TRP-ML1 Is a Lysosomal Monovalent Cation Channel ThatUndergoes Proteolytic Cleavage*

Received for publication, July 27, 2005, and in revised form, October 25, 2005 Published, JBC Papers in Press, October 27, 2005, DOI 10.1074/jbc.M508210200

Kirill Kiselyov‡§, Jin Chen‡, Youssef Rbaibi§, Daniel Oberdick§, Sandra Tjon-Kon-Sang‡, Nikolay Shcheynikov‡,Shmuel Muallem‡1, and Abigail Soyombo‡

From the ‡Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and the §Departmentof Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260

Mutations in the gene MCOLN1 coding for the TRP (transientreceptor potential) family ion channel TRP-ML1 lead to the lipidstorage disorder mucolipidosis type IV (MLIV). The function androle ofTRP-ML1arenotwell understood.We report here thatTRP-ML1 is a lysosomal monovalent cation channel. Both native andrecombinant TRP-ML1 are cleaved resulting in two products.Recombinant TRP-ML1 is detected as the full-length form and asshort N- and C-terminal forms, whereas in native cells mainly thecleaved N and C termini are detected. The N- and C-terminal frag-ments of TRP-ML1 were co-immunoprecipitated from cell lysatesand co-eluted from a Ni2� column. TRP-ML1 undergoes proteo-lytic cleavage that is inhibited by inhibitors of cathepsin B (CatB)and is altered when TRP-ML1 is expressed in CatB�/� cells. N-ter-minal sequencing of purified C-terminal fragment of TRP-ML1expressed in Sf9 cells indicates a cleavage site at Arg2002Pro201.Consequently, the conserved R200H mutation changed the cleav-age pattern of TRP-ML1. The cleavage inhibited TRP-ML1 channelactivity. This work provides the first example of inactivation bycleavage of a TRP channel. The significance of the cleavage to thefunction of TRP-ML1 is under investigation.

Mucolipidosis type IV (MLIV)2 is a lipid storage disorder character-ized by an abnormal accumulation of membranous lipids in patients’cells (reviewed in Refs. 1 and 2). Clinically, the disease manifests ascorneal clouding, degeneration of the retina, and severe psychomotorretardation (1–6). MLIV is associated with mutations in MCOLN1(TRP-ML1), a member of the TRP (transient receptor potential) familyof ion channels (7–9). The TRP family includes several members thatare implicated in human diseases, such as TRPP2 (10), TRPM1 (11), andTRPV6 (12). A critical question in MLIV pathogenesis is why do muta-tions in TRP-ML1 lead to the cellular phenotype of MLIV?Previous work on the ion selectivity and permeation of TRP-ML1

produced conflicting results. Thus, transient expression in Xenopusoocytes and in fibroblasts suggests that TRP-ML1 is targeted to thelysosomes and functions as a Ca2�-permeable channel that may regu-

late lysosomal Ca2� release and consequently agonist-evoked Ca2� sig-nals (13, 14). On the other hand, TRP-ML1 synthesized in cell-freesystem and reconstituted into planar lipid bilayers behaves as a mono-valent cations permeable, outwardly rectifying channel (15). The out-ward rectification indicates that when present in lysosomes, TRP-ML1primarily moves ions into the lysosomal lumen. The outward rectifica-tion makes it unlikely that in vivo TRP-ML1 would function as a lyso-somal Ca2� release channel, which suggested an alternative role of TRP-ML1 in lysosomal and cellular functions.In the present report we analyzed the expression pattern and channel

properties of TRP-ML1 and several disease-associated mutants. Wereport that TRP-ML1 is an outwardly rectifying monovalent cation-permeable channel that is primarily expressed in the lysosomes. In thelysosomes, TRP-ML1 is inactivated by proteolytic cleavage. These find-ings suggest a novel mechanism of regulating TRP-ML1 function.

