Skip to main content

Advertisement

SpringerLink
Log in

The tissue-specific expression of TRPML2 (MCOLN-2) gene is influenced by the presence of TRPML1

  • Ion Channels, Receptors and Transporters
  • Published:
Pflügers Archiv - European Journal of Physiology Aims and scope Submit manuscript

Abstract

Mucolipidosis type IV is a lysosomal storage disorder caused by the loss or dysfunction of the mucolipin-1 (TRPML1) protein. It has been suggested that TRPML2 could genetically compensate (i.e., become upregulated) for the loss of TRPML1. We thus investigated this possibility by first studying the expression pattern of mouse TRPML2 and its basic channel properties using the varitint-waddler (Va) model. Here, we confirmed the presence of long variant TRPML2 (TRPML2lv) and short variant (TRPML2sv) isoforms. We showed for the first time that, heterologously expressed, TRPML2lv-Va is an active, inwardly rectifying channel. Secondly, we quantitatively measured TRPML2 and TRPML3 mRNA expressions in TRPML1–/– null and wild-type (Wt) mice. In wild-type mice, the TRPML2lv transcripts were very low while TRPML2sv and TRPML3 transcripts have predominant expressions in lymphoid and kidney organs. Significant reductions of TRPML2sv, but not TRPML2lv or TRPML3 transcripts, were observed in lymphoid and kidney organs of TRPML1–/– mice. RNA interference of endogenous human TRPML1 in HEK-293 cells produced a comparable decrease of human TRPML2 transcript levels that can be restored by overexpression of human TRPML1. Conversely, significant upregulation of TRPML2sv transcripts was observed when primary mouse lymphoid cells were treated with nicotinic acid adenine dinucleotide phosphate, or N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinoline sulfonamide, both known activators of TRPML1. In conclusion, our results indicate that TRPML2 is unlikely to compensate for the loss of TRPML1 in lymphoid or kidney organs and that TRPML1 appears to play a novel role in the tissue-specific transcriptional regulation of TRPML2.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Bargal R, Goebel HH, Latta E, Bach G (2002) Mucolipidosis IV: novel mutation and diverse ultrastructural spectrum in the skin. Neuropediatrics 33:199–202

    Article  PubMed  CAS  Google Scholar 

  2. Bassi MT, Manzoni M, Monti E, Pizzo MT, Ballabio A, Borsani G (2000) Cloning of the gene encoding a novel integral membrane protein, mucolipidin-and identification of the two major founder mutations causing mucolipidosis type IV. Am J Hum Genet 67:1110–1120

    PubMed  CAS  Google Scholar 

  3. Sun M, Goldin E, Stahl S, Falardeau JL, Kennedy JC, Acierno JS Jr, Bove C, Kaneski CR, Nagle J, Bromley MC, Colman M, Schiffmann R, Slaugenhaupt SA (2000) Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel. Hum Mol Genet 9:2471–2478

    Article  PubMed  CAS  Google Scholar 

  4. Amir N, Zlotogora J, Bach G (1987) Mucolipidosis type IV: clinical spectrum and natural history. Pediatrics 79:953–959

    PubMed  CAS  Google Scholar 

  5. Bach G, Cohen MM, Kohn G (1975) Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV. Biochem Biophys Res Commun 66:1483–1490

    Article  PubMed  CAS  Google Scholar 

  6. Tellez-Nagel I, Rapin I, Iwamoto T, Johnson AB, Norton WT, Nitowsky H (1976) Mucolipidosis IV. Clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy. Arch Neurol 33:828–835

    PubMed  CAS  Google Scholar 

  7. Kiselyov K, Chen J, Rbaibi Y, Oberdick D, Tjon-Kon-Sang S, Shcheynikov N, Muallem S, Soyombo A (2005) TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage. J Biol Chem 280:43218–43223

    Article  PubMed  CAS  Google Scholar 

  8. LaPlante JM, Sun M, Falardeau J, Dai D, Brown EM, Slaugenhaupt SA, Vassilev PM (2006) Lysosomal exocytosis is impaired in mucolipidosis type IV. Mol Genet Metab 89:339–348

    Article  PubMed  CAS  Google Scholar 

  9. Pryor PR, Reimann F, Gribble FM, Luzio JP (2006) Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic. Traffic 7:1388–1398

