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1.
Yamaguchi S Jha A Li Q Soyombo AA Dickinson GD Churamani D Brailoiu E Patel S Muallem S 《The Journal of biological chemistry》2011,286(26):22934-22942
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. 相似文献
2.
Shaya Lev David A. Zeevi Ayala Frumkin Vered Offen-Glasner Gideon Bach Baruch Minke 《The Journal of biological chemistry》2010,285(4):2771-2782
The mucolipin (TRPML) ion channel proteins represent a distinct subfamily of channel proteins within the transient receptor potential (TRP) superfamily of cation channels. Mucolipin 1, 2, and 3 (TRPML1, -2, and -3, respectively) are channel proteins that share high sequence homology with each other and homology in the transmembrane domain with other TRPs. Mutations in the TRPML1 protein are implicated in mucolipidosis type IV, whereas mutations in TRPML3 are found in the varitint-waddler mouse. The properties of the wild type TRPML2 channel are not well known. Here we show functional expression of the wild type human TRPML2 channel (h-TRPML2). The channel is functional at the plasma membrane and characterized by a significant inward rectification similar to other constitutively active TRPML mutant isoforms. The h-TRPML2 channel displays nonselective cation permeability, which is Ca2+-permeable and inhibited by low extracytosolic pH but not Ca2+ regulated. In addition, constitutively active h-TRPML2 leads to cell death by causing Ca2+ overload. Furthermore, we demonstrate by functional mutation analysis that h-TRPML2 shares similar characteristics and structural similarities with other TRPML channels that regulate the channel in a similar manner. Hence, in addition to overall structure, all three TRPML channels also share common modes of regulation. 相似文献
3.
Xiping Cheng 《FEBS letters》2010,584(10):2013-2021
The mucolipin family of Transient Receptor Potential (TRPML) proteins is predicted to encode ion channels expressed in intracellular endosomes and lysosomes. Loss-of-function mutations of human TRPML1 cause type IV mucolipidosis (ML4), a childhood neurodegenerative disease. Meanwhile, gain-of-function mutations in the mouse TRPML3 result in the varitint-waddler (Va) phenotype with hearing and pigmentation defects. The broad spectrum phenotypes of ML4 and Va appear to result from certain aspects of endosomal/lysosomal dysfunction. Lysosomes, traditionally believed to be the terminal “recycling center” for biological “garbage”, are now known to play indispensable roles in intracellular signal transduction and membrane trafficking. Studies employing animal models and cell lines in which TRPML genes have been genetically disrupted or depleted have uncovered roles of TRPMLs in multiple cellular functions including membrane trafficking, signal transduction, and organellar ion homeostasis. Physiological assays of mammalian cell lines in which TRPMLs are heterologously overexpressed have revealed the channel properties of TRPMLs in mediating cation (Ca2+/Fe2+) efflux from endosomes and lysosomes in response to unidentified cellular cues. This review aims to summarize these recent advances in the TRPML field and to correlate the channel properties of endolysosomal TRPMLs with their biological functions. We will also discuss the potential cellular mechanisms by which TRPML deficiency leads to neurodegeneration. 相似文献
4.
TRPML3 is a Ca2+ permeable cation channel expressed in multiple intracellular compartments. Although TRPML3 is implicated in autophagy, how TRPML3 can regulate autophagy is not understood. To search interacting proteins with TRPML3 in autophagy, we performed split-ubiquitin membrane yeast two-hybrid (MY2H) screening with TRPML3-loop as a bait and identified GATE16, a mammalian ATG8 homologue. GST pull-down assay revealed that TRPML3 and TRPML3-loop specifically bind to GATE16, not to LC3B. Co-immunoprecipitation (co-IP) experiments showed that TRPML3 and TRPML3-loop pull down only the lipidated form of GATE16, indicating that the interaction occurs exclusively at the organellar membrane. The interaction of TRPML3 with GATE16 and GATE16-positive vesicle formation were increased in starvation induced autophagy, suggesting that the interaction facilitates the function of GATE16 in autophagosome formation. However, GATE16 was not required for TRPML3 trafficking to autophagosomes. Experiments using dominant-negative (DN) TRPML3(D458K) showed that GATE16 is localized not only in autophagosomes but also in extra-autophagosomal compartments, by contrast with LC3B. Since GATE16 acts at a later stage of the autophagosome biogenesis, our results suggest that TRPML3 plays a role in autophagosome maturation through the interaction with GATE16, by providing Ca2+ in the fusion process. 相似文献
5.
