RESUMO
Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.
Assuntos
Fusão de Membrana , Fosfatidilinositol 4,5-Difosfato , Eletricidade Estática , Água , Catálise , Lipossomos/química , Fusão de Membrana/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/farmacologia , Proteínas SNARE/metabolismo , Proteínas SNARE/química , Água/químicaRESUMO
The transient receptor potential melastatin 8 (TRPM8) channel is the primary molecular transducer responsible for the cool sensation elicited by menthol and cold in mammals. TRPM8 activation is controlled by cooling compounds together with the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Our knowledge of cold sensation and the therapeutic potential of TRPM8 for neuroinflammatory diseases and pain will be enhanced by understanding the structural basis of cooling agonist- and PIP2-dependent TRPM8 activation. We present cryo-electron microscopy structures of mouse TRPM8 in closed, intermediate, and open states along the ligand- and PIP2-dependent gating pathway. Our results uncover two discrete agonist sites, state-dependent rearrangements in the gate positions, and a disordered-to-ordered transition of the gate-forming S6-elucidating the molecular basis of chemically induced cool sensation in mammals.
Assuntos
Temperatura Baixa , Ativação do Canal Iônico , Fosfatidilinositol 4,5-Difosfato , Pirimidinonas , Canais de Cátion TRPM , Sensação Térmica , Animais , Camundongos , Microscopia Crioeletrônica , Ligantes , Mentol/química , Mentol/farmacologia , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/química , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/farmacologia , Sensação Térmica/efeitos dos fármacos , Sensação Térmica/fisiologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Conformação Proteica , Pirimidinonas/química , Pirimidinonas/farmacologiaRESUMO
Primary cilia are sensory organelles on many postmitotic cells. The ciliary membrane is continuous with the plasma membrane but differs in its phospholipid composition with phosphatidylinositol 4,5-bisposphate (PIP2) being much reduced toward the ciliary tip. In order to determine the functional significance of this difference, we used chemically induced protein dimerization to rapidly synthesize or degrade PIP2 selectively in the ciliary membrane. We observed ciliary fission when PIP2 was synthesized and a growing ciliary length when PIP2 was degraded. Ciliary fission required local actin polymerisation in the cilium, the Rho kinase Rac, aurora kinase A (AurkA) and histone deacetylase 6 (HDAC6). This pathway was previously described for ciliary disassembly before cell cycle re-entry. Activating ciliary receptors in the presence of dominant negative dynamin also increased ciliary PIP2, and the associated vesicle budding required ciliary PIP2. Finally, ciliary shortening resulting from constitutively increased ciliary PIP2 was mediated by the same actin - AurkA - HDAC6 pathway. Taken together, changes in ciliary PIP2 are a unifying point for ciliary membrane stability and turnover. Different stimuli increase ciliary PIP2 to secrete vesicles and reduce ciliary length by a common pathway. The paucity of PIP2 in the distal cilium therefore ensures ciliary stability.
Assuntos
Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/farmacologia , Actinas/genética , Actinas/metabolismo , Animais , Antibacterianos/farmacologia , Aurora Quinase A/metabolismo , Membrana Celular , Cílios , Desacetilase 6 de Histona/genética , Desacetilase 6 de Histona/metabolismo , Imuno-Histoquímica , Camundongos , Células NIH 3T3 , RNA Interferente Pequeno , Sirolimo/farmacologiaRESUMO
The neuronal-type (N-type) voltage-gated calcium (Cav) channels, which are designated Cav2.2, have an important role in the release of neurotransmitters1-3. Ziconotide is a Cav2.2-specific peptide pore blocker that has been clinically used for treating intractable pain4-6. Here we present cryo-electron microscopy structures of human Cav2.2 (comprising the core α1 and the ancillary α2δ-1 and ß3 subunits) in the presence or absence of ziconotide. Ziconotide is thoroughly coordinated by helices P1 and P2, which support the selectivity filter, and the extracellular loops (ECLs) in repeats II, III and IV of α1. To accommodate ziconotide, the ECL of repeat III and α2δ-1 have to tilt upward concertedly. Three of the voltage-sensing domains (VSDs) are in a depolarized state, whereas the VSD of repeat II exhibits a down conformation that is stabilized by Cav2-unique intracellular segments and a phosphatidylinositol 4,5-bisphosphate molecule. Our studies reveal the molecular basis for Cav2.2-specific pore blocking by ziconotide and establish the framework for investigating electromechanical coupling in Cav channels.
