RESUMO
The Na+-Cl- cotransporter (NCC) drives salt reabsorption in the kidney and plays a decisive role in balancing electrolytes and blood pressure. Thiazide and thiazide-like diuretics inhibit NCC-mediated renal salt retention and have been cornerstones for treating hypertension and edema since the 1950s. Here we determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they fit into an orthosteric site and occlude the NCC ion translocation pathway. Aberrant NCC activation by the WNKs-SPAK kinase cascade underlies Familial Hyperkalemic Hypertension, but it remains unknown whether/how phosphorylation transforms the NCC structure to accelerate ion translocation. We show that an intracellular amino-terminal motif of NCC, once phosphorylated, associates with the carboxyl-terminal domain, and together, they interact with the transmembrane domain. These interactions suggest a phosphorylation-dependent allosteric network that directly influences NCC ion translocation.
Assuntos
Hidroclorotiazida , Inibidores de Simportadores de Cloreto de Sódio , Membro 3 da Família 12 de Carreador de Soluto , Fosforilação , Membro 3 da Família 12 de Carreador de Soluto/metabolismo , Membro 3 da Família 12 de Carreador de Soluto/química , Humanos , Hidroclorotiazida/farmacologia , Hidroclorotiazida/química , Inibidores de Simportadores de Cloreto de Sódio/farmacologia , Animais , Clortalidona/metabolismo , Clortalidona/química , Clortalidona/farmacologia , Proteínas Quinases/metabolismo , Proteínas Quinases/química , Diuréticos/farmacologia , Diuréticos/química , Diuréticos/metabolismo , Tiazidas/farmacologia , Tiazidas/química , Tiazidas/metabolismo , Células HEK293 , Modelos Moleculares , Proteínas Serina-Treonina QuinasesRESUMO
Polycystin-1 (PC-1) and PC-2 form a heteromeric ion channel complex that is abundantly expressed in primary cilia of renal epithelial cells. This complex functions as a non-selective cation channel, and mutations within the polycystin complex cause autosomal dominant polycystic kidney disease (ADPKD). The spatial and temporal regulation of the polycystin complex within the ciliary membrane remains poorly understood. Using both whole-cell and ciliary patch-clamp recordings, we identify a cilia-enriched oxysterol, 7ß,27-dihydroxycholesterol (DHC), that serves as a necessary activator of the polycystin complex. We further identify an oxysterol-binding pocket within PC-2 and showed that mutations within this binding pocket disrupt 7ß,27-DHC-dependent polycystin activation. Pharmacologic and genetic inhibition of oxysterol synthesis reduces channel activity in primary cilia. In summary, our findings reveal a regulator of the polycystin complex. This oxysterol-binding pocket in PC-2 may provide a specific target for potential ADPKD therapeutics.
Assuntos
Cílios , Canais de Cátion TRPP , Cílios/metabolismo , Canais de Cátion TRPP/metabolismo , Canais de Cátion TRPP/genética , Animais , Humanos , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/patologia , Oxisteróis/metabolismo , Técnicas de Patch-Clamp , Células HEK293 , Mutação , Rim/metabolismo , Camundongos , Sítios de LigaçãoRESUMO
The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.
Assuntos
Cloretos , Água , Membro 2 da Família 12 de Carreador de Soluto/química , Membro 2 da Família 12 de Carreador de Soluto/genética , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Cloretos/metabolismo , Mutagênese , Cátions/metabolismo , Água/metabolismoRESUMO
Loop and thiazide diuretics have been cornerstones of clinical management of hypertension and fluid overload conditions for more than five decades. The hunt for their molecular targets led to the discovery of cation-chloride cotransporters (CCCs) that catalyze electroneutral movement of Cl- together with Na+ and/or K+. CCCs consist of two 1 Na+-1 K+-2 Cl- (NKCC1-2), one 1 Na+-1 Cl- (NCC), and four 1 K+-1 Cl- (KCC1-4) transporters in human. CCCs are fundamental in trans-epithelia ion secretion and absorption, homeostasis of intracellular Cl- concentration and cell volume, and regulation of neuronal excitability. Malfunction of NKCC2 and NCC leads to abnormal salt and water retention in the kidney and, consequently, imbalance in electrolytes and blood pressure. Mutations in KCC2 and KCC3 are associated with brain disorders due to impairments in regulation of excitability and possibly cell volume of neurons. A recent surge of structures of CCCs have defined their dimeric architecture, their ion binding sites, their conformational changes associated with ion translocation, and the mechanisms of action of loop diuretics and small molecule inhibitors. These breakthroughs now set the stage to expand CCC pharmacology beyond loop and thiazide diuretics, developing the next generation of diuretics with improved potency and specificity. Beyond drugging renal-specific CCCs, brain-penetrable therapeutics are sorely needed to target CCCs in the nervous system for the treatment of neurological disorders and psychiatric conditions.
