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1.
Kim HJ Li Q Tjon-Kon-Sang S So I Kiselyov K Muallem S 《The Journal of biological chemistry》2007,282(50):36138-36142
TRPML3 is a member of the TRPML subfamily of the transient receptor potential cation channel superfamily. The TRPML3(A419P) mutation causes a severe form, whereas the TRPML3(I362T/A419P) mutation results in a mild form of the varitint-waddler phenotype. The channel properties of TRPML3 and how the mutations cause each phenotype are not known. In this study, we report the first channel properties of TRPML3 as a strongly inward rectifying cation channel with a novel regulation by extracytosolic Na+. Preincubating the extracytosolic face of TRPML3 in Na+-free medium is required for channel activation, but then the channel slowly inactivates. The A419P mutation locks the channel in an open unregulated state. Similar gain of function was observed with the A419G mutation, which, like A419P, is expected to destabilize the alpha-helical fifth transmembrane domain of TRPML3. The I362T mutation results in an inactive channel, but the channel properties of TRPML3(I362T/A419P) are similar to those of TRPML3(A419P). However, the surface expression and current density of TRPML3(I362T/A419P) are lower than those of TRPML3(A419P). The A419P mutation also affects channel glycosylation and causes massive cell death. These findings show that the varitint-waddler phenotype is due to a gain of function of TRPML3(A419P) that is reduced by the TRPML3(I362T/A419P) mutant, resulting in a milder phenotype. 相似文献
2.
Hyun Jin Kim Soichiro Yamaguchi Qin Li Insuk So Shmuel Muallem 《The Journal of biological chemistry》2010,285(22):16513-16520
TRPML3 is a H+-regulated Ca2+ channel that shuttles between intracellular compartments and the plasma membrane. The A419P mutation causes the varitint-waddler phenotype as a result of gain-of-function (GOF). The mechanism by which A419P leads to GOF is not known. Here, we show that the TRPML3 pore is dynamic when conducting Ca2+ to change its conductance and permeability, which appears to be mediated by trapping Ca2+ within the pore. The pore properties can be restored by strong depolarization or by conducting Na+ through the pore. The A419P mutation results in expanded channel pore with altered permeability that limits modulation of the pore by Ca2+. This effect is specific for the A419P mutation and is not reproduced by other GOF mutations, including A419G, H283A, and proline mutations in the fifth transmembrane domain. These findings describe a novel mode of a transient receptor potential channel behavior and suggest that pore expansion by the A419P mutation may contribute to the varitint-waddler phenotype. 相似文献
3.
TRPML3, a member of the transient receptor potential (TRP) family, is an inwardly rectifying, non-selective Ca2+-permeable cation channel that is regulated by extracytosolic Na+ and H+ and can be activated by a variety of small molecules. The severe auditory and vestibular phenotype of the TRPML3(A419P) varitint-waddler mutation made this protein particularly interesting for inner ear biology. To elucidate the physiological role of murine TRPML3, we conditionally inactivated Trpml3 in mice. Surprisingly, lack of functional TRPML3 did not lead to circling behavior, balance impairment or hearing loss. 相似文献
4.
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
5.
Christian Grimm Simone J?rs Stefan Heller 《The Journal of biological chemistry》2009,284(20):13823-13831
The varitint-waddler mutation A419P renders TRPML3 constitutively active,
resulting in cationic overload, particularly in sustained influx of
Ca2+. TRPML3 is expressed by inner ear sensory hair cells, and we
were intrigued by the fact that hair cells are able to cope with expressing
the TRPML3(A419P) isoform for weeks before they ultimately die. We
hypothesized that the survival of varitint-waddler hair cells is linked to
their ability to deal with Ca2+ loads due to the abundance of
plasma membrane calcium ATPases (PMCAs). Here, we show that PMCA2
significantly reduced [Ca2+]i increase and
apoptosis in HEK293 cells expressing TRPML3(A419P). The deaf-waddler isoform
of PMCA2, operating at 30% efficacy, showed a significantly decreased ability
to rescue the Ca2+ loading of cells expressing TRPML3(A419P). When
we combined mice heterozygous for the varitint-waddler mutant allele with mice
heterozygous for the deaf-waddler mutant allele, we found severe hair bundle
defects as well as increased hair cell loss compared with mice heterozygous
for each mutant allele alone. Furthermore, 3-week-old double mutant mice
lacked auditory brainstem responses, which were present in their respective
littermates containing single mutant alleles. Likewise, heterozygous double
mutant mice exhibited severe circling behavior, which was not observed in mice
heterozygous for TRPML3(A419P) or PMCA2(G283S) alone. Our results provide a
molecular rationale for the delayed hair cell loss in varitint-waddler mice.
