RESUMEN

Ionotropic receptors are ligand-gated ion channels triggering fast neurotransmitter responses. Among them, P2X and 5-HT3 receptors have been shown to physically interact each other and functionally inducing cross inhibitory responses. Nevertheless, despite the importance of P2X4 and 5-HT3A receptors that mediate for example neuropathic pain and psychosis respectively, complementary evidence has recently started to move forward in the understanding of this interaction. In this review, we discuss current evidence supporting the mechanism of crosstalking between both receptors, from the structural to the transduction pathway level. We expect this work may guide the design of further experiments to obtain a comprehensive view for the neuropharmacological role of these interacting receptors. This article is part of the Special Issue on "The receptor-receptor interaction as a new target for therapy".

Asunto(s)

Canales Iónicos Activados por Ligandos , Receptores de Serotonina 5-HT3 , Receptores de Serotonina 5-HT3/metabolismo , Serotonina/metabolismo , Transporte de Proteínas , Unión Proteica/fisiología , Canales Iónicos Activados por Ligandos/metabolismo , Receptores Purinérgicos P2X4/metabolismo

RESUMEN

Glycine Receptors (GlyRs) are cell-surface transmembrane proteins that belong to the Cysloop ligand-gated ion channels superfamily (Cys-loop LGICs). Functional glycine receptors are conformed only by α-subunits (homomeric channels) or by α- and ß-subunits (heteromeric channels). The role of glycine as a cytoprotective is widely studied. New information about glycine modulation of vascular endothelial cells (ECs) function emerged last year. Glycine and its receptors are recognized to play a role as neurovascular protectors by a mechanism that involves α2GlyRs. Interestingly, the expression of α2GlyRs reduces after stroke injury. However, glycine reverses the inhibition of α2GlyRs by a mechanism involving the VEGF/pSTAT3 signaling. On the other hand, consistent evidence has demonstrated that ECs participate actively in the innate and adaptive immunological response. We recently reported that GlyRs are modulated by interleukin-1ß, suggesting new perspectives to explain the immune modulation of vascular function in pathological conditions such as cerebrovascular stroke. In this work, we distinguish the role of glycine and the allosteric modulation of glycine receptors as a new therapeutic target to confront post-ischemic injury.

Asunto(s)

Canales Iónicos Activados por Ligandos , Receptores de Glicina , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Glicina/metabolismo , Glicina/farmacología , Glicina/uso terapéutico , Humanos , Interleucina-1beta/metabolismo , Canales Iónicos Activados por Ligandos/metabolismo , Receptores de Glicina/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo

RESUMEN

Erwin London dedicated considerable effort to understanding lipid interactions with membrane-resident proteins and how these interactions shaped the formation and maintenance of lipid phases and domains. In this endeavor, he developed ad hoc techniques that greatly contributed to advancements in the field. We have employed and/or modified/extended some of his methodological approaches and applied them to investigate lipid interaction with the nicotinic acetylcholine receptor (nAChR) protein, the paradigm member of the superfamily of rapid pentameric ligand-gated ion channels (pLGIC). Our experimental systems ranged from purified receptor protein reconstituted into synthetic lipid membranes having known effects on receptor function, to cellular systems subjected to modification of their lipid content, e.g., varying cholesterol levels. We have often employed fluorescence techniques, including fluorescence quenching of diphenylhexatriene (DPH) extrinsic fluorescence and of nAChR intrinsic fluorescence by nitroxide spin-labeled phospholipids, DPH anisotropy, excimer formation of pyrene-phosphatidylcholine, and Förster resonance energy transfer (FRET) from the protein moiety to the extrinsic probes Laurdan, DPH, or pyrene-phospholipid to characterize various biophysical properties of lipid-receptor interactions. Some of these strategies are revisited in this review. Special attention is devoted to the anionic phospholipid phosphatidic acid (PA), which stabilizes the functional resting form of the nAChR. The receptor protein was shown to organize its PA-containing immediate microenvironment into microdomains with high lateral packing density and rigidity. PA and cholesterol appear to compete for the same binding sites on the nAChR protein.

Asunto(s)

Canales Iónicos Activados por Ligandos , Receptores Nicotínicos , Animales , Receptores Nicotínicos/química , Torpedo/metabolismo , Difenilhexatrieno , Londres , Fosfatidilcolinas/metabolismo , Colesterol/química , Ácidos Fosfatidicos/metabolismo , Pirenos

RESUMEN

Glycine receptors (GlyRs) are members of the pentameric ligand-gated ionic channel family (pLGICs) and mediate fast inhibitory neurotransmission in the brain stem and spinal cord. The function of GlyRs can be modulated by positive allosteric modulators (PAMs). So far, it is largely accepted that both the extracellular (ECD) and transmembrane (TMD) domains constitute the primary target for many of these PAMs. On the other hand, the contribution of the intracellular domain (ICD) to the PAM effects on GlyRs remains poorly understood. To gain insight about the role of the ICD in the pharmacology of GlyRs, we examined the contribution of each domain using a chimeric receptor. Two chimeras were generated, one consisting of the ECD of the prokaryotic homologue Gloeobacter violaceus ligand-gated ion channel (GLIC) fused to the TMD of the human α1GlyR lacking the ICD (Lily) and a second with the ICD (Lily-ICD). The sensitivity to PAMs of both chimeric receptors was studied using electrophysiological techniques. The Lily receptor showed a significant decrease in the sensitivity to four recognized PAMs. Remarkably, the incorporation of the ICD into the Lily background was sufficient to restore the wild-type α1GlyR sensitivity to these PAMs. Based on these data, we can suggest that the ICD is necessary to form a pLGIC having full sensitivity to positive allosteric modulators.