RESUMO
Cation-chloride cotransporters (CCCs) catalyze electroneutral symport of Cl- with Na+ and/or K+ across membranes. CCCs are fundamental in cell volume homeostasis, transepithelia ion movement, maintenance of intracellular Cl- concentration, and neuronal excitability. Here, we present a cryoelectron microscopy structure of human K+-Cl- cotransporter (KCC)1 bound with the VU0463271 inhibitor in an outward-open state. In contrast to many other amino acid-polyamine-organocation transporter cousins, our first outward-open CCC structure reveals that opening the KCC1 extracellular ion permeation path does not involve hinge-bending motions of the transmembrane (TM) 1 and TM6 half-helices. Instead, rocking of TM3 and TM8, together with displacements of TM4, TM9, and a conserved intracellular loop 1 helix, underlie alternate opening and closing of extracellular and cytoplasmic vestibules. We show that KCC1 intriguingly exists in one of two distinct dimeric states via different intersubunit interfaces. Our studies provide a blueprint for understanding the mechanisms of CCCs and their inhibition by small molecule compounds.
Assuntos
Membro 4 da Família 12 de Carreador de Soluto , Simportadores , Cátions/metabolismo , Cloretos/metabolismo , Microscopia Crioeletrônica , Humanos , Transporte de Íons , Conformação Proteica em alfa-Hélice , Membro 4 da Família 12 de Carreador de Soluto/química , Simportadores/antagonistas & inibidores , Simportadores/química , Cotransportadores de K e Cl-RESUMO
Cation-chloride cotransporters (CCCs) NKCC1 and NKCC2 catalyze electroneutral symport of 1 Na+, 1 K+, and 2 Cl- across cell membranes. NKCC1 mediates trans-epithelial Cl- secretion and regulates excitability of some neurons and NKCC2 is critical to renal salt reabsorption. Both transporters are inhibited by the so-called loop diuretics including bumetanide, and these drugs are a mainstay for treating edema and hypertension. Here, our single-particle electron cryo-microscopy structures supported by functional studies reveal an outward-facing conformation of NKCC1, showing bumetanide wedged into a pocket in the extracellular ion translocation pathway. Based on these and the previously published inward-facing structures, we define the translocation pathway and the conformational changes necessary for ion translocation. We also identify an NKCC1 dimer with separated transmembrane domains and extensive transmembrane and C-terminal domain interactions. We further define an N-terminal phosphoregulatory domain that interacts with the C-terminal domain, suggesting a mechanism whereby (de)phosphorylation regulates NKCC1 by tuning the strength of this domain association.
Assuntos
Bumetanida , Simportadores , Bumetanida/farmacologia , Cátions/metabolismo , Cloretos/metabolismo , Diuréticos/farmacologia , Membro 2 da Família 12 de Carreador de SolutoRESUMO
Mutations in the polycystin proteins, PC-1 and PC-2, result in autosomal dominant polycystic kidney disease (ADPKD) and ultimately renal failure. PC-1 and PC-2 enrich on primary cilia, where they are thought to form a heteromeric ion channel complex. However, a functional understanding of the putative PC-1/PC-2 polycystin complex is lacking due to technical hurdles in reliably measuring its activity. Here we successfully reconstitute the PC-1/PC-2 complex in the plasma membrane of mammalian cells and show that it functions as an outwardly rectifying channel. Using both reconstituted and ciliary polycystin channels, we further show that a soluble fragment generated from the N-terminal extracellular domain of PC-1 functions as an intrinsic agonist that is necessary and sufficient for channel activation. We thus propose that autoproteolytic cleavage of the N-terminus of PC-1, a hotspot for ADPKD mutations, produces a soluble ligand in vivo. These findings establish a mechanistic framework for understanding the role of PC-1/PC-2 heteromers in ADPKD and suggest new therapeutic strategies that would expand upon the limited symptomatic treatments currently available for this progressive, terminal disease.
