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Identification of a single aspartate residue critical for both fast and slow calcium-dependent inactivation of the human TRPML1 channel

  • Guangyan Wu
    Affiliations
    State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China
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  • Xue Yang
    Affiliations
    State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China
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  • Yuequan Shen
    Correspondence
    To whom correspondence should be addressed. Tel.:86-22-85358120; Fax:86-22-85358120;.
    Affiliations
    State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai University, 94 Weijin Road, Tianjin 300071, China

    Synergetic Innovation Center of Chemical Science and Engineering, 94 Weijin Road, Tianjin 300071, China
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  • Author Footnotes
    2 The abbreviations used are: TRPtransient receptor potentialLELlate endosome and lysosomeFCDIfast calcium-dependent inactivationSCDIslow calcium-dependent inactivationHEKhuman embryonic kidneyCDIcalcium-dependent inactivationNMDGN-methyl-d-glucamineBAPTA1,2-bis(o-aminophenoxy)ethane-N,N,N',N′-tetraacetic acid.
Open AccessPublished:June 08, 2018DOI:https://doi.org/10.1074/jbc.RA118.003250
      Transient receptor potential mucolipin subfamily 1 (TRPML1) is a nonselective cation channel mainly located in late endosomes and lysosomes. Mutations of the gene encoding human TRPML1 can cause severe lysosomal diseases. The activity of TRPML1 is regulated by both Ca2+ and H+, which are important for its critical physiological functions in membrane trafficking, exocytosis, autophagy, and intracellular signal transduction. However, the molecular mechanism of its dual regulation by Ca2+ and H+ remains elusive. Here, using a mutant screening method in combination with a whole-cell patch clamp technique, we identified a key TRPML1 residue, Asp-472, responsible for both fast calcium-dependent inactivation (FCDI) and slow calcium-dependent inactivation (SCDI) as well as H+ regulation. We also found that, in acidic pH, H+ can significantly delay FCDI and abolish SCDI and thereby presumably facilitate the ion conductance of the human TRPML1 channel. In summary, we have identified a key residue critical for Ca2+-induced inhibition of TRPML1 channel currents and uncovered pH-dependent regulation of this channel, providing vital information regarding the detailed mechanism of action of human TRPML1.

      Introduction

      Lysosomes, derived from membrane-enclosed compartments of late endosomes, play a critical role in a wide range of physiological functions (
      • Xu H.
      • Ren D.
      Lysosomal physiology.
      ). They can degrade macromolecules and participate in intracellular signal transduction as well as in membrane trafficking (
      • Luzio J.P.
      • Pryor P.R.
      • Bright N.A.
      Lysosomes: fusion and function.
      ,
      • Cheng X.
      • Shen D.
      • Samie M.
      • Xu H.
      Mucolipins: intracellular TRPML1–3 channels.
      ). Consequently, lysosome dysfunction causes many diseases (
      • Parkinson-Lawrence E.J.
      • Shandala T.
      • Prodoehl M.
      • Plew R.
      • Borlace G.N.
      • Brooks D.A.
      Lysosomal storage disease: revealing lysosomal function and physiology.
      ,
      • Grimm C.
      • Butz E.
      • Chen C.C.
      • Wahl-Schott C.
      • Biel M.
      From mucolipidosis type IV to Ebola: TRPML and two-pore channels at the crossroads of endo-lysosomal trafficking and disease.
      ).
      The transient receptor potential mucolipin subfamily 1 (TRPML1) channel protein is an important regulator in many lysosome-dependent cellular physiological events (
      • Cheng X.
      • Shen D.
      • Samie M.
      • Xu H.
      Mucolipins: intracellular TRPML1–3 channels.
      ,
      • Di Paola S.
      • Scotto-Rosato A.
      • Medina D.L.
      TRPML1: The Ca2+ retaker of the lysosome.
      ,
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Cao Q.
      • Yang Y.
      • Zhong X.Z.
      • Dong X.-P.
      The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin.
      ,
      • Venkatachalam K.
      • Wong C.O.
      • Zhu M.X.
      The role of TRPMLs in endolysosomal trafficking and function.
      ,
      • Li X.
      • Rydzewski N.
      • Hider A.
      • Zhang X.
      • Yang J.
      • Wang W.
      • Gao Q.
      • Cheng X.
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      A molecular mechanism to regulate lysosome motility for lysosome positioning and tubulation.
      ). It is widely expressed in every tissue in mammals (
      • Cheng X.
      • Shen D.
      • Samie M.
      • Xu H.
      Mucolipins: intracellular TRPML1–3 channels.
      ). The gene encoding TRPML1 is MCOLN1, whose mutation causes a lysosomal storage disorder called mucolipidosis type IV (
      • Di Paola S.
      • Scotto-Rosato A.
      • Medina D.L.
      TRPML1: The Ca2+ retaker of the lysosome.
      ), which is an autosomal recessive genetic disease with the typical features of psychomotor retardation, corneal opacities, retinal degeneration, strabismus, elevated blood gastrin levels, and achlorhydria (
      • Bargal R.
      • Avidan N.
      • Ben-Asher E.
      • Olender Z.
      • Zeigler M.
      • Frumkin A.
      • Raas-Rothschild A.
      • Glusman G.
      • Lancet D.
      • Bach G.
      Identification of the gene causing mucolipidosis type IV.
      ,
      • Sun M.
      • Goldin E.
      • Stahl S.
      • Falardeau J.L.
      • Kennedy J.C.
      • Acierno Jr., J.S.
      • Bove C.
      • Kaneski C.R.
      • Nagle J.
      • Bromley M.C.
      • Colman M.
      • Schiffmann R.
      • Slaugenhaupt S.A.
      Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel.
      ,
      • Lubensky I.A.
      • Schiffmann R.
      • Goldin E.
      • Tsokos M.
      Lysosomal inclusions in gastric parietal cells in mucolipidosis type IV: a novel cause of achlorhydria and hypergastrinemia.
      ).
      Similar to other TRP
      The abbreviations used are: TRP
      transient receptor potential
      LEL
      late endosome and lysosome
      FCDI
      fast calcium-dependent inactivation
      SCDI
      slow calcium-dependent inactivation
      HEK
      human embryonic kidney
      CDI
      calcium-dependent inactivation
      NMDG
      N-methyl-d-glucamine
      BAPTA
      1,2-bis(o-aminophenoxy)ethane-N,N,N',N′-tetraacetic acid.
      channels, TRPML1 is a nonselective cation channel (
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ) with permeability to Na+, K+, Ca2+, and Fe2+ but not H+. It typically shows inwardly rectifying whole-cell or whole-lysosome currents (
      • Xu H.
      • Delling M.
      • Li L.
      • Dong X.
      • Clapham D.E.
      Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice.
      ,
      • Dong X.P.
      • Cheng X.
      • Mills E.
      • Delling M.
      • Wang F.
      • Kurz T.
      • Xu H.
      The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.
      ). TRPML1 is mainly located in the membranes of late endosome and lysosome (LEL) organelle compartments (
      • Pryor P.R.
      • Reimann F.
      • Gribble F.M.
      • Luzio J.P.
      Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic.
      ,
      • Manzoni M.
      • Monti E.
      • Bresciani R.
      • Bozzato A.
      • Barlati S.
      • Bassi M.T.
      • Borsani G.
      Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization.
      ). These LEL compartments are filled with high concentrations of ions, such as Ca2+ (
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Christensen K.A.
      • Myers J.T.
      • Swanson J.A.
      pH-dependent regulation of lysosomal calcium in macrophages.
      ). Another feature of both late endosomes and lysosomes is the low luminal pH established by vacuolar-type H+-ATPase (v-ATPase), resulting in a pH of ∼ 5–6 in late endosomes and ∼ 4–5 in lysosomes (
      • Luzio J.P.
      • Pryor P.R.
      • Bright N.A.
      Lysosomes: fusion and function.
      ,
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Schwake M.
      • Schröder B.
      • Saftig P.
      Lysosomal membrane proteins and their central role in physiology.
      ,
      • Appelqvist H.
      • Wäster P.
      • Kågedal K.
      • Öllinger K.
      The lysosome: from waste bag to potential therapeutic target.
      ,
      • Mindell J.A.
      Lysosomal acidification mechanisms.
      ). Because of the special resident environment associated with both high Ca2+ and H+, TRPML1 channel activity is regulated by both Ca2+ and H+ (
      • Cheng X.
      • Shen D.
      • Samie M.
      • Xu H.
      Mucolipins: intracellular TRPML1–3 channels.
      ,
      • Di Paola S.
      • Scotto-Rosato A.
      • Medina D.L.
      TRPML1: The Ca2+ retaker of the lysosome.
      ,
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Venkatachalam K.
      • Wong C.O.
      • Zhu M.X.
      The role of TRPMLs in endolysosomal trafficking and function.
      ,
      • Li M.
      • Zhang W.K.
      • Benvin N.M.
      • Zhou X.
      • Su D.
      • Li H.
      • Wang S.
      • Michailidis I.E.
      • Tong L.
      • Li X.
      • Yang J.
      Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel.
      ). However, the molecular mechanism of TRPML1 channel regulation by Ca2+ and H+ remains elusive. In this study, using site-directed mutagenesis in combination with a whole-cell electrophysiological patch clamp technique, we identified a key aspartic acid residue that determines both FCDI and SCDI of the human TRPML1 channel in environments with different pH values.