EXPERIMENTAL PROCEDURES

Materials—The DNA-modifying enzymesN-glycosidase F (Endo F),and �-endo-N-acetylglucosaminidase H (EndoH) were fromNew Eng-land Biolabs. QuikChange site-directedmutagenesis kit was from Strat-agene. Cathepsin B inhibitors were from Calbiochem, and cathepsin Bwas from Sigma. CatB�/� cells were generously provided by Dr. Ter-ence S. Dermod (Vanderbilt University, Nashville, TN).

TRP-ML1�/� Cells—Human skin fibroblasts (HSF), clone WG0909,that is TRP-ML1�/�, and the WG0987 clone, a heterozygous relative,were obtained from the Repository for Mutant Human Cell Strains,Montreal Children’s Hospital. Fibroblasts were grown in Dulbecco’smodified Eagle’s medium supplemented with 10% fetal bovine serum,L-glutamine, and non-essential amino acids.

TRP-ML1 Expression Constructs—The full-length sequence corre-sponding to the human TRP-ML1 coding region was amplified by PCRusing IMAGE clone BF 529860 as template. The 1.7-kb amplified prod-uct was subcloned into the pCMV vectors either with no tag, an N-ter-minal HA tag, or a C-terminal Myc tag. Insert orientation and polym-erase fidelity were verified by restriction enzyme mapping andsequencing.

Site-directed Mutagenesis, Cell Transfection, and Immunoblotting—The plasmid pCMV-HA-TRP-ML1was used as a template to constructmutants using a mutagenesis kit (QuikChange, Stratagene). All muta-tions were confirmed by sequencing the entire DNA insert to verify thepresence of the desired mutation and the absence of extraneous muta-tions. HEK293 cells were transfected in 60-mm dishes with 5 �g ofplasmid DNA and 10 �l of Lipofectamine 2000 (Invitrogen).Cell extracts were prepared by sonication in homogenization buffer

containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 5 mM

MgCl2, and Complete protease inhibitor mixture tablet (Roche AppliedScience). Microsomal pellets were extracted with 1% CHAPS or 1%Triton X-100 and subjected to SDS-PAGE and immunoblotting with

* This work was supported by National Institutes of Health Grants DE12309 and DK38938(to S. M.), by a grant from the Mucolipidosis Foundation (to A. S.), and by the Pitts-burgh Life Science Greenhouse start-up fund (to K. K.). The costs of publication of thisarticle were defrayed in part by the payment of page charges. This article must there-fore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

1 To whom correspondence should be addressed: Dept. of Physiology, University ofTexas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9040.Tel.: 214-645-6008; E-mail: shmuel.muallem@utsouthwestern.edu.

2 The abbreviations used are: MLIV, mucolipidosis type IV; HA, hemagglutinin; IP, immu-noprecipitation; Ni2�-NTA, nickel-nitrilotriacetic acid; FL, full-length; TRP, transientreceptor potential; Endo F, N-glycosidase F; Endo H, �-endo-N-acetylglucosamini-dase H; HSF, human skin fibroblast; CHAPS, 3-[(3-cholamidopropyl)dimethylammo-nio]-1-propanesulfonic acid; WT, wild type; Cat, cathepsin; PM, plasma membrane.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 52, pp. 43218 –43223, December 30, 2005© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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anti-HA, anti-Myc, or anti-TRP-ML1 antibodies raised in rabbitsagainst the N-terminal sequence TAPAGPRGSETERLLTPN (�N1) oragainst the C-terminal sequence CGRDPSEEHSLLVN (�C1). Thespecificity of the anti-TRP-ML1 antibodies was verified by recognitionof the transfected protein, by blocking the signal with the peptides usedto raise the specific antibodies and by the absence of a specific signal inTRP-ML1�/� cells.

