RESUMEN
Transient Receptor Potential Mucolipin 1 (TRPML1) is a lysosomal cation channel whose loss-of-function mutations directly cause the lysosomal storage disorder mucolipidosis type IV (MLIV). TRPML1 can be allosterically regulated by various ligands including natural lipids and small synthetic molecules and the channel undergoes a global movement propagated from ligand-induced local conformational changes upon activation. In this study, we identified a functionally critical residue, Tyr404, at the C-terminus of the S4 helix, whose mutations to tryptophan and alanine yield gain- and loss-of-function channels, respectively. These allosteric mutations mimic the ligand activation or inhibition of the TRPML1 channel without interfering with ligand binding and both mutant channels are susceptible to agonist or antagonist modulation, making them better targets for screening potent TRPML1 activators and inhibitors. We also determined the high-resolution structure of TRPML1 in complex with the PI(4,5)P2 inhibitor, revealing the structural basis underlying this lipid inhibition. In addition, an endogenous phospholipid likely from sphingomyelin is identified in the PI(4,5)P2-bound TRPML1 structure at the same hotspot for agonists and antagonists, providing a plausible structural explanation for the inhibitory effect of sphingomyelin on agonist activation.
RESUMEN
Na+/Ca2+ exchangers (NCX) transport Ca2+ in or out of cells in exchange for Na+. They are ubiquitously expressed and play an essential role in maintaining cytosolic Ca2+ homeostasis. Although extensively studied, little is known about the global structural arrangement of eukaryotic NCXs and the structural mechanisms underlying their regulation by various cellular cues including cytosolic Na+ and Ca2+. Here we present the cryo-EM structures of human cardiac NCX1 in both inactivated and activated states, elucidating key structural elements important for NCX ion exchange function and its modulation by cytosolic Ca2+ and Na+. We demonstrate that the interactions between the ion-transporting transmembrane (TM) domain and the cytosolic regulatory domain define the activity of NCX. In the inward-facing state with low cytosolic [Ca2+], a TM-associated four-stranded ß-hub mediates a tight packing between the TM and cytosolic domains, resulting in the formation of a stable inactivation assembly that blocks the TM movement required for ion exchange function. Ca2+ binding to the cytosolic second Ca2+-binding domain (CBD2) disrupts this inactivation assembly which releases its constraint on the TM domain, yielding an active exchanger. Thus, the current NCX1 structures provide an essential framework for the mechanistic understanding of the ion transport and cellular regulation of NCX family proteins.
Asunto(s)
Corazón , Intercambiador de Sodio-Calcio , Humanos , Intercambiador de Sodio-Calcio/metabolismo , Transporte Iónico , Citosol/metabolismo , Calcio/metabolismoRESUMEN
Maintaining a highly acidic lysosomal pH is central to cellular physiology. Here, we use functional proteomics, single-particle cryo-EM, electrophysiology, and in vivo imaging to unravel a key biological function of human lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in regulating lysosomal pH homeostasis. Despite being widely used as a lysosomal marker, the physiological functions of the LAMP proteins have long been overlooked. We show that LAMP-1 and LAMP-2 directly interact with and inhibit the activity of the lysosomal cation channel TMEM175, a key player in lysosomal pH homeostasis implicated in Parkinson's disease. This LAMP inhibition mitigates the proton conduction of TMEM175 and facilitates lysosomal acidification to a lower pH environment crucial for optimal hydrolase activity. Disrupting the LAMP-TMEM175 interaction alkalinizes the lysosomal pH and compromises the lysosomal hydrolytic function. In light of the ever-increasing importance of lysosomes to cellular physiology and diseases, our data have widespread implications for lysosomal biology.