    Article  PubMed  CAS  Google Scholar 

  10. Thompson EG, Schaheen L, Dang H, Fares H (2007) Lysosomal trafficking functions of mucolipin-1 in murine macrophages. BMC Cell Biol 8:54

    Article  PubMed  CAS  Google Scholar 

  11. Miedel MT, Rbaibi Y, Guerriero CJ, Colletti G, Weixel KM, Weisz OA, Kiselyov K (2008) Membrane traffic and turnover in TRP-ML1-deficient cells: a revised model for mucolipidosis type IV pathogenesis. J Exp Med 205:1477–1490

    Article  PubMed  CAS  Google Scholar 

  12. Raychowdhury MK, Gonzalez-Perrett S, Montalbetti N, Timpanaro GA, Chasan B, Goldmann WH, Stahl S, Cooney A, Goldin E, Cantiello HF (2004) Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel. Hum Mol Genet 13:617–627

    Article  PubMed  CAS  Google Scholar 

  13. Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, Kiselyov K (2006) TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity. J Biol Chem 281:7294–7301

    Article  PubMed  CAS  Google Scholar 

  14. Falardeau JL, Kennedy JC, Acierno JS Jr, Sun M, Stahl S, Goldin E, Slaugenhaupt SA (2002) Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans. BMC Genomics 3:3

    Article  PubMed  Google Scholar 

  15. Di Palma F, Belyantseva IA, Kim HJ, Vogt TF, Kachar B, Noben-Trauth K (2002) Mutations in Mcoln3 associated with deafness and pigmentation defects in varitint-waddler (Va) mice. Proc Natl Acad Sci U S A 99:14994–14999

    Article  PubMed  CAS  Google Scholar 

  16. Lindvall JM, Blomberg KE, Wennborg A, Smith CI (2005) Differential expression and molecular characterisation of Lmo7, Myo1e, Sash1, and Mcoln2 genes in Btk-defective B-cells. Cell Immunol 235:46–55

    Article  PubMed  CAS  Google Scholar 

  17. Xu H, Delling M, Li L, Dong X, Clapham DE (2007) Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice. Proc Natl Acad Sci U S A 104:18321–18326

    Article  PubMed  Google Scholar 

  18. Dong XP, Cheng X, Mills E, Delling M, Wang F, Kurz T, Xu H (2008) The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel. Nature 455:992–996

    Article  PubMed  CAS  Google Scholar 

  19. Grimm C, Cuajungco MP, van Aken AF, Schnee M, Jors S, Kros CJ, Ricci AJ, Heller S (2007) A helix-breaking mutation in TRPML3 leads to constitutive activity underlying deafness in the varitint-waddler mouse. Proc Natl Acad Sci U S A 104:19583–19588

    Article  PubMed  Google Scholar 

  20. Kim HJ, Li Q, Tjon-Kon-Sang S, So I, Kiselyov K, Muallem S (2007) Gain-of-function mutation in TRPML3 causes the mouse varitint-waddler phenotype. J Biol Chem 282:36138–36142

    Article  PubMed  CAS  Google Scholar 

  21. Nagata K, Zheng L, Madathany T, Castiglioni AJ, Bartles JR, Garcia-Anoveros J (2008) The varitint-waddler (Va) deafness mutation in TRPML3 generates constitutive, inward rectifying currents and causes cell degeneration. Proc Natl Acad Sci U S A 105:353–358

    Article  PubMed  Google Scholar 

  22. Song Y, Dayalu R, Matthews SA, Scharenberg AM (2006) TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes. Eur J Cell Biol

  23. Venkatachalam K, Hofmann T, Montell C (2006) Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1. J Biol Chem 281:17517–17527

    Article  PubMed  CAS  Google Scholar 

  24. Zeevi DA, Frumkin A, Bach G (2007) TRPML and lysosomal function. Biochim Biophys Acta 1772:851–858

    PubMed  CAS  Google Scholar 

  25. Zeevi DA, Frumkin A, Offen-Glasner V, Kogot-Levin A, Bach G (2009) A potentially dynamic lysosomal role for the endogenous TRPML proteins. J Pathol