Assuntos
Analgésicos não Narcóticos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/química , Canais de Cálcio Tipo N/metabolismo , Microscopia Crioeletrônica , ômega-Conotoxinas/farmacologia , Canais de Cálcio Tipo N/ultraestrutura , Humanos , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacologia , Conformação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacosRESUMO
Cl- channels serve as key regulators of excitability and contractility in vascular, intestinal, and airway smooth muscle cells. We recently reported a Cl- conductance in detrusor smooth muscle (DSM) cells. Here, we used the whole cell patch-clamp technique to further characterize biophysical properties and physiological regulators of the Cl- current in freshly isolated guinea pig DSM cells. The Cl- current demonstrated outward rectification arising from voltage-dependent gating of Cl- channels rather than the Cl- transmembrane gradient. An exposure of DSM cells to hypotonic extracellular solution (Δ 165 mOsm challenge) did not increase the Cl- current providing strong evidence that volume-regulated anion channels do not contribute to the Cl- current in DSM cells. The Cl- current was monotonically dependent on extracellular pH, larger and lower in magnitude at acidic (5.0) and basic pH (8.5) values, respectively. Additionally, intracellularly applied phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] analog [PI(4,5)P2-diC8] increased the average Cl- current density by approximately threefold in a voltage-independent manner. The magnitude of the DSM whole cell Cl- current did not depend on the cell surface area (cell capacitance) regardless of the presence or absence of PI(4,5)P2-diC8, an intriguing finding that underscores the complex nature of Cl- channel expression and function in DSM cells. Removal of both extracellular Ca2+ and Mg2+ did not affect the DSM whole cell Cl- current, whereas Gd3+ (1 mM) potentiated the current. Collectively, our recent and present findings strongly suggest that Cl- channels are critical regulators of DSM excitability and are regulated by extracellular pH, Gd3+, and PI(4,5)P2.
Assuntos
Canais de Cloreto/fisiologia , Cloretos/metabolismo , Gadolínio/metabolismo , Potenciais da Membrana/fisiologia , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Cálcio/metabolismo , Cátions Bivalentes , Canais de Cloreto/efeitos dos fármacos , Gadolínio/farmacologia , Cobaias , Concentração de Íons de Hidrogênio , Transporte de Íons , Magnésio/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/citologia , Músculo Liso/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/análogos & derivados , Fosfatidilinositol 4,5-Difosfato/farmacologia , Cultura Primária de Células , Bexiga Urinária/citologia , Bexiga Urinária/metabolismoRESUMO
The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability, smooth muscle contraction, fluid secretion and gut motility. While opening of TMEM16A requires binding of intracellular Ca2+, prolonged Ca2+-dependent activation results in channel desensitization or rundown, the mechanism of which is unclear. Here we show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site at the cytosolic interface of transmembrane segments (TMs) 3-5. We further demonstrate that the ion-conducting pore of TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6-8 and a PIP2-binding regulatory module formed by TMs 3-5, which mediate channel activation and desensitization, respectively. PIP2 dissociation from the regulatory module results in ion-conducting pore collapse and subsequent channel desensitization. Our findings thus provide key insights into the mechanistic understanding of TMEM16 channel gating and lipid-dependent regulation.
Assuntos
Anoctamina-1/efeitos dos fármacos , Anoctamina-1/metabolismo , Cálcio/metabolismo , Agonistas dos Canais de Cloreto/metabolismo , Canais de Cloreto/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/farmacologia , Sítios de Ligação , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Modelos MolecularesRESUMO
The morphology and secondary structure of peptide fibers formed by aggregation of tubulin-associated unit (Tau) fragments (K18), in the presence of the inner cytoplasmic membrane phosphatidylinositol component (PIP2 ) or heparin sodium (HS) as cofactors, are determined with nanoscale (<10â nm) spatial resolution. By means of tip-enhanced Raman spectroscopy (TERS), the inclusion of PIP2 lipids in fibers is determined based on the observation of specific C=O ester vibration modes. Moreover, analysis of amideâ I and amideâ III bands suggests that the parallel ß-sheet secondary structure content is lower and the random coil content is higher for fibers grown from the PIP2 cofactor instead of HS. These observations highlight the occurrence of some local structural differences between these fibers. This study constitutes the first nanoscale structural characterization of Tau/phospholipid aggregates, which are implicated in deleterious mechanisms on neural membranes in Alzheimer's disease.