Electrophysiology—For conventional whole cell recording, cellsgrown on coverslips were placed in a perfusion chamber that wassecured on the stage of anOlympus IX50 invertedmicroscope equippedwith a fluorescent illuminator and filters designed to identify green flu-orescent protein-expressing cells. Transmembrane currents wererecorded using an Axopatch 200D amplifier, stored in a PC, and ana-lyzed with PClamp6 and Origin software. In the whole cell mode, thepipette solution contained (in mM) 140 cesium aspartate (to cancelendogenous K� and Cl� conductance), 5 NaCl, 5 Mg-ATP, 10 HEPES,2 EGTA, or 10 BAPTA (1,2-bis(2-aminophenoxy)ethane-N,N,N�,N�-tetraacetic acid), pH 7.2. The standard bath solution contained (inmM):140 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 10 HEPES, pH 7.5. In some experi-ments, NaCl was replaced withN-methyl-D-glucamine, and 0 or 10 mM

divalent metals were included in the bath solutions. The experimentswere performed at room temperature.

Confocal Immunocytochemistry—Cells grown on glass coverslipswere fixed and permeabilized by a 10-min incubation at �20 °C with100% methanol or were fixed by a 5-min incubation with 3.7% formal-dehyde and permeabilized by incubation with 0.01% Triton X-100 at4 °C for 5 min. After fixation, nonspecific sites were blocked by incuba-tion in 5% goat serum. Subsequently the cells were incubated with theprimary antibodies in blocking solution. Following washout of the pri-mary antibodies with phosphate-buffered saline, the cells were stainedwith fluorescent secondary antibodies and analyzed using a Bio-Rad1024 confocalmicroscope. The imageswere recordedwith a�40 objec-tive and analyzed off-line using NIH ImageTM software.

RESULTS AND DISCUSION

Localization of WT TRP-ML1 and Mutants—Expression in HeLacells suggested primarily lysosomal localization of TRP-ML1 (16). To

verify TRP-ML1 localization, HA-tagged human TRP-ML1 (HA-TRP-ML1) was expressed in HEK293 cells, HeLa cells, and HSF. Fig. 1 showsthat WT TRP-ML1 is primarily present in intracellular compartments.To identify the compartment in which TRP-ML1 is localized, cellstransfected with HA-TRP-ML1 were co-stained with anti-HA antibod-ies and antibodies against EEA1, themannose 6-phosphate receptors, orLAMP1 as markers for early endosomes, late endosomes/Golgi, andlysosomes, respectively. Fig. 1, A and B, shows that TRP-ML1 co-local-ized with mannose 6-phosphate receptors (MPR) only in the Golgi,probably because of TRP-ML1 over-expression. Significant overlap ofTRP-ML1 was found only with LAMP1, confirming its lysosomallocalization.Several disease-associatedTRP-ML1mutants have been identified (7,

8, 17, 18). The expression pattern of some of these mutants is shown inFig. 1C. The T232P and D362Ymutations resulted in retention of TRP-ML1 in the endoplasmic reticulum (Fig. 1C). Hence, these mutationscause MLIV probably because they are not targeted to the lysosomes.On the other hand the expression pattern of the F465L mutant wasidentical to that of WT TRP-ML1 (Fig. 1, D and E). As will be shownbelow, this mutation inhibits the channel activity of TRP-ML1, whichexplains why this mutation also results in MLIV.

TRP-ML1 Is Cleaved at a Post-Golgi Compartment—Expression ofTRP-ML1 tagged with HA in its N terminus (HA-TRP-ML1) inHEK293 cells resulted in two major protein products: the predictedfull-length (FL) TRP-ML1 (65–75 kDa) and a short formof 36� 1.7 kDa(Fig. 2A, left panel). Likewise, expression of TRP-ML1 tagged with Mycin its C terminus (TRP-ML1-Myc) also resulted in two glycosylatedprotein products (Fig. 2A, right panel). Hence, when over-expressed, asignificant portion of TRP-ML1 is cleaved to result in N- and C-termi-nal fragments.The large loop between the first and second transmembrane domains

(1–2 loop) of TRP-ML1 contains four putative N-glycosylation sites(Fig. 2B). To test whether TRP-ML1 is cleaved at or after exit from theendoplasmic reticulum/Golgi compartments, lysates from cellsexpressing HA-TRP-ML1 and TRP-ML1-Myc were treated either withEndo F,which cleaves allN-linked sugars, orwith EndoH,which cleavesonly highmannose-type or hybrid-type sugar groups that have not been