  26. Fares H, Greenwald I (2001) Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog. Nat Genet 28:64–68

    Article  PubMed  CAS  Google Scholar 

  27. Treusch S, Knuth S, Slaugenhaupt SA, Goldin E, Grant BD, Fares H (2004) Caenorhabditis elegans functional orthologue of human protein h-mucolipin-1 is required for lysosome biogenesis. Proc Natl Acad Sci U S A 101:4483–4488

    Article  PubMed  CAS  Google Scholar 

  28. Dietrich A, Mederos YSM, Gollasch M, Gross V, Storch U, Dubrovska G, Obst M, Yildirim E, Salanova B, Kalwa H, Essin K, Pinkenburg O, Luft FC, Gudermann T, Birnbaumer L (2005) Increased vascular smooth muscle contractility in TRPC6-/- mice. Mol Cell Biol 25:6980–6989

    Article  PubMed  CAS  Google Scholar 

  29. Selli C, Erac Y, Kosova B, Tosun M (2009) Post-transcriptional silencing of TRPC1 ion channel gene by RNA interference upregulates TRPC6 expression and store-operated Ca(2+) entry in A7r5 vascular smooth muscle cells. Vascul Pharmacol 51:96–100

    Article  PubMed  CAS  Google Scholar 

  30. Micsenyi MC, Dobrenis K, Stephney G, Pickel J, Vanier MT, Slaugenhaupt SA, Walkley SU (2009) Neuropathology of the Mcoln1(-/-) knockout mouse model of mucolipidosis type IV. J Neuropathol Exp Neurol 68:125–135

    Article  PubMed  CAS  Google Scholar 

  31. Venugopal B, Browning MF, Curcio-Morelli C, Varro A, Michaud N, Nanthakumar N, Walkley SU, Pickel J, Slaugenhaupt SA (2007) Neurologic, gastric, and opthalmologic pathologies in a murine model of mucolipidosis type IV. Am J Hum Genet 81:1070–1083

    Article  PubMed  CAS  Google Scholar 

  32. Goidin D, Mamessier A, Staquet MJ, Schmitt D, Berthier-Vergnes O (2001) Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem 295:17–21

    Article  PubMed  CAS  Google Scholar 

  33. Riccio A, Medhurst AD, Mattei C, Kelsell RE, Calver AR, Randall AD, Benham CD, Pangalos MN (2002) mRNA distribution analysis of human TRPC family in CNS and peripheral tissues. Brain Res Mol Brain Res 109:95–104

    Article  PubMed  CAS  Google Scholar 

  34. Schmittgen TD, Zakrajsek BA (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J Biochem Biophys Methods 46:69–81

    Article  PubMed  CAS  Google Scholar 

  35. Brailoiu E, Churamani D, Cai X, Schrlau MG, Brailoiu GC, Gao X, Hooper R, Boulware MJ, Dun NJ, Marchant JS, Patel S (2009) Essential requirement for two-pore channel 1 in NAADP-mediated calcium signaling. J Cell Biol 186:201–209

    Article  PubMed  CAS  Google Scholar 

  36. Cuajungco MP, Samie MA (2008) The varitint-waddler mouse phenotypes and the TRPML3 ion channel mutation: cause and consequence. Pflugers Arch 457:463–473

    Article  PubMed  CAS  Google Scholar 

  37. Beeton C, Chandy KG (2007) Preparing T cell growth factor from rat splenocytes. J Vis Exp:402

  38. Beck A, Kolisek M, Bagley LA, Fleig A, Penner R (2006) Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes. FASEB J 20:962–964

    Article  PubMed  CAS  Google Scholar 

  39. Calcraft PJ, Ruas M, Pan Z, Cheng X, Arredouani A, Hao X, Tang J, Rietdorf K, Teboul L, Chuang KT, Lin P, Xiao R, Wang C, Zhu Y, Lin Y, Wyatt CN, Parrington J, Ma J, Evans AM, Galione A, Zhu MX (2009) NAADP mobilizes calcium from acidic organelles through two-pore channels. Nature 459:596–600

    Article  PubMed  CAS  Google Scholar 

  40. Guse AH, Lee HC (2008) NAADP: a universal Ca2+ trigger. Sci Signal 1:re10

    Article  PubMed  Google Scholar 

  41. Lange I, Yamamoto S, Partida-Sanchez S, Mori Y, Fleig A, Penner R (2009) TRPM2 functions as a lysosomal Ca2+-release channel in beta cells. Sci Signal 2:ra23