Xian-ping Dong Xiang Wang Dongbiao Shen Su Chen Meiling Liu Yanbin Wang Eric Mills Xiping Cheng Markus Delling Haoxing Xu 《The Journal of biological chemistry》2009,284(46):32040-32052
The mucolipin TRP (TRPML) proteins are a family of endolysosomal cation channels with genetically established importance in humans and rodent. Mutations of human TRPML1 cause type IV mucolipidosis, a devastating pediatric neurodegenerative disease. Our recent electrophysiological studies revealed that, although a TRPML1-mediated current can only be recorded in late endosome and lysosome (LEL) using the lysosome patch clamp technique, a proline substitution in TRPML1 (TRPML1V432P) results in a large whole cell current. Thus, it remains unknown whether the large TRPML1V432P-mediated current results from an increased surface expression (trafficking), elevated channel activity (gating), or both. Here we performed systemic Pro substitutions in a region previously implicated in the gating of various 6 transmembrane cation channels. We found that several Pro substitutions displayed gain-of-function (GOF) constitutive activities at both the plasma membrane (PM) and endolysosomal membranes. Although wild-type TRPML1 and non-GOF Pro substitutions localized exclusively in LEL and were barely detectable in the PM, the GOF mutations with high constitutive activities were not restricted to LEL compartments, and most significantly, exhibited significant surface expression. Because lysosomal exocytosis is Ca2+-dependent, constitutive Ca2+ permeability due to Pro substitutions may have resulted in stimulus-independent intralysosomal Ca2+ release, hence the surface expression and whole cell current of TRPML1. Indeed, surface staining of lysosome-associated membrane protein-1 (Lamp-1) was dramatically increased in cells expressing GOF TRPML1 channels. We conclude that TRPML1 is an inwardly rectifying, proton-impermeable, Ca2+ and Fe2+/Mn2+ dually permeable cation channel that may be gated by unidentified cellular mechanisms through a conformational change in the cytoplasmic face of the transmembrane 5 (TM5). Furthermore, activation of TRPML1 in LEL may lead to the appearance of TRPML1 proteins at the PM. 相似文献
6.
Drosophila TRPML Forms PI(3,5)P2-activated Cation Channels in Both Endolysosomes and Plasma Membrane
Xinghua Feng Yu Huang Yungang Lu Jian Xiong Ching-On Wong Pu Yang Jintang Xia De Chen Guangwei Du Kartik Venkatachalam Xuefeng Xia Michael X. Zhu 《The Journal of biological chemistry》2014,289(7):4262-4272
Transient Receptor Potential mucolipin (TRPML) channels are implicated in endolysosomal trafficking, lysosomal Ca2+ and Fe2+ 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 Ca2+, Mn2+, and Fe2+, but not Fe3+. The TRPML currents were inhibited by trivalent cations Fe3+, La3+, and Gd3+. 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. 相似文献
7.
Hyun Jin Kim Abigail A. Soyombo Sandra Tjon-Kon-Sang Insuk So Shmuel Muallem 《Traffic (Copenhagen, Denmark)》2009,10(8):1157-1167
TRPML3 is an inward rectifying Ca2+ channel that is regulated by extracytosolic H+ . Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca2+ in the vicinity of cellular organelles that is necessary to regulate these cellular events. 相似文献
8.
《Cell calcium》2015,57(6):446-456
Mucolipin synthetic agonist 1 (ML-SA1) was recently identified to activate mammalian TRPML channels and shown to alleviate lipid accumulation in lysosomes of cellular models of lysosome storage diseases, mucolipidosis type IV (MLIV) and Niemann–Pick's disease type C (NPC). Owning to its potential use in complimenting genetic studies in Drosophila melanogaster to elucidate the cellular and physiological functions of TRPML channels, we examined the effect of ML-SA1 on Drosophila TRPML expressed in HEK293 cells using whole-cell, inside-out, and whole-lysosome electrophysiological recordings. We previously showed that when expressed in HEK293 cells, Drosophila TRPML was localized and functional on both plasma membrane and endolysosome. We show here that in both inside-out patches excised from the plasma membrane and whole-lysosome recordings from enlarged endolysosome vacuoles, ML-SA1 failed to activate TRPML unless exogenous phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] was applied. At 1 μM ML-SA1, the sensitivity of TRPML to PI(3,5)P2 increased approximately by 10-fold and at 10 μM ML-SA1, the deactivation of PI(3,5)P2-evoked TRPML currents was markedly slowed. On the other hand, constitutive activation of TRPML by a mutation that mimics the varitint-waddler (Va) mutation of mouse TRPML3 rendered the insect channel sensitive to activation by ML-SA1 alone. Moreover, different from the insect TRPML, mouse TRPML1 was readily activated by ML-SA1 independent of PI(3,5)P2. Thus, our data reveal that while ML-SA1 acts as a true agonist at mouse TRPML1, it behaves as an allosteric activator of the Drosophila TRPML, showing dependence on and the ability to stabilize open conformation of the insect channels. 相似文献
9.