FIGURE 1. Intracellular localization of WT andmutant TRP-ML1. A, co-localization of WT TRP-ML1 with LAMP1. HA-tagged TRP-ML1 wasexpressed in WT HSF, and the cells were co-stainedwith anti-HA and anti-LAMP1 antibodies. Theoverlap between TRP-ML1 and LAMP1 was ana-lyzed using an RGD co-localization add-on toImageJ sofware. B, limited co-localization of TRP-ML1 with the early and late endosomal markersmannose 6-phosphate receptor (MPR) and EEA.1.C, localization of the HA-tagged T232P, D363Y,and F465L mutants. D and E, co-localization of theF465L mutant with LAMP1 (D) and WT TRP-ML1tagged with Myc (E).

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digested by Golgi mannosidase II. Fig. 2A shows that the FL TRP-ML1consolidated into a single lower molecular weight band following treat-ment with Endo F, but only a small fraction of the FL was sensitive todigestionwith EndoH. Digestionwith Endo F, but not EndoH, changedthemigration of theN-terminal fragment of TRP-ML1. Therefore, boththe FL and the N-terminal fragment of TRP-ML1 are sensitive to EndoF but are partially or completely resistant to Endo H.The C-terminal fragment was sensitive to Endo F. The migration of

the entire fragment was only slightly changed by Endo H (Fig. 2A, rightpanel). This may be because the C-terminal fragment was not modifiedin the Golgi. Alternatively, because the cleavage of TRP-ML1 is pre-dicted to be between the four putative glycosylation sites (Fig. 2B), it ispossible that only one of the glycosylation sites on the C-terminal frag-ment was modified in the Golgi. To distinguish between these possibil-ities we treated transfected cells with Swainsonine, a specific inhibitor ofmedial-Golgi mannosidase II (19). In the presence of swainsonine, theN- and C-terminal fragments became completely sensitive to digestionwith Endo H. Thus, it is clear that glycosylation of the C-terminal frag-ment is modified in the Golgi, but probably only one of its glycosylationsites is modified, further confirming that the cleavage occurs betweenthe second and third TRP-ML1 glycosylation sites and indicating thatTRP-ML1 is cleaved at a post-Golgi compartment, which is likely to bethe lysosome where most TRP-ML1 is localized. Finally, in all experi-ments tested (n� 4), treatment with Swainsonine changed the cleavageofTRP-ML1 resulting in twoN-terminal fragments, which suggests thatglycosylation may affect the cleavage of the channel.To determine whether the native TRP-ML1 is also cleaved, we raised

antibodies against anN-terminal (�N1) and a C-terminal (�C1) peptidesequence of TRP-ML1. Fig. 2C shows that �N1 detected primarily theshort Endo F-sensitive product in WT fibroblasts. The specificity of�N1 is shown by a lack of specific bands in TRP-ML1�/� cells cloneWG0909. The genetic defect in cloneWG0909 is expected to be null forprotein expression (8). Transfecting WT and TRP-ML1�/� cells withTRP-ML1 increased both the FL and short forms. Although not as spe-cific as �N1, �C1 detected predominantly the C-terminal fragment ofTRP-ML1 inWT fibroblast and did not detect any form of TRP-ML1 inTRP-ML1�/� cells (Fig. 2D). Finally, the short form of TRP-ML1 waspredominantly detected in extracts fromHEK293 cells and bovine brain(Fig. 2E). The prevalence of the short form in native cells indicates thatthe native FL-TRP-ML1 is rapidly cleaved in the lysosomes.