      Results

      Ca2+ inhibits TRPML1 currents

      TRPML1 is mainly located in LEL membranes (
      • Pryor P.R.
      • Reimann F.
      • Gribble F.M.
      • Luzio J.P.
      Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic.
      ,
      • Manzoni M.
      • Monti E.
      • Bresciani R.
      • Bozzato A.
      • Barlati S.
      • Bassi M.T.
      • Borsani G.
      Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization.
      ). Therefore, it is a challenge to perform direct LEL patch clamp experiments to record WT TRPML1 currents. We instead conducted whole-cell patch clamp experiments as described previously (
      • Xu H.
      • Delling M.
      • Li L.
      • Dong X.
      • Clapham D.E.
      Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice.
      ,
      • Dong X.P.
      • Cheng X.
      • Mills E.
      • Delling M.
      • Wang F.
      • Kurz T.
      • Xu H.
      The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.
      ). Briefly, an amino acid substitution (V432P) causes the TRPML1 channel to be widely expressed on cell plasma membranes. The acquisition of TRPML1-V432P currents was proven to show the same electrophysiological properties as that of WT TRPML1. For convenience, hereafter we call the TRPML1-V432P mutant TRPML1Va (
      • Xu H.
      • Delling M.
      • Li L.
      • Dong X.
      • Clapham D.E.
      Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice.
      ,
      • Dong X.P.
      • Cheng X.
      • Mills E.
      • Delling M.
      • Wang F.
      • Kurz T.
      • Xu H.
      The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.
      ).
      It has been reported that the whole-cell currents of TRPML1Va are inhibited by Ca2+ (
      • Dong X.P.
      • Cheng X.
      • Mills E.
      • Delling M.
      • Wang F.
      • Kurz T.
      • Xu H.
      The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.
      ). To verify this result, we recorded TRPML1Va currents in the presence or absence of Ca2+ under weakly basic conditions (pH 7.4). Our results showed that Ca2+ inhibited the monovalent cation currents of the TRPML1 channel with an IC50 of 0.7 mm (Fig. 1, A and B, and Fig. S1A). It was proposed that three aspartic acid residues, Asp-111, Asp-114, and Asp-115, located between transmembrane 1 and transmembrane 2, might be responsible for Ca2+-induced inhibition of TRPML1 currents (
      • Li M.
      • Zhang W.K.
      • Benvin N.M.
      • Zhou X.
      • Su D.
      • Li H.
      • Wang S.
      • Michailidis I.E.
      • Tong L.
      • Li X.
      • Yang J.
      Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel.
      ). Therefore, we generated the TRPML1Va (3DQ) mutant, in which Asp-111, Asp-114, and Asp-115 were mutated to glutamines, to record the whole-cell currents before and after addition of 2 mm Ca2+. Our results showed that Ca2+-induced inhibition still occurred in cells expressing the TRPML1Va (3DQ) mutant (Fig. 1C and Fig. S1B), indicating that other candidate residues are crucial for Ca2+-induced inhibition of the TRPML1 channel.
      Figure thumbnail gr1
      Figure 1Ca2+-induced inhibition of TRPML1Va currents. A, Ca2+ concentration-dependent inhibition curves of TRPML1Va currents with IC50 values of 0.7 mm Ca2+ at pH 7.4 and 3.33 mm Ca2+ at pH 4.6. The curve was fit with Origin 9.0 software. Data points are representative of 4–6 independent experiments. pF, picofarad. B, Ca2+-induced inhibition of TRPML1Va WT currents at pH 7.4. Shown are current–voltage plots (left panel) for TRPML1Va and time course (right panel) for normalized TRPML1Va currents. C, Ca2+-induced inhibition of mutant TRPML1Va (3DQ) currents at pH 7.4. Shown are current–voltage plots (left panel) for TRPML1Va (3DQ) and time course (right panel) for normalized TRPML1Va (3DQ) currents. The normalization methods for all currents (including those shown below) are described under “Experimental procedures.” For all figures, numbers in brackets indicate the number of tested cells. Data points are shown as the mean ± S.E.