They also show that hair cells are able to survive for weeks with sustained
Ca2+ loading, which implies that Ca2+ loading is an
unlikely primary cause of hair cell death in ototoxic stress situations.Varitint-waddler (Va) mice express a mutant isoform (A419P) of the
transient receptor potential channel TRPML3 (murine gene symbol,
Mcoln3) that results in profound hearing loss, vestibular defects
(circling behavior, imbalance, head bobbing, waddling), pigmentation
deficiencies, sterility, and perinatal lethality in homozygous animals
(1). A second Mcoln3
variant (VaJ) that arose in the Va background
carries two mutations (I362T and A419P) and shows a phenotype with reduced
severity, particularly in heterozygous animals
(1). The A419P mutation in
Va and VaJ mice is located in
transmembrane-spanning domain
5(TM5)3 of TRPML3,
where it leads to a constitutively open channel, resulting in highly elevated
[Ca2+]i
(2-5).
In contrast to the effect of the A419P mutation on TRPML3 channel activity,
the single I362T mutation does not appear to affect
[Ca2+]i
(3,
5). When combined with the
A419P mutation, as found in VaJ mice, the constitutive
activity of this mutant TRPML3 isoform is comparable with that of A419P alone
(2-5).Here, we show that HEK293 cells expressing TRPML3-(A419P) or
TRPML3(I362T/A419P) undergo rapid apoptosis. This apoptosis is suppressed by
coexpression of plasma membrane calcium ATPase type 2 (PMCA2). In
varitint-waddler mice, sensory hair cells survive for weeks after birth
(6), which raised the question
of whether this survival could be the result of the hair cells'' ability to
deal with normally transient and localized Ca2+ influx, a feature
that is centered around the high levels of mobile Ca2+ buffers and
PMCA isoforms found in sensory hair cells
(7-10).
We decided to test this hypothesis in vivo by utilizing deaf-waddler
mice that carry a mutation (G283S) in the Atp2b2 gene encoding mutant
PMCA2. Mice homozygous for PMCA2(G283S)
(Atp2b2dfw/dfw) are deaf and have poor
balance (11). Compared with
Atp2b2 knock-out mice, deaf-waddler mice display a milder phenotype
because PMCA2(G283S) retains 30% of its biological activity compared with the
wild-type isoform (12). We
found that sensory hair cell loss, hearing loss, and vestibular dysfunction
were aggravated in mice carrying varitint-waddler and deaf-waddler alleles
compared with animals carrying the single mutant alleles. Our results reveal
that the Ca2+-buffering and Ca2+ extrusion abilities of
hair cells are powerful enough to prevent cell death for weeks, even in the
presence of constitutively active TRPML3(A419P), which is able to induce rapid
apoptosis in other cells. 相似文献
6.
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
7.
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
8.
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
9.
Lee KP Nair AV Grimm C van Zeeland F Heller S Bindels RJ Hoenderop JG 《Cell calcium》2010,48(5):275-287
TRPV5, a member of transient receptor potential (TRP) superfamily of ion channels, plays a crucial role in epithelial calcium transport in the kidney. This channel has a high selectivity for Ca(2+) and is tightly regulated by intracellular Ca(2+) concentrations. Recently it was shown that the molecular basis of deafness in varitint-waddler mouse is the result of hair cell death caused by the constitutive activity of transient receptor potential mucolipin 3 (TRPML3) channel carrying a helix breaking mutation, A419P, at the intracellular proximity of the fifth transmembrane domain (TM5). This mutation significantly elevates intracellular Ca(2+) concentration and causes rapid cell death. Here we show that substituting the equivalent location in TRPV5, the M490, to proline significantly modulates Ca(2+)-dependent inactivation of TRPV5. The single channel conductance, time constant of inactivation (τ) and half maximal inhibition constant (IC(50)) of TRPV5(M490P) were increased compared to TRPV5(WT). Moreover TRPV5(M490P) showed lower Ca(2+) permeability. Out of different point mutations created to characterize the importance of M490 in Ca(2+)-dependent inactivation, only TRPV5(M490P)-expressing cells showed apoptosis and extremely altered Ca(2+)-dependent inactivation. In conclusion, the TRPV5 channel is susceptible for helix breaking mutations and the proximal intracellular region of TM5 of this channel plays an important role in Ca(2+)-dependent inactivation. 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.