Mutual co-IP of the N and C termini of TRP-ML1 (Fig. 3A) suggeststhat they remain associated after the proteolytic cleavage. Furthermore,IP of the C terminus depleted the N terminus and IP of the N terminusdepleted the C-terminal fragments in cell extracts (Fig. 3B). The inter-action between the C- and N-terminal fragments is most likely directbecause elution of TRP-ML1-V5-His expressed in Sf9 cells after exten-sive washing of nonrelevant proteins co-eluted both fragments from theNi2�-NTA column with buffer containing 1% CHAPS (Fig. 4A).

Identification of the Cleavage Site—To determine the cleavage site,TRP-ML1-V5-His was expressed in Sf9 cells and purified on a Ni2�-NTA column. The C-terminal fragment was subjected to N-terminalsequencing. In two experiments N-terminal sequencing yielded thesequence PPPP, corresponding to residues 201–204 of TRP-ML1, sug-gesting that the cleavage was between Arg200 and Pro201. Because thepeaks were not very sharp, probably due to the glycosylation of theC-terminal, we verified the cleavage site by replacing the sequence197PPERPPPPP205 with alanines. Although these mutations did not pre-vent the cleavage, they did alter the cleavage pattern of TRP-ML1 (Fig.4A). Furthermore, the conserved substitution R200H also altered thecleavage pattern (Fig. 4B). All mutations tested around the potentialcleavage site altered but failed to prevent cleavage. This is likely becausein the lysosomes TRP-ML1 is cleaved by multiple proteases. TRP-ML1has a classical dileucine lysosomal targeting motif at the C terminus,EEHSLLVN. In an attempt to prevent targeting of TRP-ML1 to thelysosomes we deleted the LLVN sequence, which includes the criticaldileucine motif (20). Unfortunately, deletion of this sequence did notaffect TRP-ML1 localization (Fig. 4C), indicating that another sequence,yet to be identified, determines the lysosomal targeting of TRP-ML1.Nevertheless, the combination of sequencing and the mutation analysispoint to Arg2002Pro201 as a potential cleavage site. After this cleavage,the C- and N-terminal fragments become resistant to further cleavagesand accumulate in the cells.

Potential Role of CatB in TRP-ML1 Cleavage—Lysosomal localiza-tion of TRP-ML1 and the resistance of N- and C-terminal fragments toEndo H raised the possibility that TRP-ML1 is cleaved in the lysosome.To identify the lysosomal protease that cleaves TRP-ML1, HEK293 cellstransfected with TRP-ML1 were treated with 1 �M E64d, a membrane-permeable cysteine protease inhibitor, and with 0.2 �M CA-074-Me, amembrane-permeable selective cathepsin B (CatB) inhibitor (21). Thetwo inhibitors largely prevented cleavage of TRP-ML1 (Fig. 4D). As a

FIGURE 2. Native and recombinant TRP-ML1 areexpressed as cleaved proteins. A, HA-TRP-ML1(left panel) and TRP-ML1-Myc (right panel) wereexpressed in HEK293 cells, and the cells were incu-bated in the presence of absence of 2 �g/mlSwainsonine for 48 h. Extracts were then preparedand treated with Endo F or Endo H before separa-tion of proteins. The left blot was probed withanti-HA and the right blot with anti-Myc. WB, West-ern blot. B, a putative model of TRP-ML1 domains.SLS, putative serine lipase site; Out denotes extra-cellular space in the case of TRP-ML1 expressed inthe plasma membrane or lysosomal lumen for alysosome-resident channel. C, extracts preparedfrom WT and TRP-ML1�/� HSF and the same cellstransfected with TRP-ML1. The extracts weretreated with Endo F, and the blot was probed with�N1. D, extracts prepared from WT and TRP-MNL1�/� HSF were probed with anti-TRP-ML1antibodies that recognize a C-terminal sequenceof TRP-ML1. E, extracts prepared from HEK293 cellsor bovine brain were treated with Endo F and EndoH, as indicated, and probed with �N1.

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control, the cathepsin K inhibitor, CatK inhibitor II, did not affect TRP-ML1 cleavage at concentrations as high as 10 �M.