Cyntia Curcio‐Morelli Peng Zhang Bhuvarahamurthy Venugopal Florie A. Charles Marsha F. Browning Horacio F. Cantiello Susan A. Slaugenhaupt 《Journal of cellular physiology》2010,222(2):328-335
MCOLN1 encodes mucolipin‐1 (TRPML1), a member of the transient receptor potential TRPML subfamily of channel proteins. Mutations in MCOLN1 cause mucolipidosis‐type IV (MLIV), a lysosomal storage disorder characterized by severe neurologic, ophthalmologic, and gastrointestinal abnormalities. Along with TRPML1, there are two other TRPML family members, mucolipin‐2 (TRPML2) and mucolipin‐3 (TRPML3). In this study, we used immunocytochemical analysis to determine that TRPML1, TRPML2, and TRPML3 co‐localize in cells. The multimerization of TRPML proteins was confirmed by co‐immunoprecipitation and Western blot analysis, which demonstrated that TRPML1 homo‐multimerizes as well as hetero‐multimerizes with TRPML2 and TRPML3. MLIV‐causing mutants of TRPML1 also interacted with wild‐type TRPML1. Lipid bilayer re‐constitution of in vitro translated TRPML2 and TRPML3 confirmed their cation channel properties with lower single channel conductance and higher partial permeability to anions as compared to TRPML1. We further analyzed the electrophysiological properties of single channel TRPML hetero‐multimers, which displayed functional differences when compared to individual TRPMLs. Our data shows for the first time that TRPMLs form distinct functional channel complexes. Homo‐ and hetero‐multimerization of TRPMLs may modulate channel function and biophysical properties, thereby increasing TRPML functional diversity. J. Cell. Physiol. 222: 328–335, 2010. © 2009 Wiley‐Liss, Inc. 相似文献
10.
Grimm C Jörs S Guo Z Obukhov AG Heller S 《The Journal of biological chemistry》2012,287(27):22701-22708
The transient receptor potential channels TRPML2 and TRPML3 (MCOLN2 and MCOLN3) are nonselective cation channels. They are widely expressed in mammals. However, little is known about their physiological function(s) and activation mechanism(s). TRPML3 can be activated or rather de-inhibited by exposing it first to sodium-free extracellular solution and subsequently to high extracellular sodium. TRPML3 can also be activated by a variety of small chemical compounds identified in a high throughput screen and is inhibited by low pH. Furthermore, it was found that TRPML3 is constitutively active in low or no sodium-containing extracellular solution. This constitutive activity is independent of the intracellular presence of sodium, and whole-cell current densities are similar with pipette solutions containing cesium, potassium, or sodium. Here, we present mutagenesis data generated based on the hypothesis that negatively charged amino acids in the extracellular loops of TRPML3 may interfere with the observed sodium inhibition. We systematically mutated negatively charged amino acids in the first and second extracellular loops and found that mutating Glu-361 in the second loop has a significant impact on the sodium-mediated block of TRPML3. We further demonstrate that the TRPML3-related cation channel TRPML2 is also activated by lowering the extracellular sodium concentration as well as by a subset of small chemical compounds that were previously identified as activators of TRPML3, thus confirming the functional activity of TRPML2 at the plasma membrane and suggesting similar gating mechanisms for both TRPML channels. 相似文献
11.