      Asp-472 is responsible for Ca2+-induced inhibition of the TRPML1 channel

      TRP channel members generally share sequence conservation in their transmembrane domains (
      • Sun M.
      • Goldin E.
      • Stahl S.
      • Falardeau J.L.
      • Kennedy J.C.
      • Acierno Jr., J.S.
      • Bove C.
      • Kaneski C.R.
      • Nagle J.
      • Bromley M.C.
      • Colman M.
      • Schiffmann R.
      • Slaugenhaupt S.A.
      Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel.
      ,
      • Minke B.
      • Cook B.
      TRP channel proteins and signal transduction.
      ). Thus, we explored whether residues vital for Ca2+-induced inhibition were located in the TRPML1 pore region. Sequence alignment of the pore-forming regions from several TRP channel members, including human TRPML1, human TRPP2, human TRPV1, human TRPA1, and rat TRPV1 (for which structural information is available) (
      • Schmiege P.
      • Fine M.
      • Blobel G.
      • Li X.
      Human TRPML1 channel structures in open and closed conformations.
      ,
      • Grieben M.
      • Pike A.C.
      • Shintre C.A.
      • Venturi E.
      • El-Ajouz S.
      • Tessitore A.
      • Shrestha L.
      • Mukhopadhyay S.
      • Mahajan P.
      • Chalk R.
      • Burgess-Brown N.A.
      • Sitsapesan R.
      • Huiskonen J.T.
      • Carpenter E.P.
      Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).
      ,
      • Paulsen C.E.
      • Armache J.-P.
      • Gao Y.
      • Cheng Y.
      • Julius D.
      Structure of the TRPA1 ion channel suggests regulatory mechanisms.
      ,
      • Cao E.
      • Liao M.
      • Cheng Y.
      • Julius D.
      TRPV1 structures in distinct conformations reveal activation mechanisms.
      ,
      • Liao M.
      • Cao E.
      • Julius D.
      • Cheng Y.
      Structure of the TRPV1 ion channel determined by electron cryo-microscopy.
      ,
      • Wilkes M.
      • Madej M.G.
      • Kreuter L.
      • Rhinow D.
      • Heinz V.
      • De Sanctis S.
      • Ruppel S.
      • Richter R.M.
      • Joos F.
      • Grieben M.
      • Pike A.C.
      • Huiskonen J.T.
      • Carpenter E.P.
      • Kühlbrandt W.
      • Witzgall R.
      • Ziegler C.
      Molecular insights into lipid-assisted Ca2+ regulation of the TRP channel Polycystin-2.
      ,
      • Shen P.S.
      • Yang X.
      • DeCaen P.G.
      • Liu X.
      • Bulkley D.
      • Clapham D.E.
      • Cao E.
      The structure of the polycystic kidney disease channel PKD2 in lipid nanodiscs.
      ), showed that this region is highly conserved (Fig. 2A). Therefore, we performed whole-cell patch clamp experiments before and after addition of 2 mm Ca2+ in HEK293T cells expressing each human TRPML1Va mutant (single amino acid mutation from the asparagine (Asn-469) close to the selective filter to the phenylalanine (Phe-477) above the selective filter) at pH 7.4. The mutants N469L, G470A, D471Q, D472Q, M473A, F474L, V475A, T476A, and F477L were generated using a site-direct mutagenesis methodology. As shown in Fig. 2, B–J, and Fig. S2, A–I, all mutants showed Ca2+-induced inhibition properties after addition of Ca2+, except for the TRPML1Va (D472Q) mutant, which displayed significantly increased whole-cell currents (Fig. 2E and Fig. S2D). Then we examined the dose-dependent effects of Ca2+ on TRPML1Va (D472Q) mutant currents, showing significant current potentiation in a Ca2+ concentration–dependent manner (Fig. 3A). This result is in sharp contrast with Ca2+ inhibition of the TRPML1Va currents (Fig. 1A). We further utilized NMDG/Ca2+-containing solutions to test concentration-dependent effects, which also showed an obvious current potentiation phenomenon (Fig. 3B). Consequently, Asp-472 may play a key role in Ca2+-induced inhibition of TRPML1Va currents.
      Figure thumbnail gr2
      Figure 2The Asp-472 residue is important for Ca2+-induced inhibition of the TRPML1 channel. A, amino acid sequence alignment of pore regions from human TRPML1, human TRPP2, human TRPA1, human TRPV1, and rat TRPV1. MCLN1 represents TRPML1 for clarity. Highly conserved residues are colored red. B–J, current–voltage plots and corresponding time courses for normalized currents of the TRPML1Va (N469L), TRPML1Va (G470A), TRPML1Va (D471Q), TRPML1Va (D472Q), TRPML1Va (M473A), TRPML1Va (F474L), TRPML1Va (V475A), TRPML1Va (T476A), and TRPML1Va (F477L) mutants.
      Figure thumbnail gr3
      Figure 3A negative charge of Asp-472 is important for Ca2+-induced inhibition of the TRPML1 channel. A, at pH 7.4, concentration-dependent potentiation curve of Ca2+ with standard extracellular Ca2+-containing recording solutions. pF, picofarad. B, at pH 7.4, concentration-dependent potentiation curve of Ca2+ with extracellular NMDG-containing Ca2+ recording solutions. The curve was fit with Origin 9.0 software. Data points are representative of three to six independent experiments. C, current–voltage plots (left panel) and time course (right panel) for normalized TRPML1Va (D472E) currents under 0 and 2 mm Ca2+ conditions. D, current–voltage plots (left panel) and time course (right panel) for normalized TRPML1Va (D472K) currents under 0 and 2 mm Ca2+ conditions. E, current–voltage plots (left panel) and time course (right panel) for normalized TRPML1Va (D472A) currents under 0 and 2 mm Ca2+ conditions. F, time course of intracellular Ca2+ increase in HEK293T cells expressing the TRPML1Va (D472E), TRPML1Va (D472K), and TRPML1Va (D472A) mutants after addition of 2 mm Ca2+.