Asunto(s)
Enfermedad de Parkinson , Humanos , Concentración de Iones de Hidrógeno , Proteínas de Membrana de los Lisosomas/genética , Proteínas de Membrana de los Lisosomas/metabolismo , Lisosomas/metabolismo , Enfermedad de Parkinson/metabolismo , Canales de Potasio/metabolismoRESUMEN
Two-pore channels (TPCs) belong to the family of voltage-gated tetrameric cation channels and are ubiquitously expressed in organelles of animals and plants. These channels are believed to be evolutionary intermediates between homotetrameric voltage-gated potassium/sodium channels and the four-domain, single subunit, voltage-gated sodium/calcium channels. Each TPC subunit contains 12 transmembrane segments that can be divided into two homologous copies of an S1-S6 Shaker-like 6-TM domain. A functional TPC channel assembles as a dimer - the equivalent of a voltage-gated tetrameric cation channel. The plant TPC channel is localized in the vacuolar membrane and is also called the SV channel for generating the slow vacuolar (SV) current observed long before its molecular identification. Three subfamilies of mammalian TPC channels have been defined - TPC1, 2, and 3 - with the first two being ubiquitously expressed in animals and TPC3 being expressed in some animals but not in humans. Mammalian TPC1 and TPC2 are localized to the endolysosomal membrane and their functions are associated with various physiological processes. TPC3 is localized in the plasma membrane and its physiological function is not well defined.
Asunto(s)
Canales de Calcio , Canales de Sodio Activados por Voltaje , Humanos , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Activación del Canal Iónico , Endosomas/metabolismo , Cationes/metabolismo , Canales de Sodio Activados por Voltaje/metabolismo , Mamíferos/metabolismoRESUMEN
Transient receptor potential mucolipin 1 (TRPML1) is a Ca2+-permeable, nonselective cation channel ubiquitously expressed in the endolysosomes of mammalian cells and its loss-of-function mutations are the direct cause of type IV mucolipidosis (MLIV), an autosomal recessive lysosomal storage disease. TRPML1 is a ligand-gated channel that can be activated by phosphatidylinositol 3,5-bisphosphate [PI(3,5)P2] as well as some synthetic small-molecule agonists. Recently, rapamycin has also been shown to directly bind and activate TRPML1. Interestingly, both PI(3,5)P2 and rapamycin have low efficacy in channel activation individually but together they work cooperatively and activate the channel with high potency. To reveal the structural basis underlying the synergistic activation of TRPML1 by PI(3,5)P2 and rapamycin, we determined the high-resolution cryoelectron microscopy (cryo-EM) structures of the mouse TRPML1 channel in various states, including apo closed, PI(3,5)P2-bound closed, and PI(3,5)P2/temsirolimus (a rapamycin analog)-bound open states. These structures, combined with electrophysiology, elucidate the molecular details of ligand binding and provide structural insight into how the TRPML1 channel integrates two distantly bound ligand stimuli and facilitates channel opening.
Asunto(s)
Fosfatos de Fosfatidilinositol/farmacología , Sirolimus/farmacología , Canales de Potencial de Receptor Transitorio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Canales de Potencial de Receptor Transitorio/genéticaRESUMEN
Calcium is one of the most important second messengers in cells. The uptake and release of calcium ions are conducted by channels and transporters. Inside a eukaryotic cell, calcium is stored in intracellular organelles including the endoplasmic reticulum (ER), mitochondrion, and lysosome. Lysosomes are acid membrane-bounded organelles serving as the crucial degradation and recycling center of the cell. Lysosomes involve in multiple important signaling events, including nutrient sensing, lipid metabolism, and trafficking. Hitherto, two lysosomal cation channel families have been suggested to function as calcium release channels, namely the Two-pore Channel (TPC) family, and the Transient Receptor Potential Channel Mucolipin (TRPML) family. Additionally, a few plasma membrane calcium channels have also been found in the lysosomal membrane under certain circumstances. In this review, we will discuss the structural mechanism of the cation channels that may be important for lysosomal calcium release, primarily focusing on the TPCs and TRPMLs.
Asunto(s)
Calcio , Canales de Potencial de Receptor Transitorio , Calcio/metabolismo , Canales de Calcio/metabolismo , Señalización del Calcio , Cationes/metabolismo , Humanos , Lisosomas/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismoRESUMEN
Mammalian cyclic nucleotide-gated (CNG) channels are nonselective cation channels activated by cGMP or cAMP and play essential roles in the signal transduction of the visual and olfactory sensory systems. CNGA1, the principal component of the CNG channel from rod photoreceptors, can by itself form a functional homotetrameric channel and has been used as the model system in the majority of rod CNG studies. However, the native rod CNG functions as a heterotetramer consisting of three A1 and one B1 subunits and exhibits different functional properties than the CNGA1 homomer. Here we present the functional analysis of human rod CNGA1/B1 heterotetramer and its cryo-EM structures in apo, cGMP-bound, cAMP-bound, and L-cis-Diltiazem-blocked states. These structures, with resolution ranging from 2.6 to 3.3 Å, elucidate the structural mechanisms underlying the 3:1 subunit stoichiometry, the asymmetrical gating upon cGMP activation, and the unique pharmacological property of the native rod CNG channel.
