Drosophila TRPML forms PI(3,5)P2-activated cation channels in both endolysosomes and plasma membrane

Abstract

Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca(2+) and Fe(2+) release, lysosomal biogenesis, and autophagy. Mutations in human TRPML1 cause the lysosome storage disease, mucolipidosis type IV (MLIV). Unlike vertebrates, which express three TRPML genes, TRPML1-3, the Drosophila genome encodes a single trpml gene. Although the trpml-deficient flies exhibit cellular defects similar to those in mammalian TRPML1 mutants, the biophysical properties of Drosophila TRPML channel remained uncharacterized. Here, we show that transgenic expression of human TRPML1 in the neurons of Drosophila trpml mutants partially suppressed the pupal lethality phenotype. When expressed in HEK293 cells, Drosophila TRPML was localized in both endolysosomes and plasma membrane and was activated by phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) applied to the cytoplasmic side in whole lysosomes and inside-out patches excised from plasma membrane. The PI(3,5)P2-evoked currents were blocked by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), but not other phosphoinositides. Using TRPML A487P, which mimics the varitint-waddler (Va) mutant of mouse TRPML3 with constitutive whole-cell currents, we show that TRPML is biphasically regulated by extracytosolic pH, with an optimal pH about 0.6 pH unit higher than that of human TRPML1. In addition to monovalent cations, TRPML exhibits high permeability to Ca(2+), Mn(2+), and Fe(2+), but not Fe(3+). The TRPML currents were inhibited by trivalent cations Fe(3+), La(3+), and Gd(3+). These features resemble more closely to mammalian TRPML1 than TRPML2 and TRPML3, but with some obvious differences. Together, our data support the use of Drosophila for assessing functional significance of TRPML1 in cell physiology.

Keywords: Endosomes; Lysosomal Storage Disease; Lysosomes; Mucolipidosis; Mucolipin; Phosphoinositides; TRP Channels.

FIGURE 1.

Partial rescue of pupal lethality of Drosophila trpml¹ mutant by neuronal expression of human TRPML1. A, immunohistochemical staining by anti-HA antibody of larval fat-bodies from transgenic flies with UAS-hTRPML1;HA expression driven by the cg-GAL4 driver (upper panels) and control flies in which the UAS-htrpml::HA transgene was not expressed (lower panels). Confocal fluorescence images show LysoTracker Red DND-99 (violet) and Alexa Fluor 488 (green) stained acidic organelles and hTRPML1::HA, respectively. Scale bar, 10 μm and is the same for all panels. B, images of eclosed pupal case and uneclosed (dead) pupa. C, pupal lethality of trpml¹ mutant without and with pan-neuronal expression of UAS-hTRPML1;HA driven by elav-GAL4. n = 4 independent crosses. *, p < 0.001 versus trpml¹.

FIGURE 2.

Subcellular distribution of Drosophila TRPML expressed in HEK293 cells and PI(3,5)P₂-elicited current in whole-lysosome patches. A, confocal fluorescence images of TRPML-EGFP (green) and Lamp1-mCherry (red) co-transfected into HEK293 cells. Merged image indicates partial colocalization (yellow) between TRPML and Lamp1. Cells were fixed and mounted before sequential images were taken. The arrow indicates the presence of TRPML in the perimeter of the cell. Scale bar, 10 μm. B, DIC and epifluorescence images of TRPML-EGFP transfected HEK293 cells treated with vacuolin-1 (1 μm, overnight). Merged image shows presence of TRPML-EGFP in the periphery of enlarged vacuoles. Scale bar, 5 μm. C, whole-lysosome currents in HEK293 cells that expressed TRPML. Cells were treated with vacuolin-1 overnight and enlarged vacuoles were released by slicing the cell with a sharp glass pipette. The vacuole was held at 0 mV in inside-out mode for whole lysosome and currents elicited by voltage ramps from −100 mV to +100 mV in 200 ms at 1-s intervals. Shown are time courses of currents at −90 and +90 mV for a representative vacuole treated with increasing concentrations of PI(3,5)P₂ (diC8) as indicated. The dashed line indicates zero current. W/L, whole-lysosome. D, current-voltage (I-V) curves obtained by voltage ramps under conditions as indicated for the same vacuole shown in C. Similar results were obtained from two other whole-lysosome patches.

