RESUMEN
Transient receptor potential vanilloid (TRPV) channels play a critical role in calcium homeostasis, pain sensation, immunological response, and cancer progression. TRPV channels are blocked by ruthenium red (RR), a universal pore blocker for a wide array of cation channels. Here we use cryo-electron microscopy to reveal the molecular details of RR block in TRPV2 and TRPV5, members of the two TRPV subfamilies. In TRPV2 activated by 2-aminoethoxydiphenyl borate, RR is tightly coordinated in the open selectivity filter, blocking ion flow and preventing channel inactivation. In TRPV5 activated by phosphatidylinositol 4,5-bisphosphate, RR blocks the selectivity filter and closes the lower gate through an interaction with polar residues in the pore vestibule. Together, our results provide a detailed understanding of TRPV subfamily pore block, the dynamic nature of the selectivity filter and allosteric communication between the selectivity filter and lower gate.
Asunto(s)
Antineoplásicos , Canales de Potencial de Receptor Transitorio , Canales Catiónicos TRPV/genética , Rojo de Rutenio/farmacología , Microscopía por Crioelectrón , Calcio/metabolismoRESUMEN
The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.
Asunto(s)
Anoctamina-1/metabolismo , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Colesterol/metabolismo , Ácidos Grasos/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Anoctamina-1/genética , Calcio/metabolismo , Membrana Celular/genética , Colesterol/genética , Ácidos Grasos/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Fosfatidilinositol 4,5-Difosfato/genéticaRESUMEN
KEY POINTS: The calcium-activated chloride channel TMEM16A provides a pathway for chloride ion movements that are key in preventing polyspermy, allowing fluid secretion, controlling blood pressure, and enabling gastrointestinal activity. TMEM16A is opened by voltage-dependent calcium binding and regulated by permeant anions and intracellular protons. Here we show that a low proton concentration reduces TMEM16A activity while maximum activation is obtained when the external proton concentration is high. In addition, protonation conditions determine the open probability of TMEM16A without changing its calcium sensitivity. External glutamic acid 623 (E623) is key for TMEM16A's ability to respond to external protons. At physiological pH, E623 is un-protonated and TMEM16A is activated when intracellular calcium increases; however, under acidic conditions E623 is partially protonated and works synergistically with intracellular calcium to activate the channel. These findings are critical for understanding physiological and pathological processes that involve changes in pH and chloride flux via TMEM16A. ABSTRACT: Transmembrane protein 16A (TMEM16A), also known as ANO1, the pore-forming subunit of a Ca2+ -dependent Cl- channel (CaCC), is activated by direct, voltage-dependent, binding of intracellular Ca2+ . Endogenous CaCCs are regulated by extracellular protons; however, the molecular basis of such regulation remains unidentified. Here, we evaluated the effects of different extracellular proton concentrations ([H+ ]o ) on mouse TMEM16A expressed in HEK-293 cells using whole-cell and inside-out patch-clamp recordings. We found that increasing the [H+ ]o from 10-10 to 10-5.5 m caused a progressive increase in the chloride current (ICl ) that is described by titration of a protonatable site with pK = 7.3. Protons regulate TMEM16A in a voltage-independent manner, regardless of channel state (open or closed), and without altering its apparent Ca2+ sensitivity. Noise analysis showed that protons regulate TMEM16A by tuning its open probability without modifying the single channel current. We found a robust reduction of the proton effect at high [Ca2+ ]i . To identify protonation targets we mutated all extracellular glutamate and histidine residues and 4 of 11 aspartates. Most mutants were sensitive to protons. However, mutation that substituted glutamic acid (E) for glutamine (Q) at amino acid position 623 (E623Q) displayed a titration curve shifted to the left relative to wild type channels and the ICl was nearly insensitive to proton concentrations between 10-5.5 and 10-9.0 m. Additionally, ICl of the mutant containing an aspartic acid (D) to asparagine (N) substitution at position 405 (D405N) mutant was partially inhibited by a proton concentration of 10-5.5 m, but 10-9.0 m produced the same effect as in wild type. Based on our findings we propose that external protons titrate glutamic acid 623, which enables voltage activation of TMEM16A at non-saturating [Ca2+ ]i .
