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. 2011 Jul 1;286(26):22934-42.
doi: 10.1074/jbc.M110.210930. Epub 2011 May 3.

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels

Soichiro Yamaguchi  1 , Archana Jha, Qin Li, Abigail A Soyombo, George D Dickinson, Dev Churamani, Eugen Brailoiu, Sandip Patel, Shmuel Muallem
Affiliations

Transient receptor potential mucolipin 1 (TRPML1) and two-pore channels are functionally independent organellar ion channels

Soichiro Yamaguchi et al. J Biol Chem. .

Abstract

NAADP is a potent second messenger that mobilizes Ca(2+) from acidic organelles such as endosomes and lysosomes. The molecular basis for Ca(2+) release by NAADP, however, is uncertain. TRP mucolipins (TRPMLs) and two-pore channels (TPCs) are Ca(2+)-permeable ion channels present within the endolysosomal system. Both have been proposed as targets for NAADP. In the present study, we probed possible physical and functional association of these ion channels. Exogenously expressed TRPML1 showed near complete colocalization with TPC2 and partial colocalization with TPC1. TRPML3 overlap with TPC2 was more modest. TRPML1 and to some extent TRPML3 co-immunoprecipitated with TPC2 but less so with TPC1. Current recording, however, showed that TPC1 and TPC2 did not affect the activity of wild-type TRPML1 or constitutively active TRPML1(V432P). N-terminally truncated TPC2 (TPC2delN), which is targeted to the plasma membrane, also failed to affect TRPML1 and TRPML1(V432P) channel function or TRPML1(V432P)-mediated Ca(2+) influx. Whereas overexpression of TPCs enhanced NAADP-mediated Ca(2+) signals, overexpression of TRPML1 did not, and the dominant negative TRPML1(D471K) was without affect on endogenous NAADP-mediated Ca(2+) signals. Furthermore, the single channel properties of NAADP-activated TPC2delN were not affected by TRPML1. Finally, NAADP-evoked Ca(2+) oscillations in pancreatic acinar cells were identical in wild-type and TRPML1(-/-) cells. We conclude that although TRPML1 and TPCs are present in the same complex, they function as two independent organellar ion channels and that TPCs, not TRPMLs, are the targets for NAADP.