On the surface of most animal and other eukaryotic cells are small rod-like protrusions known as primary cilia. Each cilium is encased by a specialized membrane which is enriched in protein complexes that help the cell sense its local environment. Some of these complexes help transport ions in out of the cell, while others act as receptors that receive chemical signals called ligands. A unique ion channel known as the polycystin complex is able to perform both of these roles as it contains a receptor called PC-1 in addition to an ion channel called PC-2. Various mutations in the genes that code for PC-1 and PC-2 can result in autosomal dominant polycystic kidney disease (ADPKD), which is the most common monogenetic disease in humans. However, due to the small size of primary cilia which are less than a thousandth of a millimeter thick little is known about how polycystin complexes are regulated and how mutations lead to ADPKD. To overcome this barrier, Ha et al. modified kidney cells grown in the lab so that PC-1 and PC-2 form a working channel in the plasma membrane which surrounds the entire cell. As the body of a cell is around 10,000 times bigger than the cilium, this allowed the movement of ions across the polycystin complex to be studied using conventional techniques. Experiments using this newly developed assay revealed that a region at one of the ends of the PC-1 protein, named the C-type lectin domain, is essential for stimulating polycystin complexes. Ha et al. found that this domain of PC-1 is able to cut itself from the protein complex. Further experiments showed that when fragments of PC-1, which contain the C-type lectin domain, are no longer bound to the membrane, they can activate the polycystin channels in cilia as well as the plasma membrane. This suggests that this region of PC-1 may also act as a secreted ligand that can activate other polycystin channels. Some of the genetic mutations that cause ADPKD likely disrupt the activity of the polycystin complex and reduce its ability to transport ions across the cilia membrane. Therefore, the cell assay created in this study could be used to screen for small molecules that can restore the activity of these ion channels in patients with ADPKD.
Assuntos
Membrana Celular/metabolismo , Cílios/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/metabolismo , Animais , Sinalização do Cálcio , Membrana Celular/química , Membrana Celular/genética , Cílios/química , Cílios/genética , Células HEK293 , Humanos , Potenciais da Membrana , Camundongos , Modelos Moleculares , Complexos Multiproteicos , Mutação , Rim Policístico Autossômico Dominante/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Genetic variants in PKD2 which encodes for the polycystin-2 ion channel are responsible for many clinical cases of autosomal dominant polycystic kidney disease (ADPKD). Despite our strong understanding of the genetic basis of ADPKD, we do not know how most variants impact channel function. Polycystin-2 is found in organelle membranes, including the primary cilium-an antennae-like structure on the luminal side of the collecting duct. In this study, we focus on the structural and mechanistic regulation of polycystin-2 by its TOP domain-a site with unknown function that is commonly altered by missense variants. We use direct cilia electrophysiology, cryogenic electron microscopy, and superresolution imaging to determine that variants of the TOP domain finger 1 motif destabilizes the channel structure and impairs channel opening without altering cilia localization and channel assembly. Our findings support the channelopathy classification of PKD2 variants associated with ADPKD, where polycystin-2 channel dysregulation in the primary cilia may contribute to cystogenesis.
Assuntos
Cálcio/metabolismo , Cílios/patologia , Ativação do Canal Iônico , Mutação , Rim Policístico Autossômico Dominante/patologia , Canais de Cátion TRPP/metabolismo , Cílios/metabolismo , Células HEK293 , Humanos , Rim Policístico Autossômico Dominante/genética , Rim Policístico Autossômico Dominante/metabolismo , Domínios Proteicos , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genéticaRESUMO
The secondary active cation-chloride cotransporters (CCCs) utilize the existing Na+ and/or K+ gradients to move Cl- into or out of cells. NKCC1 is an intensively studied member of the CCC family and plays fundamental roles in regulating trans-epithelial ion movement, cell volume, chloride homeostasis and neuronal excitability. Here, we report a cryo-EM structure of human NKCC1 captured in a partially loaded, inward-open state. NKCC1 assembles into a dimer, with the first ten transmembrane (TM) helices harboring the transport core and TM11-TM12 helices lining the dimer interface. TM1 and TM6 helices break α-helical geometry halfway across the lipid bilayer where ion binding sites are organized around these discontinuous regions. NKCC1 may harbor multiple extracellular entryways and intracellular exits, raising the possibility that K+, Na+, and Cl- ions may traverse along their own routes for translocation. NKCC1 structure provides a blueprint for further probing structure-function relationships of NKCC1 and other CCCs.