Asunto(s)
Canales de Cloruro/fisiología , Anoctamina-1 , Calcio/fisiología , Canales de Cloruro/genética , Células HEK293 , Humanos , Modelos Moleculares , ProtonesRESUMEN
TMEM16A (ANO1), the pore-forming subunit of calcium-activated chloride channels, regulates several physiological and pathophysiological processes such as smooth muscle contraction, cardiac and neuronal excitability, salivary secretion, tumour growth and cancer progression. Gating of TMEM16A is complex because it involves the interplay between increases in intracellular calcium concentration ([Ca(2+)]i), membrane depolarization, extracellular Cl(-) or permeant anions and intracellular protons. Our goal here was to understand how these variables regulate TMEM16A gating and to explain four observations. (a) TMEM16A is activated by voltage in the absence of intracellular Ca(2+). (b) The Cl(-) conductance is decreased after reducing extracellular Cl(-) concentration ([Cl(-)]o). (c) ICl is regulated by physiological concentrations of [Cl(-)]o. (d) In cells dialyzed with 0.2 µM [Ca(2+)]i, Cl(-) has a bimodal effect: at [Cl(-)]o <30 mM TMEM16A current activates with a monoexponential time course, but above 30 mM, [Cl(-)]o ICl activation displays fast and slow kinetics. To explain the contribution of Vm, Ca(2+) and Cl(-) to gating, we developed a 12-state Markov chain model. This model explains TMEM16A activation as a sequential, direct, and Vm-dependent binding of two Ca(2+) ions coupled to a Vm-dependent binding of an external Cl(-) ion, with Vm-dependent transitions between states. Our model predicts that extracellular Cl(-) does not alter the apparent Ca(2+) affinity of TMEM16A, which we corroborated experimentally. Rather, extracellular Cl(-) acts by stabilizing the open configuration induced by Ca(2+) and by contributing to the Vm dependence of activation.
Asunto(s)
Canales de Cloruro/metabolismo , Cloruros/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Aniones/metabolismo , Anoctamina-1 , Calcio/metabolismo , Línea Celular , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Cinética , Ratones , Contracción Muscular/fisiología , Miocitos del Músculo Liso/metabolismoRESUMEN
KEY POINTS: Calcium-activated chloride channels TMEM16A and TMEM16B support important physiological processes such as fast block of polyspermy, fluid secretion, control of blood pressure and sensory transduction. Given the physiological importance of TMEM16 channels, it is important to study how incoming stimuli activate these channels. Here we study how channels open and close and how the process of gating is regulated. We show that TMEM16A and TMEM16B display fast and slow gating. These gating modes are regulated by voltage and external chloride. Dual gating explains the complex time course of the anion current. Residues within the first intracellular loop of the channel influence the slow gating mode. Dual gating is an intrinsic property observed in endogenous calcium-activated chloride channels and could be relevant to physiological processes that require sustained chloride ion movement. ABSTRACT: TMEM16A and TMEM16B are molecular components of the physiologically relevant calcium-activated chloride channels (CaCCs) present in many tissues. Their gating is dictated by membrane voltage (Vm ), intracellular calcium concentrations ([Ca(2+) ]i ) and external permeant anions. As a consequence, the chloride current (ICl ) kinetics is complex. For example, TMEM16A ICl activates slowly with a non-mono-exponential time course while TMEM16B ICl activates rapidly following a mono-exponential behaviour. To understand the underlying mechanism responsible for the complex activation kinetics, we recorded ICl from HEK-293 cells transiently transfected with either TMEM16A or TMEM16B as well as from mouse parotid acinar cells. Two distinct Vm -dependent gating modes were uncovered: a fast-mode on the millisecond time scale followed by a slow mode on the second time scale. Using long (20 s) depolarizing pulses both gating modes were activated, and a slowly rising ICl was recorded in whole-cell and inside-out patches. The amplitude of ICl at the end of the long pulse nearly doubled and was blocked by 100 µm tannic acid. The slow gating mode was strongly reduced by decreasing the [Cl(-) ]o from 140 to 30 mm and by altering the sequence of the first intracellular loop. Mutating 480 RSQ482 to AVK in the first intracellular loop of TMEM16B nearly abolished slow gating, but, mutating 448 AVK451 to RSQ in TMEM16A has little effect. Deleting 448 EAVK451 residues in TMEM16A reduced slow gating. We conclude that TMEM16 CaCCs have intrinsic Vm - and Cl(-) -sensitive dual gating that elicits complex ICl kinetics.
