Pflügers Archiv - European Journal of Physiology

, Volume 459, Issue 1, pp 79–91

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

  • Mohammad A. Samie
  • Christian Grimm
  • Jeffrey A. Evans
  • Cyntia Curcio-Morelli
  • Stefan Heller
  • Susan A. Slaugenhaupt
  • Math P. Cuajungco
Ion Channels, Receptors and Transporters

DOI: 10.1007/s00424-009-0716-5

Cite this article as:
Samie, M.A., Grimm, C., Evans, J.A. et al. Pflugers Arch - Eur J Physiol (2009) 459: 79. doi:10.1007/s00424-009-0716-5

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.

Keywords

TRP ion channel TRPML2 Mucolipidosis type IV Mucolipin-2 Lysosomal storage disorder TRPML1 knock out mouse 

Supplementary material

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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)

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High resolution image (EPS 1428 kb)
424_2009_716_Fig2_ESM.gif (201 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)

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High resolution image (EPS 7627 kb)
424_2009_716_Fig3_ESM.gif (35 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)

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High-resolution image (EPS 162 kb)
424_2009_716_Fig4_ESM.gif (72 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)

424_2009_716_Fig4_ESM.eps (367 kb)
High-resolution image (EPS 367 kb)
424_2009_716_MOESM1_ESM.doc (144 kb)
Supplementary Table S1Standard PCR and real-time QPCR primers for TRPML and housekeeping genes used in the study (DOC 143 kb)
424_2009_716_MOESM2_ESM.doc (48 kb)
Supplementary Table S2RNAi oligonucleotide sequences targeting human TRPML1 gene. (DOC 47 kb)

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Mohammad A. Samie
    • 1
  • Christian Grimm
    • 2
  • Jeffrey A. Evans
    • 1
  • Cyntia Curcio-Morelli
    • 3
  • Stefan Heller
    • 2
  • Susan A. Slaugenhaupt
    • 3
  • Math P. Cuajungco
    • 1
    • 4
  1. 1.Department of Biological Science, and Center for Applied Biotechnology StudiesCalifornia State University FullertonFullertonUSA
  2. 2.Department of Otolaryngology, and Molecular and Cellular PhysiologyStanford University School of MedicineStanfordUSA
  3. 3.Center for Human Genetic ResearchMassachusetts General Hospital and Harvard Medical SchoolBostonUSA
  4. 4.Mental Health Research InstituteMelbourneAustralia

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