    Article  PubMed  Google Scholar 

  42. Singaravelu K, Deitmer JW (2006) Calcium mobilization by nicotinic acid adenine dinucleotide phosphate (NAADP) in rat astrocytes. Cell Calcium 39:143–153

    Article  PubMed  CAS  Google Scholar 

  43. Steen M, Kirchberger T, Guse AH (2007) NAADP mobilizes calcium from the endoplasmic reticular Ca(2+) store in T-lymphocytes. J Biol Chem 282:18864–18871

    Article  PubMed  CAS  Google Scholar 

  44. Zhang F, Jin S, Yi F, Li PL (2008) TRP-ML1 Functions as a Lysosomal NAADP-Sensitive Ca(2+) Release Channel in Coronary Arterial Myocytes. J Cell Mol Med

  45. Zhang F, Li PL (2007) Reconstitution and characterization of a nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca2+ release channel from liver lysosomes of rats. J Biol Chem 282:25259–25269

    Article  PubMed  CAS  Google Scholar 

  46. Zong X, Schieder M, Cuny H, Fenske S, Gruner C, Rotzer K, Griesbeck O, Harz H, Biel M, Wahl-Schott C (2009) The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores. Pflugers Arch

  47. Chijiwa T, Mishima A, Hagiwara M, Sano M, Hayashi K, Inoue T, Naito K, Toshioka T, Hidaka H (1990) Inhibition of forskolin-induced neurite outgrowth and protein phosphorylation by a newly synthesized selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), of PC12D pheochromocytoma cells. J Biol Chem 265:5267–5272

    PubMed  CAS  Google Scholar 

  48. Vergarajauregui S, Oberdick R, Kiselyov K, Puertollano R (2008) Mucolipin 1 channel activity is regulated by protein kinase A-mediated phosphorylation. Biochem J 410:417–425

    Article  PubMed  CAS  Google Scholar 

  49. Cuajungco MP, Grimm C, Oshima K, D'Hoedt D, Nilius B, Mensenkamp AR, Bindels RJ, Plomann M, Heller S (2006) PACSINs bind to the TRPV4 cation channel. PACSIN 3 modulates the subcellular localization of TRPV4. J Biol Chem 281:18753–18762

    Article  PubMed  CAS  Google Scholar 

  50. Dong XP, Wang X, Shen D, Chen S, Liu M, Wang Y, Mills E, Cheng X, Delling M, Xu H (2009) Activating mutations of the TRPML1 channel revealed by proline scanning mutagenesis. J Biol Chem

  51. Kim HJ, Li Q, Tjon-Kon-Sang S, So I, Kiselyov K, Soyombo AA, Muallem S (2008) A novel mode of TRPML3 regulation by extracytosolic pH absent in the varitint-waddler phenotype. EMBO J 27:1197–1205

    Article  PubMed  CAS  Google Scholar 

  52. D'Hoedt D, Owsianik G, Prenen J, Cuajungco MP, Grimm C, Heller S, Voets T, Nilius B (2008) Stimulus-specific modulation of the cation channel TRPV4 by PACSIN 3. J Biol Chem 283:6272–6280

    Article  PubMed  CAS  Google Scholar 

  53. Cheng W, Yang F, Takanishi CL, Zheng J (2007) Thermosensitive TRPV channel subunits coassemble into heteromeric channels with intermediate conductance and gating properties. J Gen Physiol 129:191–207

    Article  PubMed  CAS  Google Scholar 

  54. Bai CX, Giamarchi A, Rodat-Despoix L, Padilla F, Downs T, Tsiokas L, Delmas P (2008) Formation of a new receptor-operated channel by heteromeric assembly of TRPP2 and TRPC1 subunits. EMBO Rep 9:472–479

    Article  PubMed  CAS  Google Scholar 

  55. Khan WN, Alt FW, Gerstein RM, Malynn BA, Larsson I, Rathbun G, Davidson L, Muller S, Kantor AB, Herzenberg LA et al (1995) Defective B cell development and function in Btk-deficient mice. Immunity 3:283–299