Assuntos
Fosfatidilinositol 4,5-Difosfato/farmacologia , Proteínas tau/antagonistas & inibidores , Humanos , Microscopia de Força Atômica , Tamanho da Partícula , Fosfatidilinositol 4,5-Difosfato/química , Agregados Proteicos/efeitos dos fármacos , Análise Espectral RamanRESUMO
The ability to detect and image secreted peroxynitrite (ONOO- ) along the extracellular surface of a single cell is biologically significant, as ONOO- generally exerts its function for host defense and signal transductions at the plasma membrane. However, as a result of the short lifetime and fast diffusion rate of small ONOO- , precise determination of the ONOO- level at the cell surface remains a challenging task. In this paper, the use of a membrane-anchored streptavidin-biotin-controlled binding probe (CBP), ONOO-CBP, to determine quantitatively the ONOO- level at the cell surface and to investigate the effect of different stimulants on the production of ONOO- along the plasma membrane of macrophages is reported. Our results revealed that the combination of NO synthase (iNOS) and NADPH oxidase (NOX) activators was highly effective in inducing ONOO- secretion, achieving more than a 25-fold increase in ONOO- relative to untreated cells. After 1â h of phorbol-12-myristate-13-acetate (PMA) stimulation, the amount of ONOO- secreted by RAW264.7 macrophages was similar to the condition treated with 25â µm 3-morpholinosydnonimine hydrochloride (SIN-1), which was estimated to release about 20â µm of ONOO- into Dulbecco's modified Eagle's medium (DMEM) in 1â h. This novel approach should open up new opportunities to image various reactive oxygen and nitrogen species secreted at the plasma membrane that cannot be simply achieved by conventional analytical methods.
Assuntos
Biotina/química , Membrana Celular/metabolismo , Corantes Fluorescentes/química , Ácido Peroxinitroso/análise , Estreptavidina/química , Animais , Carbocianinas/química , Ativadores de Enzimas/farmacologia , Expressão Gênica/efeitos dos fármacos , Interferon gama/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Microscopia Confocal , Microscopia de Fluorescência , Molsidomina/análogos & derivados , Molsidomina/farmacologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Ácido Peroxinitroso/química , Ácido Peroxinitroso/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacologia , Células RAW 264.7 , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
The ether-à-go-go1 (EAG1, Kv10.1) K+ channel is a member of the voltage-gated K+ channel family mainly expressed in the central nervous system and cancer cells. Membrane lipids regulate several voltage-gated K+ channels but their influence on EAG1 channels has been poorly explored. Here we have studied the regulation of hEAG1 channels by phosphatidylinositol 4,5-bisfofate (PIP2) by using different strategies to manipulate the levels of this lipid, and the patch clamp technique. We found that depletion of endogenous PIP2 by activation of the voltage-sensing phosphatase from Danio rerio (Dr-VSP) or the human muscarinic type-1 receptor (hM1R) inhibits hEAG1 currents; however, the application of exogenous PIP2 to increase the level of this lipid on the plasma membrane, also induced an inhibition of hEAG1. In summary, our results indicate that PIP2 have dual effects on hEAG1 channels and its action as activator or inhibitor depends on its initial level on the plasma membrane.
Assuntos
Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/farmacologia , Animais , Humanos , Técnicas de Patch-Clamp , Monoéster Fosfórico Hidrolases , Receptores Muscarínicos , Peixe-ZebraRESUMO
Cilia are cellular antennae that are essential for human development and physiology. A large number of genetic disorders linked to cilium dysfunction are associated with proteins that localize to the ciliary transition zone (TZ), a structure at the base of cilia that regulates trafficking in and out of the cilium. Despite substantial effort to identify TZ proteins and their roles in cilium assembly and function, processes underlying maturation of TZs are not well understood. Here, we report a role for the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2) in TZ maturation in the Drosophila melanogaster male germline. We show that reduction of cellular PIP2 levels through ectopic expression of a phosphoinositide phosphatase or mutation of the type I phosphatidylinositol phosphate kinase Skittles induces formation of longer than normal TZs. These hyperelongated TZs exhibit functional defects, including loss of plasma membrane tethering. We also report that the onion rings (onr) allele of DrosophilaExo84 decouples TZ hyperelongation from loss of cilium-plasma membrane tethering. Our results reveal a requirement for PIP2 in supporting ciliogenesis by promoting proper TZ maturation.