Asunto(s)
Canales de Cloruro/metabolismo , Activación del Canal Iónico , Células Acinares/metabolismo , Células Acinares/fisiología , Potenciales de Acción , Secuencias de Aminoácidos , Animales , Anoctamina-1 , Anoctaminas , Células Cultivadas , Canales de Cloruro/química , Canales de Cloruro/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , MutaciónRESUMEN
UNLABELLED: The helper component proteinase (HCPro) is an indispensable, multifunctional protein of members of the genus Potyvirus and other viruses of the family Potyviridae. This viral factor is directly involved in diverse steps of viral infection, such as aphid transmission, polyprotein processing, and suppression of host antiviral RNA silencing. In this paper, we show that although a chimeric virus based on the potyvirus Plum pox virus lacking HCPro, which was replaced by a heterologous silencing suppressor, caused an efficient infection in Nicotiana benthamiana plants, its viral progeny had very reduced infectivity. Making use of different approaches, here, we provide direct evidence of a previously unknown function of HCPro in which the viral factor enhances the stability of its cognate capsid protein (CP), positively affecting the yield of virions and consequently improving the infectivity of the viral progeny. Site-directed mutagenesis revealed that the ability of HCPro to stabilize CP and enhance the yield of infectious viral particles is not linked to any of its previously known activities and helped us to delimit the region of HCPro involved in this function in the central region of the protein. Moreover, the function is highly specific and cannot be fulfilled by the HCPro of a heterologous potyvirus. The importance of this novel requirement in regulating the sorting of the viral genome to be subjected to replication, translation, and encapsidation, thus contributing to the synchronization of these viral processes, is discussed. IMPORTANCE: Potyviruses form one of the most numerous groups of plant viruses and are a major cause of crop loss worldwide. It is well known that these pathogens make use of virus-derived multitasking proteins, as well as dedicated host factors, to successfully infect their hosts. Here, we describe a novel requirement for the proper yield and infectivity of potyviral progeny. In this case, such a function is performed by the extensively studied viral factor HCPro, which seems to use an unknown mechanism that is not linked to its previously described activities. To our knowledge, this is the first time that a factor different from capsid protein (CP) has been shown to be directly involved in the yield of potyviral particles. Based on the data presented here, we hypothesize that this capacity of HCPro might be involved in the coordination of mutually exclusive activities of the viral genome by controlling correct assembly of CP in stable virions.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Virus Eruptivo de la Ciruela/enzimología , Virus Eruptivo de la Ciruela/crecimiento & desarrollo , Proteínas Virales/metabolismo , Virión/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Cisteína Endopeptidasas/genética , Análisis Mutacional de ADN , Estabilidad Proteica , Nicotiana/virología , Proteínas Virales/genéticaRESUMEN
The calcium-selective TRPV5 channel activated by phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is involved in calcium homeostasis. Recently, cryoelectron microscopy (cryo-EM) provided molecular details of TRPV5 modulation by exogenous and endogenous molecules. However, the details of TRPV5 inhibition by the antifungal agent econazole (ECN) remain elusive due to the low resolution of the currently available structure. In this study, we employ cryo-EM to comprehensively examine how the ECN inhibits TRPV5. By combining our structural findings with site-directed mutagenesis, calcium measurements, electrophysiology, and molecular dynamics simulations, we determined that residues F472 and L475 on the S4 helix, along with residue W495 on the S5 helix, collectively constitute the ECN-binding site. Additionally, the structure of TRPV5 in the presence of ECN and PI(4,5)P2, which does not show the bound activator, reveals a potential inhibition mechanism in which ECN competes with PI(4,5)P2, preventing the latter from binding, and ultimately pore closure.