    Article  PubMed  CAS  Google Scholar 

  56. Karacsonyi C, Miguel AS, Puertollano R (2007) Mucolipin-2 localizes to the Arf6-associated pathway and regulates recycling of GPI-APs. Traffic 8:1404–1414

    Article  PubMed  CAS  Google Scholar 

  57. Chatterjee S, Mayor S (2001) The GPI-anchor and protein sorting. Cell Mol Life Sci 58:1969–1987

    Article  PubMed  CAS  Google Scholar 

  58. Lange I, Penner R, Fleig A, Beck A (2008) Synergistic regulation of endogenous TRPM2 channels by adenine dinucleotides in primary human neutrophils. Cell Calcium 44:604–615

    Article  PubMed  CAS  Google Scholar 

  59. Isakov N, Mally MI, Altman A (1992) Mitogen-induced human T cell proliferation is associated with increased expression of selected PKC genes. Mol Immunol 29:927–933

    Article  PubMed  CAS  Google Scholar 

  60. Martin P, Duran A, Minguet S, Gaspar ML, Diaz-Meco MT, Rennert P, Leitges M, Moscat J (2002) Role of zeta PKC in B-cell signaling and function. EMBO J 21:4049–4057

    Article  PubMed  CAS  Google Scholar 

  61. Sardiello M, Palmieri M, di Ronza A, Medina DL, Valenza M, Gennarino VA, Di Malta C, Donaudy F, Embrione V, Polishchuk RS, Banfi S, Parenti G, Cattaneo E, Ballabio A (2009) A gene network regulating lysosomal biogenesis and function. Science 325:473–477

    PubMed  CAS  Google Scholar 

  62. Venugopal B, Mesires NT, Kennedy JC, Curcio-Morelli C, Laplante JM, Dice JF, Slaugenhaupt SA (2008) Chaperone-mediated autophagy is defective in mucolipidosis type IV. J Cell Physiol

  63. Vergarajauregui S, Connelly PS, Daniels MP, Puertollano R (2008) Autophagic dysfunction in mucolipidosis type IV patients. Hum Mol Genet 17:2723–2737

    Article  PubMed  CAS  Google Scholar 

  64. Kim HJ, Soyombo AA, Tjon-Kon-Sang S, So I, Muallem S (2009) The Ca(2+) channel TRPML3 regulates membrane trafficking and autophagy. Traffic 10:1157–1167

    Article  PubMed  CAS  Google Scholar 

  65. Martina JA, Lelouvier B, Puertollano R (2009) The calcium channel mucolipin-3 is a novel regulator of trafficking along the endosomal pathway. Traffic 10:1143–1156

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We are grateful for the technical support given by Tony Ricci, Michael Schnee, Simone Jörs, and James Mull. We also thank Breck Wheelock for his help on tissue dissection and primary mouse lymphoid cell culture. We thank Dr. Rosa Puertollano (NIH/NHLBI) for providing the human TRPML2 expression plasmid and Dr. Kirill Kiselyov for the human anti-TRPML1 polyclonal antibodies. MPC is partly supported by the Howard Hughes Medical Institute under the HHMI Undergraduate Research Program and a recipient of the California State University Program for Education and Research in Biotechnology (CSUPERB) Faculty-Student Collaborative Research Grant and CSUF Junior Faculty Intramural Research Grant. M. A. S. is a Coppel Graduate Student Science scholar and is a recipient of a research grant from the CSUF Associated Students Inc. JAE is a Howell-CSUPERB scholar, and a recipient of the Nagel-CSUPERB travel award and the CSUF Associated Students Inc. research grant. SH is funded by NIH grant (DC004563). SAS is funded by NIH grant (NS39995).