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Figures

FIGURE 1.
FIGURE 1.
Localization of TRPMLs and TPCs. A and B, confocal images showing expression of mCherry-tagged TRPML1 (A) or TRPML3 (B) and GFP-tagged TPC1 (a) or TPC2 (b) in HeLa cells. Red and green images were acquired sequentially. C, the % overlaps (mean ± S.E.) were calculated as organelles expressing TRPML1 or TRPML3 relative to the total organelles expressing TPC1 or TPC2. In C, a–d summarize the combinations indicated in the figure.
FIGURE 2.
FIGURE 2.
co-IP of TRPMLs and TPCs. A–D, confocal images showing expression of mCherry-tagged TRPML1 (A and B) or TRPML3 (C and D) and GFP-tagged TPC1 (A and C) or TPC2 (B and D) in HEK293T. E and F, cells were transfected with empty vectors (controls), EGFP-tagged TRPML1, or TRPML3 and Myc-tagged TPC1 or TPC2. Cell lysates were immunoprecipitated with either anti-Myc (IP: myc shown in E) or anti-GFP (IP: GFP shown in F) and were probed with anti-GFP (E) or anti-Myc antibodies (F) to detect TRPML1/TRPML3 or TPC1/2, respectively. The migration of full length (ML1) and cleaved (*ML1) TRPML1 is highlighted. An overexposed version of the blots in F is provided in supplemental Fig. 2A to show the modest co-IP of TPC1 with TRPML1 and TRPML3.
FIGURE 3.
FIGURE 3.
TPC1 and TPC2 do not affect TRPML1 function. A and B, whole-cell current density evoked by ramp pulse protocol from −100 mV to 100 mV is shown at 10-mV intervals. HEK293T cells transfected with the indicated channels were perfused with divalent cation free NaCl-rich solution. A, cells were transfected with wild-type TRPML1 alone (●) or with TPC1 (△) or TPC2 (▿). B, cells were transfected with TRPML1(V432P) alone (●) or with TPC1 (△), TPC2 (▿), or the N-terminal truncated TPC2 (TPC2delN, ■). A (n = 3–7) and B (n = 7–8) show the mean ± S.E. C, the Ca2+ influx (340/380 ratio) elicited in cells expressing the indicated channels and bathed in Ca2+-free media when exposed to a solution containing 2 mm Ca2+. The difference of the mean between the group of TRPML1 and the other conditions were analyzed by Student's t test or Welch's test. *, p < 0.05; **, p < 0.01. ML1VP, TRPML1(V432P); ML1, TRPML1; ML1delNC, TRPML1 mutant with truncated N and C termini; TPC2delN, TPC2 mutant with truncated N terminus-targeting motif.
FIGURE 4.
FIGURE 4.
TRPML1 does not affect NAADP-mediated Ca2+ signals in SKBR3 cells. A and B, SKBR3 cells were transfected with TRPML1 (red) and either TPC1 (A) or TPC2 (B) to show the partial (A) and extensive (B) co-localization of the channels. C–F, cytosolic Ca2+ responses of individual Fura-2 loaded SKBR3 cells stimulated with NAADP. C, responses of mock-transfected cells or cells expressing TRPML1 upon microinjection of 10 nm NAADP (pipette concentration). The inset shows the response of cells expressing TPC2. D and E, responses of mock-transfected cells or cells expressing TRPML1, TPC1, or TPC2 and stimulated with a cell-permeable NAADP analog (NAADP-AM, 1 μm). 20 randomly selected cells from a typical field of view (∼50 cells) are shown in each panel. F, responses of mock-transfected cells or cells expressing the channel dead mutant TRPML1(D471K) upon microinjection of 10 μm NAADP (pipette concentration). Summary data are presented as mean ± S.E. Where appropriate, only cells that expressed the protein of interest as judged by fluorescence of fused GFP were injected with NAADP.
FIGURE 5.
FIGURE 5.
TRPML1 does not affect NAADP-mediated channel activity of TPC2. Single channel currents are from excised inside-out patches from the plasma membrane of HEK cells expressing GFP (A), TRPML1delNC (B), TPC2delN (C), or TPC2delN+TRPML1delNC (D) at a holding potential of −60 mV. Where indicated, the bath solution was supplemented with 500 nm NAADP. Traces of single or multiple channel openings are shown with expanded time course for the periods marked with 1 and 2 in the main traces. The whole-point histograms show a main channel conductance of 5 pA of TPC2 in the presence and absence of TRPML1. The bar graphs show the average TPC2 Cs+ conductance (E) and NPo (F) in the presence and absence of TRPML1. Results are means ± S.E. with the number of experiments (n) shown above each bar. NPo were calculated at a −40 mV holding potential.
FIGURE 6.
FIGURE 6.
Knock-out of TRPML1 has no effect on NAADP responses in pancreatic acinar cells. Pancreatic acinar cells were acutely isolated from the pancreas of wild type (A) or TRPML1−/− mice (B). Whole-cell current was measured at a holding membrane potential of −60 mV. NAADP response was initiated by including 50 nm NAADP in the pipette solution. As controls, the cells were stimulated with 0.1 mm carbachol at the end of each experiment. Similar results were observed in 13 control and six TRPML1−/− cells from three wild-type and two TRPML1−/− animals. In C, the amplitude of the current oscillations were averaged within each experiment and then among experiments, and D shows the average oscillation frequency. The results are the mean ± S.E., and there is no significant difference between wild-type and TRPML1−/− cells in either C or D.
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