      The negative charge of Asp-472 is important for Ca2+-induced inhibition

      To elucidate the underlying mechanism, we examined the charge effect of Asp-472 on Ca2+-induced inhibition of the TRPML1Va channel. We first mutated aspartic acid to glutamic acid at residue 472 to maintain the same negative charge properties. Similar to TRPML1Va, addition of 2 mm Ca2+ dramatically decreased the current amplitude of TRPML1Va (D472E) (Fig. 3C and Fig. S3A). At the same time, we monitored extracellular Ca2+ entry using Fura-2 dye. Our results showed that TRPML1Va (D472E) mediated the movement of extracellular Ca2+ into cells, in line with the Ca2+ conductance of TRPML1Va (Fig. 3F). We then mutated aspartic acid to lysine at residue 472 to reverse the negative charge with the positive charge. In the presence of 0 and 2 mm Ca2+, the whole-cell currents were negligible (Fig. 3D and Fig. S3B), and neither allowed extracellular Ca2+ entry (Fig. 3F), indicating that the D472K mutant lost its cation conductance. Finally, we mutated aspartic acid to alanine, which has a small and hydrophobic side chain. Surprisingly, 2 mm Ca2+ largely increased the current amplitude of TRPML1Va (D472A) (Fig. 3E and Fig. S3C), and this mutant also induced extracellular Ca2+ entry (Fig. 3F). Notably, despite the opposite effects of adding Ca2+ to the two mutants TRPML1Va (D472A) and TRPML1Va (D472E), the nearly identical elevations in their F340/F380 ratio may be attributed to the similar current levels upon 2 mm Ca2+ addition (Fig. 3, C and E, left panels). Thus, the negative charge of Asp-472 plays an essential role in mediating both the monovalent ion conductance and Ca2+-induced inhibition of the TRPML1 currents.