Assuntos
Cílios/efeitos dos fármacos , Cílios/fisiologia , Cílios/ultraestrutura , Drosophila melanogaster , Organogênese , Fosfatidilinositol 4,5-Difosfato/farmacologia , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero , Mutação em Linhagem Germinativa , Masculino , Organogênese/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genéticaRESUMO
The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca2+-triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca2+-dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca2+SIGNIFICANCE STATEMENT Syntaxin 1A (Syx) is a central protein component of the SNARE complex, which underlies neurotransmitter release. Although widely studied in relation to its participation in SNARE complex formation and its interaction with phosphoinositides, the function of Syx's polybasic juxtamembrane region (5RK) remains unclear. Previously, we showed that a conformational transition of Syx, related to calcium-triggered release, reported by a Syx-based FRET probe, is abolished upon charge neutralization of 5RK (5RK/A). Here we show that this conformational transition is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and is related to SNARE complex formation. Subsequently, we show that the 5RK/A mutation enhances spontaneous release and inhibits calcium-triggered release in neuroendocrine cells, indicating a previously unrecognized role of 5RK in neurotransmitter release.
Assuntos
Sinalização do Cálcio/fisiologia , Células Neuroendócrinas/fisiologia , Sintaxina 1/genética , Sintaxina 1/fisiologia , Animais , Sinalização do Cálcio/genética , Exocitose/fisiologia , Hipocampo/citologia , Hipocampo/fisiologia , Mutação/genética , Neurônios/fisiologia , Células PC12 , Fosfatidilinositol 4,5-Difosfato/farmacologia , Ratos , Proteínas SNARE/fisiologia , Sintaxina 1/antagonistas & inibidoresRESUMO
BACKGROUND AND PURPOSE: Ca2+ -activated Cl- channels (CaCCs) are gated open by a rise in intracellular Ca2+ concentration ([Ca2+ ]i ), typically provoked by activation of Gq -protein coupled receptors (Gq PCR). Gq PCR activation initiates depletion of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP2 ). Here, we determined whether PIP2 acts as a signalling lipid for CaCCs coded by the TMEM16A and TMEM16B genes. EXPERIMENTAL APPROACH: Patch-clamp electrophysiology, in conjunction with genetically encoded systems to control cellular PIP2 content, was used to define the mechanism of action of PIP2 on TMEM16A and TMEM16B channels. KEY RESULTS: A water-soluble PIP2 analogue (diC8-PIP2 ) activated TMEM16A channels by up to fivefold and inhibited TMEM16B by ~0.2-fold. The effects of diC8-PIP2 on TMEM16A currents were especially pronounced at low [Ca2+ ]i . In contrast, diC8-PIP2 modulation of TMEM16B channels did not vary over a broad [Ca2+ ]i range but was only detectable at highly depolarized membrane potentials. Modulation of TMEM16A and TMEM16B currents was due to changes in channel gating, while single channel conductance was unaltered. Co-expression of TMEM16A or TMEM16B with a Danio rerio voltage-sensitive phosphatase (DrVSP), which degrades PIP2 , led to reduction and enhancement of TMEM16A and TMEM16B currents respectively. These effects were abolished by an inactivating mutation in DrVSP and antagonized by simultaneous co-expression of a phosphatidylinositol-4-phosphate 5-kinase that catalyses PIP2 formation. CONCLUSIONS AND IMPLICATIONS: PIP2 acts as a modifier of TMEM16A and TMEM16B channel gating. Drugs interacting with PIP2 signalling may affect TMEM16A and TMEM16B channel gating and have potential uses in basic science and implications for therapy.