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Biological Science, and Center for Applied Biotechnology Studies, California State University Fullerton, 800 N State College Blvd, Fullerton, CA, 92831, USA

    Mohammad A. Samie, Jeffrey A. Evans & Math P. Cuajungco

  2. Department of Otolaryngology, and Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA

    Christian Grimm & Stefan Heller

  3. Center for Human Genetic Research, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA

    Cyntia Curcio-Morelli & Susan A. Slaugenhaupt

  4. Mental Health Research Institute, Parkville, Melbourne, Victoria, Australia

    Math P. Cuajungco

Authors
  1. Mohammad A. Samie
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian Grimm
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jeffrey A. Evans
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cyntia Curcio-Morelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Stefan Heller
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Susan A. Slaugenhaupt
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Math P. Cuajungco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Math P. Cuajungco.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Fig. S1

Schematic map of the mouse and human TRPML oligonucleotide sequences employed in RT-PCR, real-time QPRC, and RNAi analyses. The map illustrates the location of the primer sequences used for real-time QPCR (gray arrows) and standard RT-PCR (black arrows) for each corresponding TRPML or Mucolipin (Mcoln) gene. The open-reading frame is shown as a gray-shaded box along the complete mRNA (black line). The locations of the short-hairpin RNA oligonucleotide sequences designed to specifically target the endogenous human TRPML1 mRNA are also illustrated (dashed black arrows). The corresponding Genbank accession numbers are shown on the left of the diagram. Nucleotide base pair numbers are indicated for each Mcoln gene. (GIF 102 kb)

High resolution image (EPS 1428 kb)

Supplementary Fig. S2

Mouse TRPML2 mRNA map illustrating the gene sequence differences between TRPML2lv and TRPML2sv. The diagram shows the 5’-untranslated regions (5’-UTR; open box) of the long variant (LV) and short variant (SV). The start codon of the LV is located within its alternatively spliced exon 1 (the ATG codon is indicated by a bar on top of the text, and a solid right arrow). The start codon of SV is located within exon 2 (the ATG codon is indicated by a bar on top of the text, and a solid right arrow). Note that Exon 2 is shared by both LV and SV TRPML2 isoforms. The sequence map was generated using Lasergene 6 software (DNASTAR). (GIF 201 kb)

High resolution image (EPS 7627 kb)

Supplementary Fig. S3

Amino acid sequence alignment of the N-terminal regions of Human (Hs) TRPML2, Mouse (Mm) TRPML2lv, and Mouse (Mm) TRPML2sv proteins. The diagram shows that the N-termini of both HsTRPML2 and MmTRPML2lv proteins are similar in length, albeit divergent in amino acid sequence. In comparison with HsTRPML2 and MmTRPML2lv amino acids, the MmTRPML2sv protein does not have the twenty-eight amino acid sequence in its N-terminus. The Clustal W sequence alignment was generated using Lasergene 6 software (DNASTAR). (GIF 34 kb)

High-resolution image (EPS 162 kb)

Supplementary Fig. S4

Real-time QPCR analysis illustrating the specificity of the shRNA effect upon HsTRPML1 knockdown. HsTRPML1, HsTRPML2, and HsTRPML3 transcripts were analyzed upon co-expression of pCMV-HsTRPML1, pCMV-HsTRPML2, or pCMV-HsTRPML3 vectors (2 μg) respectively, with differing amounts of shRNA-1208 (4 μg and 8 μg). Co-expression of pCMV-HsTRPML1 vector upon RNAi knock down of HsTRPML1 resulted in dramatic reduction of over-expressed HsTRPML1 transcript levels. In contrast, co-expression of pCMV-HsTRPML2 vector upon RNAi knock down of HsTRPML1 with shRNA-1208 did not have a significant effect on over-expressed HsTRPML2 transcript levels, indicating that the shRNA specifically targeted the HsTRPML1 transcripts. This was also evident with pCMV-HsTRPML3 co-expression, since the over-expressed HsTRPML3 transcript levels did not significantly change. The cells were transfected, processed, and analyzed as described in the Materials and Methods section. Data are represented as means ± SEM, N = 3 independent trials. (GIF 72 kb)

High-resolution image (EPS 367 kb)

Supplementary Table S1

Standard PCR and real-time QPCR primers for TRPML and housekeeping genes used in the study (DOC 143 kb)

Supplementary Table S2

RNAi oligonucleotide sequences targeting human TRPML1 gene. (DOC 47 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Samie, M.A., Grimm, C., Evans, J.A. et al. The tissue-specific expression of TRPML2 (MCOLN-2) gene is influenced by the presence of TRPML1. Pflugers Arch - Eur J Physiol 459, 79–91 (2009). https://doi.org/10.1007/s00424-009-0716-5

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00424-009-0716-5

Keywords

Search

Navigation