Asunto(s)
GMP Cíclico , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Animales , Humanos , MamíferosRESUMEN
Arabidopsis thaliana two-pore channel AtTPC1 is a voltage-gated, Ca2+-modulated, nonselective cation channel that is localized in the vacuolar membrane and responsible for generating slow vacuolar (SV) current. Under depolarizing membrane potential, cytosolic Ca2+ activates AtTPC1 by binding at the EF-hand domain, whereas luminal Ca2+ inhibits the channel by stabilizing the voltage-sensing domain II (VSDII) in the resting state. Here, we present 2.8 to 3.3 Å cryoelectron microscopy (cryo-EM) structures of AtTPC1 in two conformations, one in closed conformation with unbound EF-hand domain and resting VSDII and the other in a partially open conformation with Ca2+-bound EF-hand domain and activated VSDII. Structural comparison between the two different conformations allows us to elucidate the structural mechanisms of voltage gating, cytosolic Ca2+ activation, and their coupling in AtTPC1. This study also provides structural insight into the general voltage-gating mechanism among voltage-gated ion channels.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Canales de Calcio/metabolismo , Calcio/metabolismo , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Canales de Calcio/genética , Cationes/metabolismo , Microscopía por Crioelectrón/métodos , Citosol/metabolismo , Activación del Canal Iónico , Potenciales de la Membrana/fisiología , Vacuolas/metabolismoRESUMEN
TMEM120A, also named as TACAN, is a novel membrane protein highly conserved in vertebrates and was recently proposed to be a mechanosensitive channel involved in sensing mechanical pain. Here we present the single-particle cryogenic electron microscopy (cryo-EM) structure of human TMEM120A, which forms a tightly packed dimer with extensive interactions mediated by the N-terminal coiled coil domain (CCD), the C-terminal transmembrane domain (TMD), and the re-entrant loop between the two domains. The TMD of each TMEM120A subunit contains six transmembrane helices (TMs) and has no clear structural feature of a channel protein. Instead, the six TMs form an α-barrel with a deep pocket where a coenzyme A (CoA) molecule is bound. Intriguingly, some structural features of TMEM120A resemble those of elongase for very long-chain fatty acids (ELOVL) despite the low sequence homology between them, pointing to the possibility that TMEM120A may function as an enzyme for fatty acid metabolism, rather than a mechanosensitive channel.
Asunto(s)
Coenzima A/metabolismo , Elongasas de Ácidos Grasos/química , Ácidos Grasos/química , Canales Iónicos/química , Canales Iónicos/metabolismo , Proteínas Portadoras , Fenómenos Electrofisiológicos , Ácidos Grasos/clasificación , Ácidos Grasos/metabolismo , Células HEK293 , Humanos , Canales Iónicos/genética , Metabolismo de los Lípidos , Proteínas de la Membrana , Membranas , Unión ProteicaRESUMEN
Mammalian cyclic nucleotide-gated (CNG) channels play an essential role in the signal transduction of the visual and olfactory sensory systems. Here we reveal the structural mechanism of ligand gating in human rod CNGA1 channel by determining its cryo-EM structures in both the apo closed and cGMP-bound open states. Distinct from most other members of voltage-gated tetrameric cation channels, CNGA1 forms a central channel gate in the middle of the membrane, occluding the central cavity. Structural analyses of ion binding profiles in the selectivity filters of the wild-type channel and the E365Q filter mutant allow us to unambiguously define the two Ca2+ binding sites inside the selectivity filter, providing structural insights into Ca2+ blockage and permeation in CNG channels. The structure of the E365Q mutant also reveals two alternative side-chain conformations at Q365, providing a plausible explanation for the voltage-dependent gating of CNG channel acquired upon E365 mutation.