Inhibition of cleavage by CA-074-Me suggested that CatB is involvedin the cleavage of TRP-ML1. A more direct role for CatB was obtainedby expressing human TRP-ML1 inmouse CatB�/� fibroblasts (Fig. 4E).We were not able to analyze the fate of the native mouse TRP-ML1 inthe CatB�/� fibroblasts because �N1 only recognized the human iso-form. Human TRP-ML1 was cleaved in CatB�/� fibroblasts; however,the cleavage was at a site different from that observed in WT mouse

fibroblasts as evident from the generation of an �3-kDa longer N-ter-minal fragment in TRP-ML1�/� cells.In aggregate, the findings shown in Fig. 4 indicate that TRP-ML1 is

targeted to the lysosomes where it is cleaved by CatB or a CatB-depend-ent pathway. Another possibility is that TRP-ML1 is cleaved bymultiplelysosomal proteases and the final cleavage, mediated by CatB, occurs atthe Arg2002Pro201 site. Inhibition of CatB and expression of TRP-ML1in CatB�/� cells prevent the final cleavage by CatB and unmask thecleavage by other lysosomal proteases, resulting in the different cleavagepatterns illustrated in Fig. 4. In either case, cleavage by CatB plays aprominent role in the cleavage of TRP-ML1 in the lysosomes.That the cleavage occurs after the exit of TRP-ML1 from the endo-

plasmic reticulum is further confirmed by the differential processing ofthemutants shown in Fig. 5A. Full-length T232P andD362Ywere com-pletely sensitive to digestion by both Endo F and Endo H, confirmingthat these mutants are retained in the endoplasmic reticulum or cis-Golgi (Fig. 1). Consequently, these mutants were also not cleaved. Bycontrast, the F465L mutant was cleaved and the cleaved product wasresistant to Endo H, consistent with normal targeting and processing ofthis mutant. The finding that targeting and processing of the F465Lmutant was normal suggests that the channel function of TRP-ML1 isnot required for its targeting.

The Channel Function ofWTTRP-ML1 andMutants—Although themajority of TRP-ML1 is expressed in intracellular compartments, sur-face biotinylation showed that some of the over-expressed WT andF465LTRP-ML1was targeted to the plasmamembrane (Fig. 5B). Underthis moderate over-expression condition, no T232P and minimalamounts of the D362Y mutants were found at the plasma membrane.Saturation of the protein trafficking pathway by marked over-expres-sion forced expression of significant amounts of the FL and short formsof TRP-ML1 and only the FL form of the T232P mutant at the plasmamembrane (PM) (Fig. 5C). Such PMmistargetingwas necessary to studyTRP-ML1 channel properties using the whole cell configuration.TRP-ML1 conductance was measured by whole cell current record-

ing with intracellular solution containing cesium aspartate and extra-cellular solution containing sodiumaspartate orNaCl.Under these con-ditions, TRP-ML1 showed characteristic strongly outwardly rectifyingcurrent (Fig. 6, A and B). The strong outward rectification indicatespreferential transport of ions from the cytoplasm into the lysosomal

FIGURE 3. Interaction of the C and N termini of TRP-ML1. A, extracts prepared fromHEK293 cells expressing HA-TRP-ML1 or TRP-ML1-Myc were used for IP with anti-HA(left), anti-Myc (middle), or �N1 (right), and the precipitated proteins were probed with�N1 (left and middle) or anti-Myc (right). WB, Western blot. B, for the immunodepletionexperiments, extract from cells expressing TRP-ML1-Myc was used for IP with anti-Myc(left) or �N1 (right), and the residual TRP-ML1 in the supernatant was analyzed. Note thatIP with anti-Myc depleted the N-terminal fragment and IP with �N1 depleted the C-ter-minal fragment, indicating that the N and C termini remain associated.

