubMedCrossRefPubMedCentralGoogle Scholar
  48. 48.
    Wildman SS, Boone M, Peppiatt-Wildman CM, Contreras-Sanz A, King BF, Shirley DG, Deen PM, Unwin RJ (2009) Nucleotides downregulate aquaporin 2 via activation of apical P2 receptors. J Am Soc Nephrol 20(7):1480–1490. doi: 10.1681/ASN.2008070686 PubMedCrossRefPubMedCentralGoogle Scholar
  49. 49.
    Wildman SS, Kang ES, King BF (2009) ENaC, renal sodium excretion and extracellular ATP. Purinergic Signal 5(4):481–489. doi: 10.1007/s11302-009-9150-6 PubMedCrossRefPubMedCentralGoogle Scholar
  50. 50.
    Wildman SS, Marks J, Churchill LJ, Peppiatt CM, Chraibi A, Shirley DG, Horisberger JD, King BF, Unwin RJ (2005) Regulatory interdependence of cloned epithelial Na+ channels and P2X receptors. J Am Soc Nephrol 16(9):2586–2597. doi: 10.1681/ASN.2005020130 PubMedCrossRefGoogle Scholar
  51. 51.
    Wildman SS, Marks J, Turner CM, Yew-Booth L, Peppiatt-Wildman CM, King BF, Shirley DG, Wang W, Unwin RJ (2008) Sodium-dependent regulation of renal amiloride-sensitive currents by apical P2 receptors. J Am Soc Nephrol 19(4):731–742. doi: 10.1681/ASN.2007040443 PubMedCrossRefPubMedCentralGoogle Scholar
  52. 52.
    Woudenberg-Vrenken TE, Sukinta A, van der Kemp AW, Bindels RJ, Hoenderop JG (2011) Transient receptor potential melastatin 6 knockout mice are lethal whereas heterozygous deletion results in mild hypomagnesemia. Nephron Physiol 117(2):p11–19. doi: 10.1159/000320580 PubMedCrossRefGoogle Scholar
  53. 53.
    Yamamoto T, Suzuki Y (2002) Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon. Am J Physiol Gastrointest Liver Physiol 283(2):G300–308. doi: 10.1152/ajpgi.00541.2001 PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Jeroen H. F. de Baaij
    • 1
  • Maxime G. Blanchard
    • 1
  • Marla Lavrijsen
    • 1
  • Jens Leipziger
    • 2
  • René J. M. Bindels
    • 1
  • Joost G. J. Hoenderop
    • 1
  1. 1.Department of Physiology, Radboud Institute for Molecular Life SciencesRadboud University Medical CenterNijmegenThe Netherlands
  2. 2.Department of Biomedicine, Physiology and BiophysicsAarhus UniversityAarhusDenmark

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