FIGURE 3.

PI(3,5)P₂-evoked current in inside-out patches excised from plasma membrane of HEK293 cells expressing Drosophila TRPML. A, representative time courses of currents at −100 mV and +100 mV recorded using voltage ramps from an inside-out (I/O) patch excised from a TRPML-transfected cell. diC8 PI(3,5)P₂ (1 μm) was bath applied multiple times as indicated to the cytoplasmic side. Note the time-dependent run-up of the PI(3,5)P₂-elicited currents. B, I-V curves obtained by voltage ramps at the indicated time points in A. C, representative current traces for the same patch as shown in A elicited by voltage steps (−100 to +90 mV, with 10 mV increments, a step duration of 1 s and an interval of 2 s between consecutive steps) from the holding potential of 0 mV in the absence (upper) and presence (lower) of 1 μm diC8 PI(3,5)P₂. D, concentration dependence of diC8 PI(3,5)P₂-evoked currents from inside-out patches. Currents were measured using the similar protocol as in A, with varying concentrations of diC8 PI(3,5)P₂ after the currents elicited by 1 μm diC8 PI(3,5)P₂ had stabilized. Current amplitudes at −100 mV were normalized to that elicited by 1 μm diC8 PI(3,5)P₂. Data points (means ± S.E., n = 7) were fitted with the Hill equation, which yielded an EC₅₀ of 0.39 ± 0.07 μm. E, similar to A, but with consecutive application of diC8 PI(3,5)P₂ and diC16 PI(3,5)P₂ (both at 3 μm) as indicated. Note the slow rise of current elicited by diC16 PI(3,5)P₂. F, I-V curves obtained by voltage ramps at the indicated time points in E.

FIGURE 4.

Inhibition of TRPML by PI(4,5)P₂. A, representative time courses of currents at −100 mV and +100 mV recorded using voltage ramps from an inside-out (I/O) patch excised from a TRPML-transfected cell. PI(3,5)P₂ or PI(4,5)P₂ (both diC8 and at 1 μm) was bath applied to the cytoplasmic side as indicated. B, summary data (means ± S.E.) for currents at −100 mV under basal (no phosphoinositide applied), PI(3,5)P₂- and PI(4,5)P₂-treated conditions. C, inhibition of PI(3,5)P₂-elicited currents by PI(4,5)P₂. Similar to A, but with different concentrations of PI(4,5)P₂ applied while the patch was exposed to 0.3 μm PI(3,5)P₂, demonstrating concentration-dependent inhibition of the PI(3,5)P₂-elicited currents by PI(4,5)P₂. D, I-V curves obtained by voltage ramps at the indicated time points in C. E, summary (n = 4) of currents at −100 mV normalized to that activated by 0.3 μm PI(3,5)P₂. Data points were fitted with the a dose response equation: y = 1/(1 + 10^(Log(IC₅₀)-x)ⁿ). F, summary (means ± S.E.) of the effects of indicated phosphoinositides on PI(3,5)P₂-evoked TRPML current in inside-out patches. Phosphoinositides were used at 3 μm and PI(3,5)P₂ was at 0.3 μm. Current amplitudes at −100 mV were normalized to that evoked by PI(3,5)P₂ alone. *, p < 0.01 versus PI(3,5)P₂ alone.

FIGURE 5.