FIGURE 4. Identification of the TRP-ML1 cleavage site. A, TRP-ML1-V5-His expressed inSf9 cells was purified using Ni2�-NTA column. The purified material resulted in elution ofboth cleaved products, although the His tag is only at the C terminus. N-terminalsequencing of the purified C-terminal half resulted in the sequence PPPP (shown in bold).B, TRP-ML1(197AAAAAAAAA205) (left blot) and TRP-ML1(R200H) (right blot) were trans-fected in HEK293 cells. Note the altered cleavage pattern. C, expression pattern of(�LLVN)TRP-ML1. D, cells expressing TRP-ML1 were treated with 2 �M E64d, 0.2 �M

CA-74-Me, or 10 �M CatK inhibitor II, and the proteins were analyzed by blotting with�N1. The -fold change in FL and short TRP-ML1 of cell treated with E64d from 3–5 exper-iments is shown in the columns. E, TRP-ML1 was expressed in WT and CatB�/� mousefibroblasts. Note the altered cleavage pattern and increased level of FL TRP-ML1 inCatB�/� cells.

FIGURE 5. Expression pattern of TRP-ML1 and mutants. A, sensitivity of the TRP-ML1mutants to endoglycosidases was determined by treating extracts prepared fromHEK293 cells expressing the indicated mutants with Endo F or Endo H, and the proteinswere probed with �N1. B and C, surface expression of TRP-ML1 and the F465L, T232P, andD362Y mutants moderately (B) or markedly (C) over-expressed was determined bybiotinylation.

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lumen at moderately negative and positive membrane potentials. Thecurrent-voltage characteristic of TRP-ML1 expressed inHEK293 cells issimilar to the current measured with cell-free synthesized TRP-ML1reconstituted into lipid bilayers (15).Another important finding, as shown in Fig. 6A, is that expression of

the F465Lmutant did not result in channel activity, although expressionand PM targeting of the F465L mutant was similar to that of WT-TRP-ML1. Phe465 is predicted to be in the pore region of TRP-ML1, on thebasis of sequence comparison with other members of the TRP channelfamily (22, 23), and thus the F465L mutation likely impaired channelpermeability. Hence, although the T232P and D362Y are processingmutants, the F465L is a channel permeability mutant, leading to thedisease phenotype.

TRP-ML1 Is Inactivated by Proteolytic Cleavage—The finding thatthe two fragments of TRP-ML1 remained associated (Fig. 4) and that FLand short TRP-ML1 were present at the PM (Fig. 5, B and C) raised thequestion of which form of TRP-ML1 mediates the current and what isthe role of the channel cleavage. The first clue that the cleavage inacti-vates TRP-ML1was obtained bymeasuring the current in cells express-ing the T232Pmutant. Cells over-expressing the T232Pmutant showedsmall TRP-ML1-specific current (Fig. 6,A andC), although not as oftenas cells over-expressingWTTRP-ML1. Because only the FL T232P waspresent at the PM (Fig. 5C), the TRP-ML1 current must be mediated bythe FL protein.Further evidence that CatB-mediated cleavage inactivates TRP-ML1

is presented in Fig. 7. In the first series of experiments, TRP-ML1expressed in HEK293 cells was treated with CatB while recording thewhole cell current. Fig. 7, A and B, shows that within 50–60 s of treat-ment with 0.2 or 0.5 units/ml recombinant CatB at pH 5, the currentdecreased by 30–60%. The cells were incubated at pH 5 for severalminutes before CatB application. No run-down of TRP-ML1 channelactivity was detected during this treatment. Another TRP channel,

TRPC3, was used as a control for the specificity of the effect of CatB.Cells expressing TRPC3 were pretreated with CatB at pH 5, and theamplitude of the current activated by stimulation of the P2Y2 receptorswith UTP was compared in control cells and cells treated with CatB.Treatment with CatB did not affect the amplitude of the UTP-stimu-lated TRPC3-dependent current (Fig. 7C).In the second set of experiments HEK293 cells expressing TRP-

ML1 were treated with the CatB inhibitor E64d. Biotinylationshowed that inhibition of CatB, which reduces the cleavage toincrease the level of FL TRP-ML1 (Fig. 4D), increased the level of FLTRP-ML1 at the plasma membrane (Fig. 7B). This correlated with anincreased TRP-ML1-mediated current (Fig. 7B). Hence, the com-bined results with the T232P mutant, treatment with CatB, and theeffect of the CatB inhibitor suggest that the cleavage served to inac-tivate TRP-ML1 channel function.