Assuntos
Membro 2 da Família 12 de Carreador de Soluto/química , Sítios de Ligação , Microscopia Crioeletrônica , Humanos , Transporte de Íons , Modelos Moleculares , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Imagem Individual de Molécula , Membro 2 da Família 12 de Carreador de Soluto/genéticaRESUMO
Transient receptor potential (TRP) ion channels are evolutionarily ancient sensory proteins that detect and integrate a wide range of physical and chemical stimuli. TRP channels are fundamental for numerous biological processes and are therefore associated with a multitude of inherited and acquired human disorders. In contrast to many other major ion channel families, high-resolution structures of TRP channels were not available before 2013. Remarkably, however, the subsequent "resolution revolution" in cryo-EM has led to an explosion of TRP structures in the last few years. These structures have confirmed that TRP channels assemble as tetramers and resemble voltage-gated ion channels in their overall architecture. But beyond the relatively conserved transmembrane core embedded within the lipid bilayer, each TRP subtype appears to be endowed with a unique set of soluble domains that may confer diverse regulatory mechanisms. Importantly, TRP channel TR structures have revealed sites and mechanisms of action of numerous synthetic and natural compounds, as well as those for endogenous ligands such as lipids, Ca2+, and calmodulin. Here, I discuss these recent findings with a particular focus on the conserved transmembrane region and how these structures may help to rationally target this important class of ion channels for the treatment of numerous human conditions.
Assuntos
Canais de Potencial de Receptor Transitório/química , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Humanos , Ligantes , Domínios Proteicos/fisiologiaRESUMO
The original version of this Article contained an error in the spelling of the author David Bulkley, which was incorrectly given as David Bulkey. This has now been corrected in both the PDF and HTML versions of the Article.
RESUMO
PKD2 and PKD1 genes are mutated in human autosomal dominant polycystic kidney disease. PKD2 can form either a homomeric cation channel or a heteromeric complex with the PKD1 receptor, presumed to respond to ligand(s) and/or mechanical stimuli. Here, we identify a two-residue hydrophobic gate in PKD2L1, and a single-residue hydrophobic gate in PKD2. We find that a PKD2 gain-of-function gate mutant effectively rescues PKD2 knockdown-induced phenotypes in embryonic zebrafish. The structure of a PKD2 activating mutant F604P by cryo-electron microscopy reveals a π- to α-helix transition within the pore-lining helix S6 that leads to repositioning of the gate residue and channel activation. Overall the results identify hydrophobic gates and a gating mechanism of PKD2 and PKD2L1.
Assuntos
Canais de Cálcio/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Receptores de Superfície Celular/metabolismo , Canais de Cátion TRPP/metabolismo , Regulação Alostérica , Sequência de Aminoácidos , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Microscopia Crioeletrônica , Feminino , Técnicas de Silenciamento de Genes , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico , Modelos Moleculares , Mutação , Rim Policístico Autossômico Dominante/genética , Conformação Proteica , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPP/química , Canais de Cátion TRPP/genética , Xenopus , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/genéticaRESUMO
The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.
Assuntos
Rim Policístico Autossômico Dominante/metabolismo , Canais de Cátion TRPP/química , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , Microscopia Crioeletrônica , Células HEK293 , Humanos , Bicamadas Lipídicas/química , Mutação de Sentido Incorreto , Nanoestruturas/química , Rim Policístico Autossômico Dominante/genética , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Canais de Cátion TRPP/genéticaRESUMO
When integral membrane proteins are visualized in detergents or other artificial systems, an important layer of information is lost regarding lipid interactions and their effects on protein structure. This is especially relevant to proteins for which lipids have both structural and regulatory roles. Here we demonstrate the power of combining electron cryo-microscopy with lipid nanodisc technology to ascertain the structure of the rat TRPV1 ion channel in a native bilayer environment. Using this approach, we determined the locations of annular and regulatory lipids and showed that specific phospholipid interactions enhance binding of a spider toxin to TRPV1 through formation of a tripartite complex. Furthermore, phosphatidylinositol lipids occupy the binding site for capsaicin and other vanilloid ligands, suggesting a mechanism whereby chemical or thermal stimuli elicit channel activation by promoting the release of bioactive lipids from a critical allosteric regulatory site.
Assuntos
Bicamadas Lipídicas/metabolismo , Nanoestruturas/química , Venenos de Aranha/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Sítio Alostérico/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Capsaicina/metabolismo , Microscopia Crioeletrônica , Ligantes , Bicamadas Lipídicas/química , Proteínas de Membrana/química , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Dados de Sequência Molecular , Nanoestruturas/ultraestrutura , Fosfatos de Fosfatidilinositol/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ratos , Venenos de Aranha/química , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/ultraestrutura , TemperaturaRESUMO
The transient receptor potential (TRP) ion channel family is large and functionally diverse, second only to potassium channels. Despite their prominence within the animal kingdom, TRP channels have resisted crystallization and structural determination for many years. This barrier was recently broken when the three-dimensional structure of the vanilloid receptor 1 (TRPV1) was determined by single particle electron cryo-microscopy (cryo-EM). Moreover, this is the first example in which the near atomic resolution structure of an integral membrane protein was elucidated by this technique and in a manner not requiring crystals, demonstrating the transformative power of single particle cryo-EM for revealing high-resolution structures of integral membrane proteins, particularly those of mammalian origin. Here we summarize technical advances, in both biochemistry and cryo-EM, that led to this major breakthrough.