Asunto(s)
Antifúngicos , Econazol , Canales Catiónicos TRPV , Antifúngicos/farmacología , Calcio/metabolismo , Microscopía por Crioelectrón , Econazol/farmacología , Simulación de Dinámica Molecular , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/químicaRESUMEN
Long-chain acyl-coenzyme A (LC-CoA) is a crucial metabolic intermediate that plays important cellular regulatory roles, including activation and inhibition of ion channels. The structural basis of ion channel regulation by LC-CoA is not known. Transient receptor potential vanilloid 5 and 6 (TRPV5 and TRPV6) are epithelial calcium-selective ion channels. Here, we demonstrate that LC-CoA activates TRPV5 and TRPV6 in inside-out patches, and both exogenously supplied and endogenously produced LC-CoA can substitute for the natural ligand phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in maintaining channel activity in intact cells. Utilizing cryo-electron microscopy, we determined the structure of LC-CoA-bound TRPV5, revealing an open configuration with LC-CoA occupying the same binding site as PI(4,5)P2 in previous studies. This is consistent with our finding that PI(4,5)P2 could not further activate the channels in the presence of LC-CoA. Our data provide molecular insights into ion channel regulation by a metabolic signaling molecule.
Asunto(s)
Acilcoenzima A , Canales de Calcio , Microscopía por Crioelectrón , Sitios de Unión , Ciclo CelularRESUMEN
Transient Receptor Potential Vanilloid 5 and 6 (TRPV5 and TRPV6) are Ca2+ selective epithelial ion channels. They are the products of a relatively recent gene duplication in mammals, and have high sequence homology to each other. Their functional properties are also much more similar to each other than to other members of the TRPV subfamily. They are both constitutively active, and this activity depends on the endogenous cofactor phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Both channels undergo Ca2+-induced inactivation, which is mediated by direct binding of the ubiquitous Ca2+ binding protein calmodulin (CaM) to the channels, and by a decrease in PI(4,5)P2 levels by Ca2+ -induced activation of phospholipase C (PLC). Recent cryo electron microscopy (cryo-EM) and X-ray crystallography structures provided detailed structural information for both TRPV5 and TRPV6. This review will discuss this structural information in the context of the function of these channels focusing on the mechanism of CaM inhibition, activation by PI(4,5)P2 and binding of pharmacological modulators.
Asunto(s)
Calcio , Canales Catiónicos TRPV , Animales , Calcio/metabolismo , Calmodulina/metabolismo , Microscopía por Crioelectrón , Mamíferos/metabolismo , Fosfatidilinositoles , Canales Catiónicos TRPV/metabolismoRESUMEN
Numerous essential physiological processes depend on the TMEM16A-mediated Ca2+-activated chloride fluxes. Extensive structure-function studies have helped to elucidate the Ca2+ gating mechanism of TMEM16A, revealing a Ca2+-sensing element close to the anion pore that alters conduction. However, substrate selection and the substrate-gating relationship in TMEM16A remain less explored. Here, we study the gating-permeant anion relationship on mouse TMEM16A expressed in HEK 293 cells using electrophysiological recordings coupled with site-directed mutagenesis. We show that the apparent Ca2+ sensitivity of TMEM16A increased with highly permeant anions and SCN- mole fractions, likely by stabilizing bound Ca2+. Conversely, mutations at crucial gating elements, including the Ca2+-binding site 1, the transmembrane helix 6 (TM6), and the hydrophobic gate, impaired the anion permeability and selectivity of TMEM16A. Finally, we found that, unlike anion-selective wild-type channels, the voltage dependence of unselective TMEM16A mutant channels was less sensitive to SCN-. Therefore, our work identifies structural determinants of selectivity at the Ca2+ site, TM6, and hydrophobic gate and reveals a reciprocal regulation of gating and selectivity. We suggest that this regulation is essential to set ionic selectivity and the Ca2+ and voltage sensitivities in TMEM16A.
Asunto(s)
Calcio , Canales de Cloruro , Animales , Aniones/metabolismo , Anoctamina-1/genética , Calcio/metabolismo , Canales de Cloruro/química , Canales de Cloruro/genética , Células HEK293 , Humanos , Activación del Canal Iónico , Ratones , Proteínas de Neoplasias/metabolismoRESUMEN
The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure-function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.
