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

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.

Fig. 1

Real-time QPCR analyses of mouse (Mm) TRPML2lv and TRPML2sv from organs and tissues of wild type (WT) mice. The transcript ratio of TRPML2lv were very low compared with TRPML2sv. The transcript ratio of TRPML2sv showed a tissue-specific expression pattern with normalized values markedly higher in lymphoid (thymus and spleen) and kidney organs followed by heart, lung, liver, and stomach. Data are represented as means ± SEM, N=6 wild-type mice. The samples were processed and analyzed as described in the Materials and methods section

Fig. 2

Constitutive channel activity of TRPML varitint-waddler (Va) mutant proteins. a Sequence alignment (clustal W) of the inner half of the TM5 domain of the three TRPML proteins. Conserved amino-acid residues are highlighted in black. Arrow indicates the position of the proline substitution from the varitint-waddler mutation A419P in TRPML3 and equivalent positions for TRPML1 and TRPML2 proteins. b Ca²⁺-imaging experiments showing intracellular Ca²⁺ levels of HEK-293 cells expressing either wild-type TRPML1, TRPML2lv (long variant), TRPML2sv (short variant), or TRPML3, and TRPML-Va mutant versions TRPML1-V432P, TRPML2lvA424P, TRPML2sv-A396P, and TRPML3-A419P. All experiments were performed 15-20 h after transfection due to cytotoxic effect of the mutation. Data are represented as means ± SEM, N=number in parenthesis. c Steady-state current-voltage plots of constitutively active whole-cell currents elicited by TRPML1-V432P, TRPML2svA396P, TRPML2lv-A424P, and TRPML3-A419P mutant proteins compared with their respective wild-type versions in response to 10 ms voltage steps from a holding potential of +10 mV between −200 mV and +100 mV in 20 mV incremental steps and normalized by cell capacitance (pF). All TRPML-Va mutants showed inward rectification while wild-type TRPMLs did not elicit any response. d Average inward current densities at −80 mV of all TRPML-Va mutant and wild-type proteins shown in panel c and normalized by pF. The TRPML3-Va mutation showed higher current density compared with the other TRPML-Va mutants. Data are represented as means ±SEM, N=numbers in parenthesis. All experimental procedures are outlined in the Materials and methods section

Fig. 3

Real-time QPCR analyses of a TRPML2lv, b TRPML2sv, and c TRPML3 mRNA expression levels from various organs and tissues taken from TRPML1−/− null mice and their wild-type littermates. In a, TRPML2lv transcript levels consistently showed low transcript ratio across all samples obtained from both TRPML1−/− mice and wild-type littermates. In b, TRPML2sv levels exhibited a predominantly tissue-specific expression in wild-type but were significantly reduced in lymphoid and kidney organs of TRPML1−/− mice. In c, TRPML3 transcripts showed a similar tissue-specific distribution pattern, although no significant changes were observed in TRPML3 transcript levels between TRPML1−/− mice and wild-type littermates. Real-time QPCR analyses were performed as described in the Materials and methods section. Data are represented as means ±SEM, N=6 TRPML1−/− mice, and N=6 wild-type mice. *p<0.005, Student's t-test, two-tailed

Fig. 3

Real-time QPCR analyses of a TRPML2lv, b TRPML2sv, and c TRPML3 mRNA expression levels from various organs and tissues taken from TRPML1−/− null mice and their wild-type littermates. In a, TRPML2lv transcript levels consistently showed low transcript ratio across all samples obtained from both TRPML1−/− mice and wild-type littermates. In b, TRPML2sv levels exhibited a predominantly tissue-specific expression in wild-type but were significantly reduced in lymphoid and kidney organs of TRPML1−/− mice. In c, TRPML3 transcripts showed a similar tissue-specific distribution pattern, although no significant changes were observed in TRPML3 transcript levels between TRPML1−/− mice and wild-type littermates. Real-time QPCR analyses were performed as described in the Materials and methods section. Data are represented as means ±SEM, N=6 TRPML1−/− mice, and N=6 wild-type mice. *p<0.005, Student's t-test, two-tailed

Fig. 4

RNAi-mediated knockdown of endogenous HsTRPML1 and subsequent rescue of endogenous HsTRPML2 mRNA expression levels. a Real-time QPCR analyses of control (untreated) and RNAi-treated samples. Endogenous HsTRPML1 expression levels were markedly reduced upon shRNA-1208 treatment. A concomitant decrease of HsTRPML2 transcripts was also observed but not with HsTRPML3 transcripts. Data are represented as means ±SEM, N =3 independent trials. *p< 0.05, Student's t test, two-tailed. b Real-time QPCR of endogenous HsTRPML2 transcript levels upon coexpression of pCMV HsTRPML1 with varying concentrations of shRNA-1208 (4μg and 8μg). The decrease of endogenous HsTRPML2 levels upon RNAi treatment was rescued by overexpressing a pCMV-HsTRPML1 construct. Data are represented as means ± SEM, N=3 independent trials. Real-time QPCR reactions were performed as described in the Materials and methods section

Fig. 5

Effects of H89 and NAADP on TRPML2sv expression in primary mouse lymphoid (splenocyte) cells. The transcript ratios of mouse TRPML2sv (MmTRPML2sv) following incubation with H89 (10μM) or NAADP (1μM) were significantly increased when compared with untreated controls. No effects were observed upon analyses of mouse TRPML1 (MmTRPML1) and TRPML3 (MmTRPML3) expression levels upon treatment of the same compounds. Data are represented as means ± SEM, N=5 independent trials. *p<0.05 , Student's t test, two-tailed. Real-time QPCR reaction was performed as described in the Materials and methods section