Asunto(s)
Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Activación del Canal Iónico/fisiología , Sitios de Unión , Calcio/metabolismo , GMP Cíclico/metabolismo , Células HEK293 , Humanos , Conformación ProteicaRESUMEN
Mitochondrial Ca2+ uptake is mediated by an inner mitochondrial membrane protein called the mitochondrial calcium uniporter. In humans, the uniporter functions as a holocomplex consisting of MCU, EMRE, MICU1 and MICU2, among which MCU and EMRE form a subcomplex and function as the conductive channel while MICU1 and MICU2 are EF-hand proteins that regulate the channel activity in a Ca2+-dependent manner. Here, we present the EM structures of the human mitochondrial calcium uniporter holocomplex (uniplex) in the presence and absence of Ca2+, revealing distinct Ca2+ dependent assembly of the uniplex. Our structural observations suggest that Ca2+ changes the dimerization interaction between MICU1 and MICU2, which in turn determines how the MICU1-MICU2 subcomplex interacts with the MCU-EMRE channel and, consequently, changes the distribution of the uniplex assemblies between the blocked and unblocked states.
Asunto(s)
Canales de Calcio/química , Canales de Calcio/metabolismo , Calcio/química , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/metabolismo , Humanos , Proteínas de Transporte de Membrana Mitocondrial/química , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Conformación Proteica , Multimerización de ProteínaRESUMEN
ZnT8 is a Zn2+/H+ antiporter that belongs to SLC30 family and plays an essential role in regulating Zn2+ accumulation in the insulin secretory granules of pancreatic ß cells. However, the Zn2+/H+ exchange mechanism of ZnT8 remains unclear due to the lack of high-resolution structures. Here, we report the cryo-EM structures of human ZnT8 (HsZnT8) in both outward- and inward-facing conformations. HsZnT8 forms a dimeric structure with four Zn2+ binding sites within each subunit: a highly conserved primary site in transmembrane domain (TMD) housing the Zn2+ substrate; an interfacial site between TMD and C-terminal domain (CTD) that modulates the Zn2+ transport activity of HsZnT8; and two adjacent sites buried in the cytosolic domain and chelated by conserved residues from CTD and the His-Cys-His (HCH) motif from the N-terminal segment of the neighboring subunit. A comparison of the outward- and inward-facing structures reveals that the TMD of each HsZnT8 subunit undergoes a large structural rearrangement, allowing for alternating access to the primary Zn2+ site during the transport cycle. Collectively, our studies provide the structural insights into the Zn2+/H+ exchange mechanism of HsZnT8.
Asunto(s)
Transportador 8 de Zinc/ultraestructura , Secuencia de Aminoácidos , Animales , Antiportadores/metabolismo , Sitios de Unión , Microscopía por Crioelectrón , Células HEK293 , Humanos , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Alineación de Secuencia , Zinc/metabolismo , Transportador 8 de Zinc/metabolismoRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
A family of plant nuclear ion channels, including DMI1 (Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca2+ spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K+) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca2+-gated K+ (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca2+ sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca2+ binding sites to the physiological function of CASTOR as well as DMI1.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Lotus , Micorrizas , Rhizobium , Simbiosis , Activación del Canal Iónico , Membrana Nuclear/metabolismo , Proteínas de Plantas/metabolismo , Dominios ProteicosRESUMEN
Mitochondrial calcium uptake is crucial to the regulation of eukaryotic Ca2+ homeostasis and is mediated by the mitochondrial calcium uniporter (MCU). While MCU alone can transport Ca2+ in primitive eukaryotes, metazoans require an essential single membrane-spanning auxiliary component called EMRE to form functional channels; however, the molecular mechanism of EMRE regulation remains elusive. Here, we present the cryo-EM structure of the human MCU-EMRE complex, which defines the interactions between MCU and EMRE as well as pinpoints the juxtamembrane loop of MCU and extended linker of EMRE as the crucial elements in the EMRE-dependent gating mechanism among metazoan MCUs. The structure also features the dimerization of two MCU-EMRE complexes along an interface at the N-terminal domain (NTD) of human MCU that is a hotspot for post-translational modifications. Thus, the human MCU-EMRE complex, which constitutes the minimal channel components among metazoans, provides a framework for future mechanistic studies on MCU.