Asunto(s)
Canales de Cloruro/química , Activación del Canal Iónico , Secuencia de Aminoácidos , Animales , Canales de Cloruro CLC-2 , Bovinos , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Cloruros/metabolismo , Humanos , Ratones , Mutación , Estructura Terciaria de Proteína , Alineación de Secuencia , Relación Estructura-ActividadRESUMEN
Anoctamin-1 (ANO1 or TMEM16A) is a homo-dimeric Ca2+-activated Cl- channel responsible for essential physiological processes. Each monomer harbours a pore and a Ca2+-binding pocket; the voltage-dependent binding of two intracellular Ca2+ ions to the pocket gates the pore. However, in the absence of intracellular Ca2+ voltage activates TMEM16A by an unknown mechanism. Here we show voltage-activated anion currents that are outwardly rectifying, time-independent with fast or absent tail currents that are inhibited by tannic and anthracene-9-carboxylic acids. Since intracellular protons compete with Ca2+ for binding sites in the pocket, we hypothesized that voltage-dependent titration of these sites would induce gating. Indeed intracellular acidification enabled activation of TMEM16A by voltage-dependent protonation, which enhanced the open probability of the channel. Mutating Glu/Asp residues in the Ca2+-binding pocket to glutamine (to resemble a permanent protonated Glu) yielded channels that were easier to activate at physiological pH. Notably, the response of these mutants to intracellular acidification was diminished and became voltage-independent. Thus, voltage-dependent protonation of glutamate/aspartate residues (Glu/Asp) located in the Ca2+-binding pocket underlines TMEM16A activation in the absence of intracellular Ca2+.
Asunto(s)
Anoctamina-1/metabolismo , Calcio/metabolismo , Cloruros/metabolismo , Proteínas Recombinantes de Fusión/genética , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Anoctamina-1/antagonistas & inhibidores , Anoctamina-1/genética , Antracenos/farmacología , Cationes Bivalentes , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Transporte Iónico/efectos de los fármacos , Ratones , Mutación , Técnicas de Placa-Clamp , Plásmidos/química , Plásmidos/metabolismo , Protones , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-Actividad , Taninos/farmacología , TransfecciónRESUMEN
It has been reported that muscarinic type-2 receptors (M2R) are voltage sensitive in an agonist-specific manner. In this work, we studied the effects of membrane potential on the interaction of M2R with the superagonist iperoxo (IXO), both functionally (using the activation of the ACh-gated K+ current (IKACh) in cardiomyocytes) and by molecular dynamics (MD) simulations. We found that IXO activated IKACh with remarkable high potency and clear voltage dependence, displaying a larger effect at the hyperpolarized potential. This result is consistent with a greater affinity, as validated by a slower (τ = 14.8 ± 2.3 s) deactivation kinetics of the IXO-evoked IKACh than that at the positive voltage (τ = 6.7 ± 1.2 s). The voltage-dependent M2R-IXO interaction induced IKACh to exhibit voltage-dependent features of this current, such as the 'relaxation gating' and the modulation of rectification. MD simulations revealed that membrane potential evoked specific conformational changes both at the external access and orthosteric site of M2R that underlie the agonist affinity change provoked by voltage on M2R. Moreover, our experimental data suggest that the 'tyrosine lid' (Y104, Y403, and Y426) is not the previously proposed voltage sensor of M2R. These findings provide an insight into the structural and functional framework of the biased signaling induced by voltage on GPCRs.
Asunto(s)
Activación del Canal Iónico/efectos de los fármacos , Isoxazoles/farmacología , Simulación de Dinámica Molecular , Compuestos de Amonio Cuaternario/farmacología , Receptor Muscarínico M2/fisiología , Acetilcolina/farmacología , Animales , Gatos , Células Cultivadas , Estimulación Eléctrica , Femenino , Activación del Canal Iónico/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Modelos Moleculares , Agonistas Muscarínicos/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/fisiología , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Conformación Proteica , Receptor Muscarínico M2/química , Receptor Muscarínico M2/metabolismo , Xenopus laevisRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.