Whole-cell currents of Drosophila TRPML^Va expressed in HEK293 cells and its dependence on extracytosolic pH. A, alignment of putative transmembrane segment 5 of Drosophila TRPML with that of mouse TRPML1, TRPML2, and TRPML3. Names in parentheses are Gene Product ID in UniProtKB database. The amino acids mutated in the Va mutants are boxed and shaded in gray. The asterisk indicates Ala-487 of Drosophila TRPML, which was replaced with a proline in TRPML^Va. B, summary (means ± S.E.) of current amplitudes at −100 mV from whole-cell recordings of basal activity in normal Tyrode's bath solution by voltage ramps in HEK293 cells expressing TRPML^Va or untransfected cells. C, representative I-V curves obtained by voltage ramps of whole-cell recording of an HEK293 cell expressing TRPML^Va in normal Tyrode's bath solution (basal), NMDG bath, and pH 4.6 Tyrode's bath. W/C, whole-cell. D, representative traces of whole-cell currents elicited by voltage steps (−80 mV to +80 mV, with 10-mV increments and a step duration of 400 ms) from the holding potential of 0 mV in an HEK293 cell expressing TRPML^Va in normal Tyrode's (basal, left), NMDG (middle), and pH 4.6 Tyrode's (right) bath solutions. E, representative time courses of currents at −100 mV and +100 mV recorded using voltage ramps by whole-cell recording of an HEK293 cell expressing TRPML^Va. Constitutive basal currents were seen in normal Tyrode's bath. NMDG and Tyrode's solutions with different pH were applied as indicated through perfusion. F, I-V curves obtained by voltage ramps for selected conditions shown in E. G, summary of pH-dependent modifications of TRPML^Va and human TRPML1^Va (hTRPML1^Va). Currents at −100 mV were normalized to that recorded in pH 5.0 Tyrode's bath. Data points (means ± S.E., n = 5 for each) were fitted with Gaussian function.

FIGURE 6.

Cation permeability of Drosophila TRPML^Va. A, representative time courses of currents at −100 mV and +100 mV recorded using voltage ramps by whole-cell recording of an HEK293 cell expressing TRPML^Va. Constitutive basal currents were seen in normal Tyrode's bath. Divalent-free (DVF) isotonic NMDG, K⁺, Na⁺, and Cs⁺ solutions, as well as an isotonic Ca²⁺ solution, were applied as indicated. B, I-V curves obtained by voltage ramps in different extracellular solutions as indicated, for the same cell as in A. C, I-V curves of whole-cell currents obtained by voltage ramps for another cell exposed to normal Tyrode's, DVF isotonic Na⁺ and NMDG, and Fe²⁺ (50 mm) solutions. D, summary (means ± S.E.) of current amplitudes at −100 mV in DVF isotonic K⁺ and Cs⁺, isotonic Ca²⁺ and Mn²⁺, and 50 mm Fe²⁺ normalized to that in DVF isotonic Na⁺. E, I-V curves of whole-cell currents obtained by voltage ramps for an HEK293 cell expressing TRPML^Va exposed sequentially to normal Tyrode's, NMDG, 50 mm Fe²⁺, 35 mm Fe³⁺ solutions. The current recovered after washout in 50 mm Fe²⁺. pH was 4.6 for all solutions except for normal Tyrode's and NMDG solutions, which had pH of 7.4.

FIGURE 7.

Inhibition of TRPML^Va current by trivalent cations. A, representative time courses of currents at −100 mV and +100 mV recorded using voltage ramps by whole-cell recording of an HEK293 cell expressing TRPML^Va. The cell was exposed to normal Tyrode's NMDG, pH 4.6 Tyrode's, and pH 4.6 Tyrode's solution containing 1 and 10 mm FeCl₃ as indicated, showing concentration dependent inhibition by Fe³⁺. B, I-V curves obtained by voltage ramps under conditions shown in A. C and D, similar to A and B, but pH 7.2 Tyrode's solution was used, and 1 mm LaCl₃ and 1 mm GdCl₃ in the pH 7.2 Tyrode's were applied as shown. E and F, I-V curves obtained by voltage ramps from whole-cell recording of two different TRPML^Va-transfected cells exposed to pH 4.6 Tyrode's solution without or with 1 mm LaCl₃ (E), or GdCl₃ (F). G, summary data (means ± S.E.) for % inhibition by La³⁺ and Gd³⁺ (both at 1 mm) of whole-cell currents at −100 mV in HEK293 cells that expressed either TRPML^Va or hTRPML1^Va. The extracellular solution had pH either at 4.6 or 7.2 as indicated.