Conclusions—We report here that TRP-ML1 is a lysosomal, mono-valent, cation channel that is inactivated by cleavage. The cleavagemay be mediated by multiple lysosomal proteases, one of which isCatB, which appears to mediate the critical or final cleavage. Impor-tantly, native TRP-ML1 is also cleaved. Furthermore, the cleavedform is the predominant form of the channel found in native cells.The cleavage serves to inactivate the channel function of TRP-ML1.The question that arises is why do cells need to inactivate TRP-ML1?

TRP-ML1 has a central role in lipid hydrolysis and processing, as evi-dent from the disease phenotype and as will be shown in subsequentpublications. It is possible that inactivation by cleavage constitutes aregulatory mechanism to limit the duration of TRP-ML1 channel activ-ity. Unregulated TRP-ML1 activity may be detrimental to lipid process-ing. TRP-ML1 is probably targeted enmasse to the lysosomes, where itsfunction is rapidly completed and it is inactivated by cleavage to preventdisruption of lysosomal ionic homeostasis, which results in the accumu-lation of the cleaved form of the channel.

B. C.

A.

FIGURE 6. Ionic currents by TRP-ML1 and mutants. A, the whole cell currents in controlHEK293 cells, HEK293 cells expressing WT-TRP-ML1, and the indicated mutants weremeasured using voltage pulses from �100 to �100 mV, 250 ms in duration, appliedevery 2 s. The patch pipette contained 140 mM cesium aspartate. Extracellular solutioncontained 140 mM sodium gluconate, 2 mM CaCl2, and 1 mM MgCl2. This is representativeof 5–10 experiments. B, representative I/V relationship recorded from control cells andcells expressing TRP-ML1. Solutions are as described in A. C, the average outward currentrecorded from cells expressing WT and mutant TRP-ML1 was normalized to the currentrecorded with WT TRP-ML1.

FIGURE 7. Inactivation of TRP-ML1 by CatB-mediated cleavage. A, an HEK293 cellexpressing TRP-ML1 was analyzed using whole cell current recording. The amplitude ofthe outward current recorded at �80 mV is plotted as a function of time. Shortly after thewhole cell configuration was established, the extracellular solution was changed to pH 5.About 60 s later the cell was treated with 0.5 units/ml recombinant CatB. The average ofcurrent inhibition by 0.2 and 0.5 units/ml CatB recorded from at least 5 cells is shown inthe columns. In B, HEK293 cells expressing TRP-ML1 were treated with E64d, and theeffect of reduced cleavage on plasma membrane expression of TRP-ML1 was evaluatedby biotinylation,and the effect on the extent of the current was measured in 8 –15 cells bywhole cell current recording. C, HEK293 cells transfected with TRPC3 (both traces) weretreated with CatB (solid trace) exactly as described in A, and the outward current wasmeasured before and after stimulation with 100 �M UTP. The columns in C show theaverage of at least five cells.

Mucolipin 1 Is Regulated by Proteolytic Cleavage

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Acknowledgment—We thank Dr. Ora Weisz for discussion.

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Mucolipin 1 Is Regulated by Proteolytic Cleavage

DECEMBER 30, 2005 • VOLUME 280 • NUMBER 52 JOURNAL OF BIOLOGICAL CHEMISTRY 43223

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Nikolay Shcheynikov, Shmuel Muallem and Abigail SoyomboKirill Kiselyov, Jin Chen, Youssef Rbaibi, Daniel Oberdick, Sandra Tjon-Kon-Sang,

CleavageTRP-ML1 Is a Lysosomal Monovalent Cation Channel That Undergoes Proteolytic

doi: 10.1074/jbc.M508210200 originally published online October 27, 20052005, 280:43218-43223.J. Biol. Chem. 

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