Asunto(s)
Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio Shab/metabolismo , Células HEK293 , Humanos , Activación del Canal Iónico/fisiología , Técnicas de Placa-Clamp/métodos , Canales de Potasio con Entrada de Voltaje/metabolismo , Receptores Muscarínicos/metabolismoRESUMEN
The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked GlcNAc (O-GlcNAc). While Arabidopsis has two O-GlcNAc transferases, SECRET AGENT (SEC) and SPINDLY (SPY), previous work suggests that SEC modifies PPV-CP and that the modification plays a role in the infection process. Here, we show that when co-expressed in Escherichia coli SEC modifies PPV-CP. Deletion mapping and site-directed mutagenesis identified three threonine and a serine located near the N-terminus of PPV-CP that are modified by SEC. Two of these threonines have recently been shown to be modified in virus from plants suggesting that SEC has the same specificity in plants and E. coli.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Proteínas de la Cápside/metabolismo , N-Acetilglucosaminiltransferasas/metabolismo , Virus Eruptivo de la Ciruela/metabolismo , Sustitución de Aminoácidos , Arabidopsis/enzimología , Secuencia de Bases , Sitios de Unión , Proteínas de la Cápside/química , Proteínas de la Cápside/genética , ADN Viral/genética , Glicosilación , Mutagénesis Sitio-Dirigida , Virus Eruptivo de la Ciruela/genética , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Serina/química , Treonina/químicaRESUMEN
A large number of O-linked N-acetylglucosamine (O-GlcNAc) residues have been mapped in vertebrate proteins, however targets of O-GlcNAcylation in plants still have not been characterized. We show here that O-GlcNAcylation of the N-terminal region of the capsid protein of Plum pox virus resembles that of animal proteins in introducing O-GlcNAc monomers. Thr-19 and Thr-24 were specifically O-GlcNAcylated. These residues are surrounded by amino acids typical of animal O-GlcNAc acceptor sites, suggesting that the specificity of O-GlcNAc transferases is conserved among plants and animals. In laboratory conditions, mutations preventing O-GlcNAcylation of Thr-19 and Thr-24 did not have noticeable effects on PPV competence to infect Prunus persicae or Nicotiana clevelandii. However, the fact that Thr-19 and Thr-24 are highly conserved among different PPV strains suggests that their O-GlcNAc modification could be relevant for efficient competitiveness in natural conditions.
Asunto(s)
Proteínas de la Cápside/química , Virus Eruptivo de la Ciruela/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Secuencia de Bases , Sitios de Unión , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , ADN Viral/genética , Glicosilación , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Enfermedades de las Plantas/virología , Virus Eruptivo de la Ciruela/genética , Virus Eruptivo de la Ciruela/patogenicidad , Procesamiento Proteico-Postraduccional , Prunus/virología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Treonina/química , Nicotiana/virologíaRESUMEN
CLC-2 channels are dimeric double-barreled chloride channels that open in response to hyperpolarization. Hyperpolarization activates protopore gates that independently regulate the permeability of the pore in each subunit and the common gate that affects the permeability through both pores. CLC-2 channels lack classic transmembrane voltage-sensing domains; instead, their protopore gates (residing within the pore and each formed by the side chain of a glutamate residue) open under repulsion by permeant intracellular anions or protonation by extracellular H(+). Here, we show that voltage-dependent gating of CLC-2: (a) is facilitated when permeant anions (Cl(-), Br(-), SCN(-), and I(-)) are present in the cytosolic side; (b) happens with poorly permeant anions fluoride, glutamate, gluconate, and methanesulfonate present in the cytosolic side; (c) depends on pore occupancy by permeant and poorly permeant anions; (d) is strongly facilitated by multi-ion occupancy; (e) is absent under likely protonation conditions (pHe = 5.5 or 6.5) in cells dialyzed with acetate (an impermeant anion); and (f) was the same at intracellular pH 7.3 and 4.2; and (g) is observed in both whole-cell and inside-out patches exposed to increasing [Cl(-)]i under unlikely protonation conditions (pHe = 10). Thus, based on our results we propose that hyperpolarization activates CLC-2 mainly by driving intracellular anions into the channel pores, and that protonation by extracellular H(+) plays a minor role in dislodging the glutamate gate.