Bhuvarahamurthy Venugopal Nicholas T. Mesires John C. Kennedy Cyntia Curcio‐Morelli Janice M. LaPlante J. Fred Dice Susan A. Slaugenhaupt 《Journal of cellular physiology》2009,219(2):344-353
Mucolipidosis type IV (MLIV) is a lysosomal storage disorder caused by mutations in the MCOLN1 gene, a member of the transient receptor potential (TRP) cation channel gene family. The encoded protein, transient receptor potential mucolipin‐1 (TRPML1), has been localized to lysosomes and late endosomes but the pathogenic mechanism by which loss of TRPML1 leads to abnormal cellular storage and neuronal cell death is still poorly understood. Yeast two‐hybrid and co‐immunoprecipitation (coIP) experiments identified interactions between TRPML1 and Hsc70 as well as TRPML1 and Hsp40. Hsc70 and Hsp40 are members of a molecular chaperone complex required for protein transport into the lysosome during chaperone‐mediated autophagy (CMA). To determine the functional relevance of this interaction, we compared fibroblasts from MLIV patients to those from sex‐ and age‐matched controls and show a defect in CMA in response to serum withdrawal. This defect in CMA was subsequently confirmed in purified lysosomes isolated from control and MLIV fibroblasts. We further show that the amount of lysosomal‐associated membrane protein type 2A (LAMP‐2A) is reduced in lysosomal membranes of MLIV fibroblasts. As a result of decreased CMA, MLIV fibroblasts have increased levels of oxidized proteins compared to control fibroblasts. We hypothesize that TRPML1 may act as a docking site for intralysosomal Hsc70 (ly‐Hsc70) allowing it to more efficiently pull in substrates for CMA. It is also possible that TRPML1 channel activity may be required for CMA. Understanding the role of TRPML1 in CMA will undoubtedly help to characterize the pathogenesis of MLIV. J. Cell. Physiol. 219: 344–353, 2009. © 2008 Wiley‐Liss, Inc. 相似文献
12.
《Cell calcium》2018
Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections are associated with a significant mortality rate, and existing drugs show poor efficacy. Identifying novel targets/pathways required for MERS infectivity is therefore important for developing novel therapeutics. As an enveloped virus, translocation through the endolysosomal system provides one pathway for cellular entry of MERS-CoV. In this context, Ca2+-permeable channels within the endolysosomal system regulate both the luminal environment and trafficking events, meriting investigation of their role in regulating processing and trafficking of MERS-CoV. Knockdown of endogenous two-pore channels (TPCs), targets for the Ca2+ mobilizing second messenger NAADP, impaired infectivity in a MERS-CoV spike pseudovirus particle translocation assay. This effect was selective as knockdown of the lysosomal cation channel mucolipin-1 (TRPML1) was without effect. Pharmacological inhibition of NAADP-evoked Ca2+ release using several bisbenzylisoquinoline alkaloids also blocked MERS pseudovirus translocation. Knockdown of TPC1 (biased endosomally) or TPC2 (biased lysosomally) decreased the activity of furin, a protease which facilitates MERS fusion with cellular membranes. Pharmacological or genetic inhibition of TPC1 activity also inhibited endosomal motility impairing pseudovirus progression through the endolysosomal system. Overall, these data support a selective, spatially autonomous role for TPCs within acidic organelles to support MERS-CoV translocation. 相似文献
13.
The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels.
Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies:
TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like);
the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell
types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis.
Most TRPs are non-selective cation channels, only few are highly Ca2+ selective, some are even permeable for highly hydrated Mg2+ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage
and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization
of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca2+ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca2+ and Mg2+), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function
as intracellular Ca2+ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been
implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and
pain, and ongoing research may help find new therapies for treatments of related diseases. 相似文献
14.
Kiselyov K Colletti GA Terwilliger A Ketchum K Lyons CW Quinn J Muallem S 《Cell calcium》2011,50(3):288-294
Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca2+-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe2+, Ca2+ and Zn2+ transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death. 相似文献
15.
Ellen Spooner Brooke M. McLaughlin Talya Lepow Tyler A. Durns Justin Randall Cameron Upchurch Katherine Miller Erin M. Campbell Hanna Fares 《PloS one》2013,8(2)
Mucolipidosis type IV is a lysosomal storage disorder resulting from mutations in the MCOLN1 gene, which encodes the endosomal/lysosomal Transient Receptor Potential channel protein mucolipin-1/TRPML1. Cells isolated from Mucolipidosis type IV patients and grown in vitro and in in vivo models of this disease both show several lysosome-associated defects. However, it is still unclear how TRPML1 regulates the transport steps implicated by these defects. Identifying proteins that associate with TRPML1 will facilitate the elucidation of its cellular and biochemical functions. We report here two saturation screens for proteins that interact with TRPML1: one that is based on immunoprecipitation/mass spectrometry and the other using a genetic yeast two-hybrid approach. From these screens, we identified largely non-overlapping proteins, which represent potential TRPML1-interactors., Using additional interaction assays on some of the potential interactors from each screen, we validated some proteins as candidate TRPML1 interactors In addition, our analysis indicates that each of the two screens not only identified some false-positive interactors, as expected from any screen, but also failed to uncover potential TRPML1 interactors. Future studies on the true interactors, first identified in these screens, will help elucidate the structure and function of protein complexes containing TRPML1. 相似文献
16.