      Asp-472 plays key role in both FCDI and SCDI of Ca2+ inhibition

      To further verify that Asp-472 is important for Ca2+-induced inhibition, we employed NMDG+ solution with Ca2+ as the only permeable ion. First, the whole-cell currents were recorded as the control experiment in cells expressing WT TRPML1Va before and after addition of 2 mm Ca2+. Fairly small Ca2+ currents were observed after addition of Ca2+ (Fig. 4A and Fig. S4A). Then the currents of the TRPML1Va (D472Q) and TRPML1Va (D472A) mutants were acquired in the absence and presence of 2 mm Ca2+, both showing much larger Ca2+ currents than the control experiment (Fig. 4, B and C, and Fig. S4, B and C). We also added a Ca2+ chelator (10 mm EGTA or 10 mm BAPTA) to the intracellular pipette solution to record the whole-cell currents of the TRPML1Va (D472Q) mutant after addition of 2 mm Ca2+. The resulting currents are similar to those without a Ca2+ chelator (Fig. 4, D and E, and Fig. S4, D and E), indicating that the Ca2+ current potentiation of the TRPML1Va (D472Q) mutant is not regulated by intracellular Ca2+.
      Figure thumbnail gr4
      Figure 4Ca2+ modulates both FCDI and SCDI in the TRPML1 channel through residue Asp-472. A–C, at pH 7.4, current–voltage plots of TRPML1Va, TRPML1Va (D472Q), and TRPML1Va (D472A) with extracellular NMDG/2 mm Ca2+ recording solution. D and E, at pH 7.4, current–voltage plots of TRPML1Va (D472Q) with NMDG/2 mm Ca2+ recording solution in the presence of 10 mm EGTA or 10 mm BAPTA pipette solutions. F and G, at pH 7.4, FCDI current traces of TRPML1Va, TRPML1Va (D472Q), and TRPML1Va (D472A), with NMDG/2 mm Ca2+ recording solution induced by a −100-mV step potential. H, at pH 7.4, FCDI current traces of TRPML1Va and TRPML1Va (D472Q) with NMDG/2 mm Ca2+ recording solution induced by a −100-mV step potential in the presence of 10 mm BAPTA pipette solution. I, at pH 7.4, normalized SCDI current curves of TRPML1Va and TRPML1Va (D472Q) with NMDG/2 mm Ca2+ recording solution with or without 10 mm EGTA or 10 mm BAPTA in pipette solution (left panel) and statistical analysis of decay time constant for SCDI (right panel). The curve was fit with Origin 9.0 software. ***, p < 0.001; NS, no statistical significance.
      Ca2+ channels, such as store-operated calcium channels (
      • Prakriya M.
      • Lewis R.S.
      Store-operated calcium channels.
      ,
      • Parekh A.B.
      • Putney Jr., J.W.
      Store-operated calcium channels.
      ) and voltage-gated calcium channels (
      • Ben-Johny M.
      • Yue D.T.
      Calmodulin regulation (calmodulation) of voltage-gated calcium channels.
      ), can be modulated by Ca2+ to cause inactivation called calcium-dependent inactivation (CDI). CDI comprises Ca2+-dependent fast inactivation (FCDI) and Ca2+-dependent slow inactivation (SCDI), which have distinct spatial and temporal mechanisms; the former at the millisecond level and the latter at the second level (
      • Parekh A.B.
      Regulation of CRAC channels by Ca2+-dependent inactivation.
      ,
      • Li X.
      • Wu G.
      • Yang Y.
      • Fu S.
      • Liu X.
      • Kang H.
      • Yang X.
      • Su X.-C.
      • Shen Y.
      Calmodulin dissociates the STIM1-Orai1 complex and STIM1 oligomers.
      ). However, for the TRPML1 channel, FCDI and SCDI processes have not yet been described. We therefore further explored the CDI of the TRPML1Va channel. To verify that monovalent cations cannot induce the CDI phenomenon in the TRPML1Va channel, the currents were first recorded with the 0 Ca2+ solution, showing no FCDI-like currents (Fig. S4F). Then obvious FCDI was observed after replacement of the 0 Ca2+ solution with 2 mm Ca2+ solution (Fig. S4F). Ba2+ caused few FCDI currents (Fig. S4F). Therefore, Ca2+ indeed induces the classic CDI of the TRPML1Va channel.
      To further investigate the CDI of the TRPML1Va channel, NMDG/Ca2+ solution was used. The current trace of TRPML1Va was first recorded in the NMDG with 2 mm Ca2+ solution, showing a small Ca2+ current with the classic FCDI phenomenon, with two time constants, including the fast τ value of 1.29 ± 0.14 ms (n = 7) and the slow τ value of 12.73 ± 1.51 ms (n = 7) (Fig. 4F). TRPML1Va also showed a decreased current amplitude, by 18.8% ± 3.8% (n = 7), from the start to the end position (Fig. 4F). Interestingly, under the same conditions, the mutant TRPML1Va (D472Q) not only presented a much larger current amplitude than TRPML1Va but also dramatically abolished the FCDI, showing an increased current amplitude, by 2.8% ± 0.7% (n = 6), from the start to the end position (Fig. 4F). This was also true for the mutant TRPML1Va (D472A), which showed an increased current amplitude, by 12.1% ± 2% (n = 8), from the start to end position (Fig. 4G). In the presence of 10 mm BAPTA in the pipette solution, FCDI abrogation by mutant TRPML1Va (D472Q) was not affected, showing an increased current amplitude, by 4.5% ± 0.4% (n = 4), from the start to end position, suggesting that abrogation was not attributed to the intracellular Ca2+ increase (Fig. 4H). It has been noted that the time constants of TRPML1va (D472Q), TRPML1va (D472Q) with 10 mm BAPTA, and TRPML1va (D472A) were not calculated because their FCDI current traces were best fit with the linear function. The SCDI process of TRPML1Va was also studied with elapsed time through one repetitive depolarized potential. As illustrated in Fig. 4I, the current amplitude of the TRPML1Va channel showed a time-dependent decay with a small time constant (∼9 s) in the presence of 2 mm Ca2+. However, the time-dependent decay curve of the current amplitude of the TRPML1Va (D472Q) mutant was significantly right-shifted, showing a much larger time constant than TRPML1Va (Fig. 4I). Addition of 10 mm EGTA or BAPTA to the pipette solution had no obvious effect on the decay time constant of the SCDI of the TRPML1Va (D472Q) mutant (Fig. 4I). These results suggest that Asp-472 is a key site to modulate both FCDI and SCDI of the TRPML1 channel.