Assuntos
Anoctamina-1/metabolismo , Anoctaminas/antagonistas & inibidores , Fosfatidilinositol 4,5-Difosfato/farmacologia , Animais , Anoctaminas/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Camundongos , Fosfatidilinositol 4,5-Difosfato/química , Relação Estrutura-AtividadeRESUMO
TWIK-related two-pore domain K(+) channels 1 and 2 (TREKs) are activated under various physicochemical conditions. However, the directions in which they are regulated by PI(4,5)P2 and intracellular ATP are not clearly presented yet. In this study, we investigated the effects of ATP and PI(4,5)P2 on overexpressed TREKs (HEK293T and COS-7) and endogenously expressed TREK-2 (mouse astrocytes and WEHI-231 B cells). In all of these cells, both TREK-1 and TREK-2 currents were spontaneously increased by dialysis with ATP-free pipette solution for whole-cell recording (ITREK-1,w-c and ITREK-2w-c) or by membrane excision for inside-out patch clamping without ATP (ITREK-1,i-o and ITREK-2,i-o). Steady state ITREK-2,i-o was reversibly decreased by 3 mM ATP applied to the cytoplasmic side, and this reduction was prevented by wortmannin, a PI-kinase inhibitor. An exogenous application of PI(4,5)P2 inhibited the spontaneously increased ITREKs,i-o, suggesting that intrinsic PI(4,5)P2 maintained by intracellular ATP and PI kinase may set the basal activity of TREKs in the intact cells. The inhibition of intrinsic TREK-2 by ATP was more prominent in WEHI-231 cells than astrocytes. Interestingly, unspecific screening of negative charges by poly-L-lysine also inhibited ITREK-2,i-o. Application of PI(4,5)P2 after the poly-L-lysine treatment showed dose-dependent dual effects, initial activation and subsequent inhibition of ITREK-2,i-o at low and high concentrations, respectively. In HEK293T cells coexpressing TREK-2 and a voltage-sensitive PI(4,5)P2 phosphatase, sustained depolarization increased ITREK-2,w-c initially (<5 s) but then decreased the current below the control level. In HEK293T cells coexpressing TREK-2 and type 3 muscarinic receptor, application of carbachol induced transient activation and sustained suppression of ITREK-2,w-c and cell-attached ITREK-2. The inhibition of TREK-2 by unspecific electrostatic quenching, extensive dephosphorylation, or sustained hydrolysis of PI(4,5)P2 suggests the existence of dual regulatory modes that depend on PI(4,5)P2 concentration.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Trifosfato de Adenosina/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Feminino , Células HEK293 , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Voltage-gated Ca(2+) (CaV) channels are dynamically modulated by G protein-coupled receptors (GPCR). The M1 muscarinic receptor stimulation is known to enhance CaV2.3 channel gating through the activation of protein kinase C (PKC). Here, we found that M1 receptors also inhibit CaV2.3 currents when the channels are fully activated by PKC. In whole-cell configuration, the application of phorbol 12-myristate 13-acetate (PMA), a PKC activator, potentiated CaV2.3 currents by â¼two-fold. After the PMA-induced potentiation, stimulation of M1 receptors decreased the CaV2.3 currents by 52 ± 8%. We examined whether the depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is responsible for the muscarinic suppression of CaV2.3 currents by using two methods: the Danio rerio voltage-sensing phosphatase (Dr-VSP) system and the rapamycin-induced translocatable pseudojanin (PJ) system. First, dephosphorylation of PI(4,5)P2 to phosphatidylinositol 4-phosphate (PI(4)P) by Dr-VSP significantly suppressed CaV2.3 currents, by 53 ± 3%. Next, dephosphorylation of both PI(4)P and PI(4,5)P2 to PI by PJ translocation further decreased the current by up to 66 ± 3%. The results suggest that CaV2.3 currents are modulated by the M1 receptor in a dual mode-that is, potentiation through the activation of PKC and suppression by the depletion of membrane PI(4,5)P2. Our results also suggest that there is rapid turnover between PI(4)P and PI(4,5)P2 in the plasma membrane.