Eric Hosy Julien P. Dupuis Michel Vivaudou 《The Journal of biological chemistry》2010,285(5):3084-3091
The function of the ATP-sensitive potassium (KATP) channel relies on the proper coupling between its two subunits: the pore-forming Kir6.2 and the regulator SUR. The conformation of the interface between these two subunits can be monitored using a rhodamine 123 (Rho) protection assay because Rho blocks Kir6.2 with an efficiency that depends on the relative position of transmembrane domain (TMD) 0 of the associated SUR (Hosy, E., Dérand, R., Revilloud, J., and Vivaudou, M. (2007) J. Physiol. 582, 27–39). Here we find that the natural and synthetic KATP channel activators MgADP, zinc, and SR47063 induced a Rho-insensitive conformation. The activating mutation F132L in SUR1, which causes neonatal diabetes, also rendered the channel resistant to Rho block, suggesting that it stabilized an activated conformation by uncoupling TMD0 from the rest of SUR1. At a nearby residue, the SUR1 mutation E128K impairs trafficking, thereby reducing surface expression and causing hyperinsulinism. To augment channel density at the plasma membrane to investigate the effect of mutating this residue on channel function, we introduced the milder mutation E126A at the matching residue of SUR2A. Mutation E126A imposed a hypersensitive Rho phenotype indicative of a functional uncoupling between TMD0 and Kir6.2. These results suggest that the TMD0-Kir6.2 interface is mobile and that the gating modes of Kir6.2 correlate with distinct positions of TMD0. They further demonstrate that the second intracellular loop of SUR, which contains the two residues studied here, is a key structural element of the TMD0-Kir6.2 interface. 相似文献
12.
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
13.
Extracellular sodium interacts with the HERG channel at an outer pore site 总被引:3,自引:0,他引:3
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Mullins FM Stepanovic SZ Desai RR George AL Balser JR 《The Journal of general physiology》2002,120(4):517-537
Most voltage-gated K(+) currents are relatively insensitive to extracellular Na(+) (Na(+)(o)), but Na(+)(o) potently inhibits outward human ether-a-go-go-related gene (HERG)-encoded K(+) channel current (Numaguchi, H., J.P. Johnson, Jr., C.I. Petersen, and J.R. Balser. 2000. Nat. Neurosci. 3:429-30). We studied wild-type (WT) and mutant HERG currents and used two strategic probes, intracellular Na(+) (Na(+)(i)) and extracellular Ba(2+) (Ba(2+)(o)), to define a site where Na(+)(o) interacts with HERG. Currents were recorded from transfected Chinese hamster ovary (CHO-K1) cells using the whole-cell voltage clamp technique. Inhibition of WT HERG by Na(+)(o) was not strongly dependent on the voltage during activating pulses. Three point mutants in the P-loop region (S624A, S624T, S631A) with intact K(+) selectivity and impaired inactivation each had reduced sensitivity to inhibition by Na(+)(o). Quantitatively similar effects of Na(+)(i) to inhibit HERG current were seen in the WT and S624A channels. As S624A has impaired Na(+)(o) sensitivity, this result suggested that Na(+)(o) and Na(+)(i) act at different sites. Extracellular Ba(2+) (Ba(2+)(o)) blocks K(+) channel pores, and thereby serves as a useful probe of K(+) channel structure. HERG channel inactivation promotes relief of Ba(2+) block (Weerapura, M., S. Nattel, M. Courtemanche, D. Doern, N. Ethier, and T. Hebert. 2000. J. Physiol. 526:265-278). We used this feature of HERG inactivation to distinguish between simple allosteric and pore-occluding models of Na(+)(o) action. A remote allosteric model predicts that Na(+)(o) will speed relief of Ba(2+)(o) block by promoting inactivation. Instead, Na(+)(o) slowed Ba(2+) egress and Ba(2+) relieved Na(+)(o) inhibition, consistent with Na(+)(o) binding to an outer pore site. The apparent affinities of the outer pore for Na(+)(o) and K(+)(o) as measured by slowing of Ba(2+) egress were compatible with competition between the two ions for the channel pore in their physiological concentration ranges. We also examined the role of the HERG closed state in Na(+)(o) inhibition. Na(+)(o) inhibition was inversely related to pulsing frequency in the WT channel, but not in the pore mutant S624A. 相似文献
14.