      H+ alleviates Ca2+-induced inhibition

      Late endosomes and lysosomes are acidic compartments filled with high concentrations of H+ (
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Appelqvist H.
      • Wäster P.
      • Kågedal K.
      • Öllinger K.
      The lysosome: from waste bag to potential therapeutic target.
      ). Therefore, we investigated the effect of pH on the Ca2+ concentration-dependent inhibition of the TRPML1Va channel. First, the effect of Ca2+-induced inhibition was alleviated 5-fold with an IC50 value of 3.33 mm Ca2+ at pH 4.6 compared with that at pH 7.4 (Fig. 1A). Second, in the absence of Ca2+, the whole-cell currents of TRPML1Va were similar at pH 7.4 and pH 4.6 (Fig. 5A and Fig. S5A), indicating that H+ did not potentiate the monovalent cation conductance of TRPML1Va. In contrast, in the presence of 2 mm Ca2+, the whole-cell currents of TRPML1Va largely increased as the solution pH changed from 7.4 to 4.6 (Fig. 5B and Fig. S5B), indicating that H+ alleviated the Ca2+-induced inhibition associated with TRPML1Va currents. To further verify whether the negative charge of Asp-472 is involved in the H+ regulation of TRPML1 currents, we used the TRPML1Va mutant (D472Q). In the presence of 0 and 2 mm Ca2+, the whole-cell currents of the mutant TRPML1Va (D472Q) similarly decreased as the pH decreased from 7.4 to 4.6 (Fig. 5, C and D, and S5, C and D), indicating that the residue Asp-472 indeed significantly contributes to H+ regulation of the TRPML1 channel.
      Figure thumbnail gr5
      Figure 5Residue Asp-472 plays a key role in H+ alleviation of Ca2+-induced inhibition of TRPML1 currents. A and B, in the presence of 0 and 2 mm Ca2+, current–voltage plots and time courses for normalized currents of TRPML1Va before and after acidification (pH 4.6). C and D, in the presence of 0 and 2 mm Ca2+, current–voltage plots and time courses for normalized currents of the mutant TRPML1Va (D472Q) before and after acidification. E, with NMDG/2 mmCa2+ solution, FCDI current traces of TRPML1Va at pH 7.4 and at pH 4.6. F, with NMDG/2 mm Ca2+ solution, normalized SCDI current curves for TRPML1Va at pH 7.4 and 4.6. G, cartoon model of double regulation of TRPML1 channel conductance by Ca2+ and H+. The selectivity filter is shown in green. Residues Asp-472, Asp-471, and Gly-470 are shown in red, green, and orange, respectively. The monovalent cation, Ca2+, and H+ are shown in blue, red, and black, respectively.
      We then investigated the reciprocal regulation of both CDI and H+. As illustrated in Fig. 5E, the FCDI at the physiological pH 4.6 was significantly slowed compared with the pH 7.4 condition. At pH 4.6, TRPML1Va showed only one time constant value for the fast τ of 2.19 ± 0.39 ms (n = 4), and the current amplitude decreased by 10.7% ± 1.1% (n = 4) (Fig. 5E). We further compared SCDI processes at pH 7.4 and pH 4.6. Surprisingly, SCDI was completely abolished at pH 4.6 (Fig. 5F). In summary, H+ can alleviate the Ca2+-induced inhibition of the TRPML1 channel.