Assuntos
Canais de Cálcio Tipo R/metabolismo , Membrana Celular/química , Potenciais da Membrana , Proteína Quinase C/metabolismo , Receptor Muscarínico M1/metabolismo , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoAssuntos
Membrana Celular/efeitos dos fármacos , Fosfatos de Inositol/farmacologia , Fosfatidilinositol 4,5-Difosfato/farmacologia , Canais de Cátion TRPM/metabolismo , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Humanos , Fosfatos de Inositol/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Técnicas de Patch-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Fosfatidilinositóis/farmacologia , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/genéticaRESUMO
AIM: Pirt is a two-transmembrane domain protein that regulates the function of a variety of ion channels. Our previous study indicated that Pirt acts as a positive endogenous regulator of the TRPM8 channel. The aim of this study was to investigate the mechanism underlying the regulation of TRPM8 channel by Pirt. METHODS: HEK293 cells were transfected with TRPM8+Pirt or TRPM8 alone. Menthol (1 mmol/L) was applied through perfusion to induce TRPM8-mediated voltage-dependent currents, which were recorded using a whole-cell recording technique. PIP2 (10 µmol/L) was added into the electrode pipettes (PI was taken as a control). Additionally, cell-attached single-channel recordings were conducted in CHO cells transfected with TRPM8+Pirt or TRPM8 alone, and menthol (1 mmol/L) was added into the pipette solution. RESULTS: Either co-transfection with Pirt or intracellular application of PIP2 (but not PI) significantly enhanced menthol-induced TRPM8 currents. Furthermore, Pirt and PIP2 synergistically modulated menthol-induced TRPM8 currents. Single-channel recordings revealed that co-transfection with Pirt significantly increased the single channel conductance. CONCLUSION: Pirt and PIP2 synergistically enhance TRPM8 channel activity, and Pirt regulates TRPM8 channel activity by increasing the single channel conductance.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Membrana/metabolismo , Canais de Cátion TRPM/metabolismo , Proteínas de Transporte/genética , Células HEK293 , Humanos , Ativação do Canal Iônico , Proteínas de Membrana/genética , Mentol/farmacologia , Fosfatidilinositol 4,5-Difosfato/farmacologia , Canais de Cátion TRPM/genéticaRESUMO
The transient receptor potential melastatin (TRPM)-3 channel is critical for various physiologic processes. In somatosensory neurons, TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, whereas in pancreatic ß-cells the channel has been linked to glucose-induced insulin release. As a typical representative of the TRP family, TRPM3 is highly polymodal. In cells, it is activated by heat and chemical agonists, including pregnenolone sulfate (PS) and nifedipine (Nif). To define the nuances of TRPM3 channel activity and its modulators, we succeeded in incorporating the TRPM3 protein into planar lipid bilayers. We found that phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole is necessary for channel opening by PS. Unlike PS, the presence of Nif alone sufficed to induce TRPM3 activity and demonstrated distinct gating behavior. We also performed an extensive thermodynamic analysis of TRPM3 activation and found that TRPM3 exhibited slight temperature sensitivity in the bilayers. In the absence of other agonists TRPM3 channels remained closed upon heat-induced stimulation, but opened in the presence of PIP2, although with only a low open-probability profile. Together, our results elucidate the details peculiar to TRPM3 channel function in an isolated system. We confirmed its direct gating by PS and PIP2, but found a lack of the strong intrinsic temperature sensitivity common to other thermosensitive TRP channels.
Assuntos
Ativação do Canal Iônico/fisiologia , Bicamadas Lipídicas/metabolismo , Canais de Cátion TRPM/metabolismo , Linhagem Celular , Clotrimazol/farmacologia , Células HEK293 , Temperatura Alta , Humanos , Hiperalgesia/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Nifedipino/farmacologia , Fosfatidilinositol 4,5-Difosfato/farmacologia , Pregnenolona/farmacologiaRESUMO
The mechanisms by which n-3 polyunsaturated fatty acids (n-3 PUFA), abundant in fish oil, exert their anti-inflammatory effects have not been rigorously defined. We have previously demonstrated that n-3 PUFA decrease the amount of phosphatidylinositol-(4,5)-bisphosphate, [PI(4,5)P2], in CD4(+) T cells, leading to suppressed actin remodeling upon activation. Since discrete pools of PI(4,5)P2 exist in the plasma membrane, we determined whether n-3 PUFA modulate spatial organization of PI(4,5)P2 relative to raft and non-raft domains. We used Förster resonance energy transfer (FRET) to demonstrate that lipid raft mesodomains in the plasma membrane of CD4(+) T cells enriched in n-3 PUFA display increased co-clustering of Lck(N10) and LAT(ΔCP), markers of lipid rafts. CD4(+) T cells enriched in n-3 PUFA also exhibited a depleted plasma membrane non-raft PI(4,5)P2 pool as detected by decreased co-clustering of Src(N15), a non-raft marker, and PH(PLC-δ), a PI(4,5)P2 reporter. Incubation with exogenous PI(4,5)P2 rescued the effects on the non-raft PI(4,5)P2 pool, and reversed the suppression of T cell proliferation in CD4(+) T cells enriched with n-3 PUFA. Furthermore, CD4(+) T cells isolated from mice fed a 4% docosahexaenoic acid (DHA)-enriched diet exhibited a decrease in the non-raft pool of PI(4,5)P2, and exogenous PI(4,5)P2 reversed the suppression of T cell proliferation. Finally, these effects were not due to changes to post-translational lipidation, since n-3 PUFA did not alter the palmitoylation status of signaling proteins. These data demonstrate that n-3 PUFA suppress T cell proliferation by altering plasma membrane topography and the spatial organization of PI(4,5)P2.