Math P. Cuajungco Luigi C. Basilio Joshua Silva Thomas Hart Jonathan Tringali Cheng‐Chang Chen Martin Biel Christian Grimm 《Traffic (Copenhagen, Denmark)》2014,15(11):1247-1265
Mucolipidosis type IV (MLIV) is caused by loss of function mutations in the TRPML1 ion channel. We previously reported that tissue zinc levels in MLIV were abnormally elevated; however, the mechanism behind this pathologic accumulation remains unknown. Here, we identify transmembrane (TMEM)‐163 protein, a putative zinc transporter, as a novel interacting partner for TRPML1. Evidence from yeast two‐hybrid, tissue expression pattern, co‐immunoprecipitation, mass spectrometry and confocal microscopy studies confirmed the physical association of TMEM163 with TRPML1. This interaction is disrupted when a part of TMEM163's N‐terminus was deleted. Further studies to define the relevance of their interaction revealed that the plasma membrane (PM) levels of TMEM163 significantly decrease when TRPML1 is co‐expressed in HEK‐293 cells, while it mostly localizes within the PM when co‐expressed with a mutant TRPML1 that distributes mostly in the PM. Meanwhile, co‐expression of TMEM163 does not alter TRPML1 channel activity, but its expression levels in MLIV patient fibroblasts are reduced, which correlate with marked accumulation of zinc in lysosomes when these cells are acutely exposed to exogenous zinc (100 μM). When TMEM163 is knocked down or when TMEM163 and TRPML1 are co‐knocked down in HEK‐293 cells treated overnight with 100 nm zinc, the cells have significantly higher intracellular zinc levels than untreated control. Overall, these findings suggest that TMEM163 and TRPML1 proteins play a critical role in cellular zinc homeostasis, and thus possibly explain a novel mechanism for the pathological overload of zinc in MLIV disease. 相似文献
17.
18.
TRPML3 and TRPV5 are members of the mucolipin (TRPML) and TRPV subfamilies of transient receptor potential (TRP) cation channels. Based on sequence similarities of the pore forming regions and on structure-function evidence, we hypothesized that the pore forming domains of TRPML and TRPV5/TRPV6 channels have similarities that indicate possible functional interactions between these TRP channel subfamilies. Here we show that TRPML3 and TRPV5 associate to form a novel heteromeric ion channel. This novel conductance is detectable under conditions that do not activate either TRPML3 or TRPV5. It has pharmacological similarity with TRPML3 and requires functional TRPML3 as well as functional TRPV5. Single channel analyses revealed that TRPML3 and TRPV5 heteromers have different features than the respective homomers, and furthermore, that they occur in potentially distinct stoichiometric configurations. Based on overlapping expression of TRPML3 and TRPV5 in the kidney and the inner ear, we propose that TRPML3 and TRPV5 heteromers could have a biological function in these organs. 相似文献
19.
A Novel Calcium Binding Site in the Slow Vacuolar Cation Channel TPC1 Senses Luminal Calcium Levels 总被引:2,自引:0,他引:2
Beata Dadacz-Narloch Diana Beyhl Christina Larisch Enrique J. López-Sanjurjo Ralf Reski Kazuyuki Kuchitsu Thomas D. Müller Dirk Becker Gerald Sch?nknecht Rainer Hedrich 《The Plant cell》2011,23(7):2696-2707
Cytosolic calcium homeostasis is pivotal for intracellular signaling and requires sensing of calcium concentrations in the cytosol and accessible stores. Numerous Ca2+ binding sites have been characterized in cytosolic proteins. However, little is known about Ca2+ binding inside organelles, like the vacuole. The slow vacuolar (SV) channel, encoded by Arabidopsis thaliana TPC1, is regulated by luminal Ca2+. However, the D454/fou2 mutation in TPC1 eliminates vacuolar calcium sensitivity and increases store calcium content. In a search for the luminal calcium binding site, structure modeling indicated a possible coordination site formed by residues Glu-450, Asp-454, Glu-456, and Glu-457 on the luminal side of TPC1. Each Glu residue was replaced by Gln, the modified genes were transiently expressed in loss-of-TPC1-function protoplasts, and SV channel responses to luminal calcium were recorded by patch clamp. SV channels lacking any of the four negatively charged residues appeared altered in calcium sensitivity of channel gating. Our results indicate that Glu-450 and Asp-454 are directly involved in Ca2+ binding, whereas Glu-456 and Glu-457 are probably involved in connecting the luminal Ca2+ binding site to the channel gate. This novel vacuolar calcium binding site represents a potential tool to address calcium storage in plants. 相似文献
20.
Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1 总被引:1,自引:0,他引:1
Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa. 相似文献