      Discussion

      TRPML1 is mainly located in late endosomes and lysosomes, whose compartments are filled with high concentrations of Ca2+ and H+ (
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Appelqvist H.
      • Wäster P.
      • Kågedal K.
      • Öllinger K.
      The lysosome: from waste bag to potential therapeutic target.
      ). Its special dwelling environment determines the regulation of its functional activity by both Ca2+ and H+ (
      • Xu H.
      • Delling M.
      • Li L.
      • Dong X.
      • Clapham D.E.
      Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice.
      ,
      • Dong X.P.
      • Cheng X.
      • Mills E.
      • Delling M.
      • Wang F.
      • Kurz T.
      • Xu H.
      The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.
      ,
      • Li M.
      • Zhang W.K.
      • Benvin N.M.
      • Zhou X.
      • Su D.
      • Li H.
      • Wang S.
      • Michailidis I.E.
      • Tong L.
      • Li X.
      • Yang J.
      Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel.
      ). In this study, we identified a key residue, Asp-472, that is important for Ca2+-induced inhibition of TRPML1 channel currents. We showed that the TRPML1Va (D472E) mutant had similar ion channel properties as WT TRPML1Va. However, the TRPML1Va (D472K) mutant, which included a positively charged amino acid substitution, completely abolished the channel conductance of TRPML1. The neutral amino acid substitution mutant TRPML1Va (D472A) abrogated Ca2+-induced inhibition of the TRPML1 channel activity but retained the cation conductance of TRPML1.
      Calcium-dependent inactivation is an important negative feedback regulation mechanism to prevent excessive Ca2+ entry into the cytoplasm. FCDI occurs rapidly after Ca2+ influx, whereas SCDI occurs upon slow global Ca2+ rise (
      • Parekh A.B.
      Regulation of CRAC channels by Ca2+-dependent inactivation.
      ,
      • Zweifach A.
      • Lewis R.S.
      Slow calcium-dependent inactivation of depletion-activated calcium current: store-dependent and -independent mechanisms.
      ,
      • Parekh A.B.
      Slow feedback inhibition of calcium release-activated calcium current by calcium entry.
      ). Generally, there are different regulators to manage FCDI and SCDI. Among these regulating factors, Ca2+-bound calmodulin is the most widely reported regulator (
      • Ben-Johny M.
      • Yue D.T.
      Calmodulin regulation (calmodulation) of voltage-gated calcium channels.
      ,
      • Li X.
      • Wu G.
      • Yang Y.
      • Fu S.
      • Liu X.
      • Kang H.
      • Yang X.
      • Su X.-C.
      • Shen Y.
      Calmodulin dissociates the STIM1-Orai1 complex and STIM1 oligomers.
      ,
      • Rosenmund C.
      • Feltz A.
      • Westbrook G.L.
      Calcium-dependent inactivation of synaptic NMDA receptors in hippocampal neurons.
      ,
      • Peterson B.Z.
      • DeMaria C.D.
      • Adelman J.P.
      • Yue D.T.
      Calmodulin is the Ca2+ sensor for Ca2+-dependent inactivation of L-type calcium channels.
      ,
      • Zühlke R.D.
      • Pitt G.S.
      • Deisseroth K.
      • Tsien R.W.
      • Reuter H.
      Calmodulin supports both inactivation and facilitation of L-type calcium channels.
      ,
      • Grant L.
      • Fuchs P.
      Calcium- and calmodulin-dependent inactivation of calcium channels in inner hair cells of the rat cochlea.
      ,
      • Zhang S.
      • Ehlers M.D.
      • Bernhardt J.P.
      • Su C.-T.
      • Huganir R.L.
      Calmodulin mediates calcium-dependent inactivation of N-methyl-d-aspartate receptors.
      ). However, the TRPML1 channel showed unconventional behavior in both FCDI and SCDI regulation. The single residue Asp-472 itself can regulate both the FCDI and SCDI of the TRPML1 channel. Ca2+-dependent calmodulin regulation seemed not to be involved in the regulation of the TRPML1 channel because the presence or absence of the Ca2+ chelator BAPTA caused no significant effects on both FCDI and SCDI. The FCDI induced by Ca2+ can be well-explained by the special localization of Asp-472 near the selectivity filter (
      • Schmiege P.
      • Fine M.
      • Blobel G.
      • Li X.
      Human TRPML1 channel structures in open and closed conformations.
      ), parallel to the spatial and temporal mechanism of FCDI occurrence (
      • Parekh A.B.
      Regulation of CRAC channels by Ca2+-dependent inactivation.
      ,
      • Zweifach A.
      • Lewis R.S.
      Slow calcium-dependent inactivation of depletion-activated calcium current: store-dependent and -independent mechanisms.
      ,
      • Hoth M.
      • Penner R.
      Calcium release-activated calcium current in rat mast cells.
      ). Strikingly, Ca2+ also facilitated the SCDI process through residue Asp-472 because the mutant TRPML1Va (D472Q) significantly prolonged the decay time constant of SCDI. Importantly, under physiological pH 4.6 conditions, we showed that Asp-472 also played a critical role in the H+ alleviation of the Ca2+-induced inhibition of TRPML1 channel currents. The D472Q mutant fully abrogated the H+ attenuation effects on Ca2+-induced inhibition. Further studies showed that H+ was able to not only significantly delay FCDI but also fully abolish SCDI. This unusual regulation of CDI can be further emphasized compared with other TRP channels adopting the Ca2+-bound calmodulin regulation approach (
      • Lambers T.T.
      • Weidema A.F.
      • Nilius B.
      • Hoenderop J.G.
      • Bindels R.J.
      Regulation of the mouse epithelial Ca2+ channel TRPV6 by the Ca2+ sensor calmodulin.
      ,
      • de Groot T.
      • Kovalevskaya N.V.
      • Verkaart S.
      • Schilderink N.
      • Felici M.
      • van der Hagen E.A.
      • Bindels R.J.
      • Vuister G.W.
      • Hoenderop J.G.
      Molecular mechanisms of calmodulin action on TRPV5 and modulation by parathyroid hormone.
      ,
      • Niemeyer B.A.
      • Bergs C.
      • Wissenbach U.
      • Flockerzi V.
      • Trost C.
      Competitive regulation of CaT-like-mediated Ca2+ entry by protein kinase C and calmodulin.
      ,
      • Zhu M.X.
      Multiple roles of calmodulin and other Ca2+-binding proteins in the functional regulation of TRP channels.
      ). This is probably an important molecular regulation mechanism for TRPML1-dependent cellular physiological events. Because lysosomes and late endosomes are filled with highly concentrated Ca2+ and H+ (
      • Dong X.-P.
      • Wang X.
      • Xu H.
      TRP channels of intracellular membranes.
      ,
      • Appelqvist H.
      • Wäster P.
      • Kågedal K.
      • Öllinger K.
      The lysosome: from waste bag to potential therapeutic target.
      ), the opposite regulation between Ca2+ and H+ is required to synergistically manage TRPML1 Ca2+ release. These results together indicated that the residue Asp-472 is indispensable for both TRPML1 ion conductance and its regulation by Ca2+ and H+.
      Based on these results, we propose a possible molecular model for the regulation of the TRPML1 channel current by Ca2+ and H+ (Fig. 5G). Under weakly basic conditions (pH 7.4), Asp-472 is deprotonated and able to effectively aggregate Ca2+ to form a positively charged microdomain, causing obvious CDI of the TRPML1 channel, thereby inducing Ca2+ inhibition of conductance. Additionally, the positively charged microdomain may also interfere with the access of monovalent cations as well as Ca2+ to the selective filter because of repulsive interactions. Consequently, a Ca2+-induced inhibition phenomenon is observed. However, under acidic conditions (pH 4.6), Asp-472 is protonated and weakens the binding ability of Ca2+. The FCDI process, especially SCDI, is clearly inhibited. Therefore, access of monovalent cations as well as Ca2+ to the selective filter is similar, with no Ca2+-induced inhibition phenomenon in the TRPML1 channel.
      A previous report showed that Ca2+ and H+ can regulate TRPML1 channel activity through three aspartic acid residues (D111Q, D114Q, and D115Q) (
      • Li M.
      • Zhang W.K.
      • Benvin N.M.
      • Zhou X.
      • Su D.
      • Li H.
      • Wang S.
      • Michailidis I.E.
      • Tong L.
      • Li X.
      • Yang J.
      Structural basis of dual Ca2+/pH regulation of the endolysosomal TRPML1 channel.
      ). However, these three residues seem to be dispensable for the regulation of the TRPML1 channel by Ca2+ and H+ in our system. The discrepancy may require further clarification in the future. Notably, we used a constitutively activated TRPML1Va mutant (
      • Xu H.
      • Delling M.
      • Li L.
      • Dong X.
      • Clapham D.E.
      Activating mutation in a mucolipin transient receptor potential channel leads to melanocyte loss in varitint-waddler mice.
      ) to conduct this research. Although the basic electrophysiological properties of this mutant are identical to those of WT TRPML1 (
      • Dong X.P.
      • Cheng X.
      • Mills E.
      • Delling M.
      • Wang F.
      • Kurz T.
      • Xu H.
      The type IV mucolipidosis-associated protein TRPML1 is an endolysosomal iron release channel.
      ), it will be necessary to perform relevant direct late endosome or lysosome patch clamp experiments under physiological conditions in the future.

      Experimental procedures

      Plasmids

      The human TRPML1 gene (accession number NM_020533) was synthesized by Genewiz and cloned into the pcDNA-EGFP vector in the BamH1 and Xho1 restriction sites using transcription PCR. The mutants, including TRPML1Va, TRPML1Va(D472E), TRPML1Va (D472K), TRPML1Va (D472A), TRPML1Va (D111Q, D114Q, D115Q), TRPML1Va (N469L), TRPML1Va (G470A), TRPML1Va (D471Q), TRPML1Va (D472Q), TRPML1Va (M473A), TRPML1Va (F474L), TRPML1Va (V475A), TRPML1Va (T476A), and TRPML1Va (F477L), were generated with the corresponding mutation primers listed in Table S1.