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Gorduras na Dieta/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/química , Linfócitos T CD4-Positivos/citologia , Expressão Gênica , Vetores Genéticos , Lentivirus/genética , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/farmacologia , Fosfolipase C delta/genética , Fosfolipase C delta/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Cultura Primária de Células , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Cardiac KCNQ1/KCNE1 channels (IKs) are dependent on the concentration of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) and on cytosolic ATP by two distinct mechanisms. In this study we measured IKs and FRET between PH-PLCδ-based fluorescent PIP2 sensors in a stable KCNQ1/KCNE1 CHO cell line. Effects of activating either a muscarinic M3 receptor or the switchable phosphatase Ci-VSP on IKs were analyzed. Recovery of IKs from inhibition induced by muscarinic stimulation was incomplete despite full PIP2 resynthesis. Recovery of IKs was completely suppressed under ATP-free conditions, but partially restored by the ATP analog AMP-PCP, providing evidence that depletion of intracellular ATP inhibits IKs independent of PIP2-depletion. Simultaneous patch-clamp and FRET measurements in cells co-expressing Ci-VSP and the PIP2-FRET sensor revealed a component of IKs inhibition directly related to dynamic PIP2-depletion. A second component of inhibition was independent of acute changes in PIP2 and could be mimicked by ATP-free pipette solution, suggesting that it results from intracellular ATP-depletion. The reduction of intracellular ATP upon Ci-VSP activation appears to be independent of its activity as a phosphoinositide phosphatase. Our data demonstrate that ATP-depletion slowed IKs activation but had no short-term effect on PIP2 regeneration, suggesting that impaired PIP2-resynthesis cannot account for the rapid IKs inhibition by ATP-depletion. Furthermore, the second component of IKs inhibition by Ci-VSP was reduced by AMP-PCP in the pipette filling solution, indicating that direct binding of ATP to the KCNQ1/KCNE1 complex is required for voltage activation of IKs. We suggest that fluctuations of the cellular metabolic state regulate IKs in parallel with Gq-coupled PLC activation and PIP2-depletion.
Assuntos
Trifosfato de Adenosina/farmacologia , Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Acetilcolina/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Sinergismo Farmacológico , Transferência Ressonante de Energia de Fluorescência , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Canal de Potássio KCNQ1/genética , Potenciais da Membrana/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Receptor Muscarínico M3/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Transient receptor potential (TRP) channel melastatin subfamily member 4 (TRPM4) is a broadly expressed nonselective monovalent cation channel. TRPM4 is activated by membrane depolarization and intracellular Ca(2+), which is essential for the activation. The Ca(2+) sensitivity is known to be regulated by calmodulin and membrane phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). Although these regulators must play important roles in controlling TRPM4 activity, mutation analyses of the calmodulin-binding sites have suggested that Ca(2+) binds to TRPM4 directly. However, the intrinsic binding sites in TRPM4 remain to be elucidated. Here, by using patch clamp and molecular biological techniques, we show that there are at least two functionally different divalent cation-binding sites, and the negatively charged amino acids near and in the TRP domain in the C-terminal tail of TRPM4 (Asp-1049 and Glu-1062 of rat TRPM4) are required for maintaining the normal Ca(2+) sensitivity of one of the binding sites. Applications of Co(2+), Mn(2+), or Ni(2+) to the cytosolic side potentiated TRPM4 currents, increased the Ca(2+) sensitivity, but were unable to evoke TRPM4 currents without Ca(2+). Mutations of the acidic amino acids near and in the TRP domain, which are conserved in TRPM2, TRPM5, and TRPM8, deteriorated the Ca(2+) sensitivity in the presence of Co(2+) or PI(4,5)P2 but hardly affected the sensitivity to Co(2+) and PI(4,5)P2. These results suggest a novel role of the TRP domain in TRPM4 as a site responsible for maintaining the normal Ca(2+) sensitivity. These findings provide more insights into the molecular mechanisms of the regulation of TRPM4 by Ca(2+).