Asunto(s)
Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Complejos Multiproteicos/metabolismo , Multimerización de Proteína/fisiología , Canales de Calcio/genética , Células HEK293 , Humanos , Complejos Multiproteicos/genética , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
The otopetrin (OTOP) proteins were recently characterized as proton channels. Here we present the cryo-EM structure of OTOP3 from Xenopus tropicalis (XtOTOP3) along with functional characterization of the channel. XtOTOP3 forms a homodimer with each subunit containing 12 transmembrane helices that can be divided into two structurally homologous halves; each half assembles as an α-helical barrel that could potentially serve as a proton conduction pore. Both pores open from the extracellular half before becoming occluded at a central constriction point consisting of three highly conserved residues - Gln232/585-Asp262/Asn623-Tyr322/666 (the constriction triads). Mutagenesis shows that the constriction triad from the second pore is less amenable to perturbation than that of the first pore, suggesting an unequal contribution between the two pores to proton transport. We also identified several key residues at the interface between the two pores that are functionally important, particularly Asp509, which confers intracellular pH-dependent desensitization to OTOP channels.
Asunto(s)
Canales Iónicos/metabolismo , Canales Iónicos/ultraestructura , Xenopus , Animales , Microscopía por Crioelectrón , Análisis Mutacional de ADN , Canales Iónicos/genética , Mutagénesis , Conformación Proteica , Multimerización de ProteínaRESUMEN
Mammalian two-pore channels (TPCs) regulate the physiological functions of the endolysosome. Here we present cryo-EM structures of human TPC2 (HsTPC2), a phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-activated, Na+ selective channel, in the ligand-bound and apo states. The apo structure captures the closed conformation, while the ligand-bound form features the channel in both open and closed conformations. Combined with functional analysis, these structures provide insights into the mechanism of PI(3,5)P2-regulated gating of TPC2, which is distinct from that of TPC1. Specifically, the endolysosome-specific PI(3,5)P2 binds at the first 6-TM and activates the channel - independently of the membrane potential - by inducing a structural change at the pore-lining inner helix (IS6), which forms a continuous helix in the open state but breaks into two segments at Gly317 in the closed state. Additionally, structural comparison to the voltage-dependent TPC1 structure allowed us to identify Ile551 as being responsible for the loss of voltage dependence in TPC2.
Asunto(s)
Canales de Calcio/química , Fosfolípidos/química , Microscopía por Crioelectrón , Endosomas/química , Células HEK293 , Humanos , Activación del Canal Iónico , Iones , Ligandos , Lisosomas/química , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
Immune checkpoint blockade therapy has been successful in treating some types of cancer but has not shown clinical benefits for treating leukaemia1. This result suggests that leukaemia uses unique mechanisms to evade this therapy. Certain immune inhibitory receptors that are expressed by normal immune cells are also present on leukaemia cells. Whether these receptors can initiate immune-related primary signalling in tumour cells remains unknown. Here we use mouse models and human cells to show that LILRB4, an immunoreceptor tyrosine-based inhibition motif-containing receptor and a marker of monocytic leukaemia, supports tumour cell infiltration into tissues and suppresses T cell activity via a signalling pathway that involves APOE, LILRB4, SHP-2, uPAR and ARG1 in acute myeloid leukaemia (AML) cells. Deletion of LILRB4 or the use of antibodies to block LILRB4 signalling impeded AML development. Thus, LILRB4 orchestrates tumour invasion pathways in monocytic leukaemia cells by creating an immunosuppressive microenvironment. LILRB4 represents a compelling target for the treatment of monocytic AML.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Receptores de Superficie Celular/metabolismo , Transducción de Señal , Escape del Tumor/inmunología , Animales , Apolipoproteínas E/metabolismo , Arginasa/metabolismo , Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Tolerancia Inmunológica/inmunología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Receptores Inmunológicos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Escape del Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this Article, ref. 15 has been replaced and references 32 to 50 have been renumbered online.
RESUMEN
The mitochondrial calcium uniporter (MCU) is a highly selective calcium channel localized to the inner mitochondrial membrane. Here, we describe the structure of an MCU orthologue from the fungus Neosartorya fischeri (NfMCU) determined to 3.8 Å resolution by phase-plate cryo-electron microscopy. The channel is a homotetramer with two-fold symmetry in its amino-terminal domain (NTD) that adopts a similar structure to that of human MCU. The NTD assembles as a dimer of dimers to form a tetrameric ring that connects to the transmembrane domain through an elongated coiled-coil domain. The ion-conducting pore domain maintains four-fold symmetry, with the selectivity filter positioned at the start of the pore-forming TM2 helix. The aspartate and glutamate sidechains of the conserved DIME motif are oriented towards the central axis and separated by one helical turn. The structure of NfMCU offers insights into channel assembly, selective calcium permeation, and inhibitor binding.