      Cell culture and transfection

      HEK293T cells were grown in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (HyClone). HEK293T cells were cultured at 37 °C with 5% CO2. All plasmids were transfected into HEK293T cells using polyethyleneimine.

      Whole-cell recordings and data analysis

      The recording pipettes were pulled from borosilicate glass using a P-97 glass microelectrode puller (Sutter Instrument) and polished with an MF-830 (Narishige). The pipettes had a resistance of 3–5 megaohms after being filled with the internal recording solution containing 120 mm cesium methanesulfonate, 4 mm NaCl, 2 mm MgCl2, 2 mm Na2-ATP, 10 mm EGTA, and 20 mm HEPES (pH adjusted to 7.2 with CsOH). This internal solution was used for all non-NMDG solution recordings. After establishment of the whole-cell configuration, the currents were recorded using an Axopatch 700B amplifier (Molecular Devices) and digitized using a Digidata 1550A (Molecular Devices). The voltage protocol included 50-ms voltage steps to −100 mV from a holding potential of 0 mV, followed by a voltage ramp increasing from −100 to + 100 mV in 50 ms with a frequency of 0.5 Hz. A 100-ms step potential from 0 mV to −100 mV was applied for investigation of the FCDI process. All currents were sampled at 10 kHz and filtered at 2 kHz by the low-pass filter. Current recordings were acquired through pClamp software (Molecular Devices). The standard extracellular 0 Ca2+ recording solution contained 153 mm NaCl, 5 mm KCl, 1 mm MgCl2, 10 mm glucose, and 20 mm HEPES (pH 7.4 adjusted with NaOH). The standard extracellular Ca2+-containing recording solutions were made in different concentrations by diluting the 1 m CaCl2 stock solution in the standard extracellular 0 Ca2+ recording solution. The pH 4.6 extracellular 0 Ca2+ recording solution contained 153 mm sodium gluconate, 5 mm KCl, 1 mm MgCl2, 10 mm MES, 10 mm HEPES, and 10 mm glucose (pH 4.6 adjusted with HCl). The pH 4.6 extracellular Ca2+-containing recording solutions were made in different concentrations by diluting the 1 m CaCl2 stock solution in the pH 4.6 extracellular 0 Ca2+ recording solution. The pH 7.4 extracellular NMDG/0 Ca2+ recording solution contained 160 mm NMDG, 20 mm HEPES, and 10 mm glucose (pH 7.4 adjusted with HCl). The pH 7.4 extracellular NMDG/Ca2+-containing recording solutions were made in different concentrations by diluting the 1 m CaCl2 stock solution in the pH 7.4 extracellular NMDG/0 Ca2+ recording solution. The pH 4.6 extracellular NMDG/0 Ca2+ recording solution contained 160 mm NMDG, 10 mm HEPES, 10 mm MES, and 10 mm glucose (pH 4.6 adjusted with HCl). The pH 4.6 extracellular NMDG/Ca2+-containing recording solutions were made in different concentrations through diluting the 1 m CaCl2 stock solution in the pH 4.6 extracellular NMDG/0 Ca2+ recording solution. To eliminate the obvious external currents recorded with the above internal pipette solution, we applied the following pipette solution containing 160 mm NMDG, 20 mm HEPES, and 10 mm glucose (pH 7.2 adjusted with HCl) for all NMDG-containing solution recordings. 10 mm glucose was replaced with 10 mm EGTA or BAPTA for the EGTA- or BAPTA-containing pipette solutions (pH 7.2 adjusted with HCl). The solution exchange was performed using a peristaltic pump and was accomplished within several seconds. Data were analyzed using pClamp and Origin software (OriginLab Corp.). All experiments were conducted at room temperature. The method for the normalization of the recorded currents was as follows. The very beginning current density was set as −1.0, the current densities during the measurement were displayed as the ratio of the very beginning current density to obtain the normalized current densities, and these densities were plotted as a function of the recording time. The time constants of FCDI were obtained through the nonlinear function fit (ExpDec 1 or ExpDec 2) in Origin 9.0 software. Statistical analysis was executed in SPSS Statistics 20 (Statistical Product and Service Solutions, IBM Corp.) by one-way analysis of variance. p < 0.05 represents statistical significance. Data points represent the mean ± S.E.

      Intracellular Ca2+ measurements

      HEK293T cells expressing the TRPML1Va, TRPML1Va (D472E), TRPML1Va (D472K), and TRPML1Va (D472A) mutants were plated on glass-bottom dishes coated with poly-l-lysine (Sigma-Aldrich). Twenty-four hours after transfection, the cells were loaded with 5 μm Fura-2/AM (Invitrogen) in the standard extracellular 0 Ca2+ recording solution (pH 7.4) at room temperature for 30 min. After loading, the cells were transferred to Fura-2/AM–free solution for 30 min. Fluorescence imaging was undertaken at room temperature using a Leica DMI6000B microscope with the LAS software before and after addition of 2 mm Ca2+. Consecutive excitation occurred at 340 and 380 nm every 2 s, and the emission was collected at 510 nm. The intracellular Ca2+ concentration change is shown as the 340/380 ratio. Data points are shown as the mean ± S.E.

      Author contributions

      G. W., X. Y., and Y. S. data curation; G. W. investigation; G. W. and Y. S. writing-original draft; X. Y. and Y. S. supervision; X. Y. and Y. S. project administration; Y. S. conceptualization; Y. S. funding acquisition; Y. S. writing-review and editing.

      Acknowledgment

      We thank Xiangchen Guan for excellent technical service.

      Supplementary Material

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