Assuntos
Microscopia Crioeletrônica/métodos , Mamíferos , Canais de Cátion TRPV/química , Animais , Canais de Cátion TRPV/metabolismoRESUMO
Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 Å resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5-6 (S5-S6) and the intervening pore loop, which is flanked by S1-S4 voltage-sensor-like domains. TRPV1 has a wide extracellular 'mouth' with a short selectivity filter. The conserved 'TRP domain' interacts with the S4-S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function.
Assuntos
Microscopia Crioeletrônica , Modelos Moleculares , Canais de Cátion TRPV/química , Animais , Repetição de Anquirina , Células HEK293 , Humanos , Estrutura Terciária de Proteína , RatosRESUMO
Transient receptor potential (TRP) channels are polymodal signal detectors that respond to a wide range of physical and chemical stimuli. Elucidating how these channels integrate and convert physiological signals into channel opening is essential to understanding how they regulate cell excitability under normal and pathophysiological conditions. Here we exploit pharmacological probes (a peptide toxin and small vanilloid agonists) to determine structures of two activated states of the capsaicin receptor, TRPV1. A domain (consisting of transmembrane segments 1-4) that moves during activation of voltage-gated channels remains stationary in TRPV1, highlighting differences in gating mechanisms for these structurally related channel superfamilies. TRPV1 opening is associated with major structural rearrangements in the outer pore, including the pore helix and selectivity filter, as well as pronounced dilation of a hydrophobic constriction at the lower gate, suggesting a dual gating mechanism. Allosteric coupling between upper and lower gates may account for rich physiological modulation exhibited by TRPV1 and other TRP channels.
Assuntos
Modelos Moleculares , Canais de Cátion TRPV/química , Canais de Cátion TRPV/fisiologia , Animais , Mutação , Estrutura Terciária de Proteína , Ratos , Canais de Cátion TRPV/genéticaRESUMO
The capsaicin receptor, TRPV1, is regulated by phosphatidylinositol-4,5-bisphosphate (PIP(2)), although the precise nature of this effect (i.e., positive or negative) remains controversial. Here, we reconstitute purified TRPV1 into artificial liposomes, where it is gated robustly by capsaicin, protons, spider toxins, and, notably, heat, demonstrating intrinsic sensitivity of the channel to both chemical and thermal stimuli. TRPV1 is fully functional in the absence of phosphoinositides, arguing against their proposed obligatory role in channel activation. Rather, introduction of various phosphoinositides, including PIP(2), PI4P, and phosphatidylinositol, inhibits TRPV1, supporting a model whereby phosphoinositide turnover contributes to thermal hyperalgesia by disinhibiting the channel. Using an orthogonal chemical strategy, we show that association of the TRPV1 C terminus with the bilayer modulates channel gating, consistent with phylogenetic data implicating this domain as a key regulatory site for tuning stimulus sensitivity. Beyond TRPV1, these findings are relevant to understanding how membrane lipids modulate other "receptor-operated" TRP channels.
Assuntos
Temperatura Alta/efeitos adversos , Canais Iônicos/metabolismo , Lipídeos/fisiologia , Fosfatidilinositóis/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Capsaicina/farmacologia , Células Cultivadas , Células Sf9/metabolismo , SpodopteraRESUMO
Programmed death-1 (PD-1) is a member of the CD28/B7 superfamily that delivers negative signals upon interaction with its two ligands, PD-L1 or PD-L2. The high-resolution crystal structure of the complex formed by the complete ectodomains of murine PD-1 and PD-L2 revealed a 1:1 receptor:ligand stoichiometry and displayed a binding interface and overall molecular organization distinct from that observed in the CTLA-4/B7 inhibitory complexes. Furthermore, our structure also provides insights into the association between PD-1 and PD-L1 and highlights differences in the interfaces formed by the two PD-1 ligands (PD-Ls) Mutagenesis studies confirmed the details of the proposed PD-1/PD-L binding interfaces and allowed for the design of a mutant PD-1 receptor with enhanced affinity. These studies define spatial and organizational constraints that control the localization and signaling of PD-1/PD-L complexes within the immunological synapse and provide a basis for manipulating the PD-1 pathways for immunotherapy.