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
15.
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
16.
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
17.
《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
18.
Miller S Bartlett W Chandrasekaran S Simpson S Edwards M Booth IR 《The EMBO journal》2003,22(1):36-46
The major structural features of the Escherichia coli MscS mechanosensitive channel protein have been explored using alkaline phosphatase (PhoA) fusions, precise deletions and site-directed mutations. PhoA protein fusion data, combined with the positive-inside rule, strongly support a model in which MscS crosses the membrane three times, adopting an N(out)-C(in) configuration. Deletion data suggest that the C-terminal domain of the protein is essential for the stability of the MscS channel, whereas the protein will tolerate small deletions at the N-terminus. Four mutants that exhibit either gain-of-function (GOF) or loss-of-function have been identified: a double mutation I48D/S49P inactivates MscS, whereas the MscS mutants T93R, A102P and L109S cause a strong GOF phenotype. The similarity of MscS to the last two domains of MscK (formerly KefA) is reinforced by the demonstration that expression of a truncated MscK protein can substitute for MscL and MscS in downshock survival assays. The data derived from studies of the organization, conservation and the influence of mutations provide significant insights into the structure of the MscS channel. 相似文献
19.
The alpha-factor pheromone receptor (STE2) activates a G protein signal pathway that induces conjugation of the yeast Saccharomyces cerevisiae. Previous studies implicated the third intracellular loop of this receptor in G protein activation. Therefore, the roles of transmembrane domains five and six (TMD5 and -6) that bracket the third intracellular loop were analyzed by scanning mutagenesis in which each residue was substituted with cysteine. Out of 42 mutants examined, four constitutive mutants and two strong loss-of-function mutants were identified. Double mutants combining Cys substitutions in TMD5 and TMD6 gave a broader range of phenotypes. Interestingly, a V223C mutation in TMD5 caused constitutive activity when combined with the L247C, L248C, or S251C mutations in TMD6. Also, the L226C mutation in TMD5 caused constitutive activity when combined with either the M250C or S251C mutations in TMD6. The residues affected by these mutations are predicted to fall on one side of their respective helices, suggesting that they may interact. In support of this, cysteines substituted at position 223 in TMD5 and position 247 in TMD6 formed a disulfide bond, providing the first direct evidence of an interaction between these transmembrane domains in the alpha-factor receptor. Altogether, these results identify an important region of interaction between conserved hydrophobic regions at the base of TMD5 and TMD6 that is required for the proper regulation of receptor signaling. 相似文献
20.
The interactions of chi-conopeptide MrIA with the human norepinephrine transporter (hNET) were investigated by determining the effects of hNET point mutations on the inhibitory potency of MrIA. The mutants were produced by site-directed mutagenesis and expressed in COS-7 cells. The potency of MrIA was greater for inhibition of uptake by hNET of [3H]norepinephrine (Ki 1.89 microM) than [3H]dopamine (Ki 4.33 microM), and the human dopamine transporter and serotonin transporter were not inhibited by MrIA (to 7 microM). Of 18 mutations where hNET amino acid residues were exchanged with those of the human dopamine transporter, MrIA had increased potency for inhibition of [3H]norepinephrine uptake for three mutations (in predicted extracellular loops 3 and 4 and transmembrane domain (TMD) 8) and decreased potency for one mutation (in TMD6 and intracellular loop (IL) 3). Of the 12 additional mutations in TMDs 2, 4, 5, and 11 and IL1, three mutations (in TMD2 and IL1) had reduced MrIA inhibitory potency. All of the other mutations tested had no influence on MrIA potency. A comparison of the results with previous data for desipramine and cocaine inhibition of norepinephrine uptake by the mutant hNETs reveals that MrIA binding to hNET occurs at a site that is distinct from but overlaps with the binding sites for tricyclic antidepressants and cocaine. 相似文献