Asunto(s)

Regulación Alostérica/fisiología , Receptores de Glicina/fisiología , Regulación Alostérica/efectos de los fármacos , Células Cultivadas , Depresores del Sistema Nervioso Central/farmacología , Quimera , Cianobacterias , Etanol/farmacología , Espacio Extracelular/fisiología , Humanos , Concentración de Iones de Hidrógeno , Membranas Intracelulares/fisiología , Isoflurano/farmacología , Canales Iónicos Activados por Ligandos/fisiología , Potenciales de la Membrana/efectos de los fármacos

RESUMEN

Zinc is an essential metal to life. This transition metal is a structural component of many proteins and is actively involved in the catalytic activity of cell enzymes. In either case, these zinc-containing proteins are metalloproteins. However, the amino acid residues that serve as ligands for metal coordination are not necessarily the same in structural proteins compared to enzymes. While crystals of structural proteins that bind zinc reveal a higher preference for cysteine sulfhydryls rather than histidine imidazole rings, catalytic enzymes reveal the opposite, i.e., a greater preference for the histidines over cysteines for catalysis, plus the influence of carboxylic acids. Based on this paradigm, we reviewed the putative ligands of zinc in ionotropic receptors, where zinc has been described as an allosteric modulator of channel receptors. Although these receptors do not strictly qualify as metalloproteins since they do not normally bind zinc in structural domains, they do transitorily bind zinc at allosteric sites, modifying transiently the receptor channel's ion permeability. The present contribution summarizes current information showing that zinc allosteric modulation of receptor channels occurs by the preferential metal coordination to imidazole rings as well as to the sulfhydryl groups of cysteine in addition to the carboxyl group of acid residues, as with enzymes and catalysis. It is remarkable that most channels, either voltage-sensitive or transmitter-gated receptor channels, are susceptible to zinc modulation either as positive or negative regulators.

Asunto(s)

Canales Iónicos Activados por Ligandos/química , Metaloproteínas/química , Zinc/química , Regulación Alostérica/fisiología , Animales , Humanos , Canales Iónicos Activados por Ligandos/metabolismo , Metaloproteínas/metabolismo , Dominios Proteicos , Relación Estructura-Actividad , Zinc/metabolismo

RESUMEN

Ion channels allow for rapid ion diffusion through the plasma membrane. In some conditions, ion channels induce changes in the critical plasma membrane permeability that permit 900-Da solutes to enter cells. This process is known as the pore phenomenon. Some transient receptor potential (TRP) channel subtypes have been highlighted such as the P2X7 receptor, plasma membrane VDAC-1 channel, and pannexin hemichannels. The TRP ion channels are considered multimodal transducers that respond to several kinds of stimuli. In addition, many TRP channel subtypes are involved in physiological and pathophysiological processes such as inflammation, pain, and cancer. The TRPA1, TRPM8, and TRPV1-4 subtypes have been shown to promote large-molecular-weight solute uptake, including impermeable fluorescent dyes, QX-314 hydrophilic lidocaine derivative, gabapentin, and antineoplastic drugs. This review discusses the current knowledge of TRP-associated pores and encourages scientists to study their features and explore them as novel therapeutic tools.

Asunto(s)

Permeabilidad de la Membrana Celular , Canales Iónicos Activados por Ligandos/metabolismo , Potenciales de la Membrana , Canales Aniónicos Dependientes del Voltaje/metabolismo , Animales , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Transporte Iónico , Proteínas de Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Dolor/tratamiento farmacológico , Dolor/metabolismo

RESUMEN

Ligand-gated ion channels (LGICs) are cell surface integral proteins that mediate the fast neurotransmission in the nervous system. LGICs require auxiliary subunits for their trafficking, assembly and pharmacological modulation. Auxiliary subunits do not form functional homomeric receptors, but are reported to assemble with the principal subunits in order to modulate their pharmacological profiles. For example, nACh receptors are built at least by co-assemble of α and ß subunits, and the neuronal auxiliary subunits ß3 and α5 and muscle type ß, δ, γ, and ϵ determine the agonist affinity of these receptors. Serotonergic 5-HT3B, 5-HT3C, 5-HT3D and 5-HT3E are reported to assemble with the 5-HT3A subunit to modulate its pharmacological profile. Functional studies evaluating the role of γ2 and δ auxiliary subunits of GABAA receptors have made important advances in the understanding of the action of benzodiazepines, ethanol and neurosteroids. Glycine receptors are composed principally by α1-3 subunits and the auxiliary subunit ß determines their synaptic location and their pharmacological response to propofol and ethanol. NMDA receptors appear to be functional as heterotetrameric channels. So far, the existence of NMDA auxiliary subunits is controversial. On the other hand, Kainate receptors are modulated by NETO 1 and 2. AMPA receptors are modulated by TARPs, Shisa 9, CKAMP44, CNIH2-3 auxiliary proteins reported that controls their trafficking, conductance and gating of channels. P2X receptors are able to associate with auxiliary Pannexin-1 protein to modulate P2X7 receptors. Considering the pharmacological relevance of different LGICs auxiliary subunits in the present work we will highlight the therapeutic potential of these modulator proteins.

Asunto(s)

Canales Iónicos Activados por Ligandos/efectos de los fármacos , Animales , Humanos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos Activados por Ligandos/química , Canales Iónicos Activados por Ligandos/metabolismo , Modelos Moleculares , Subunidades de Proteína , Receptores AMPA/química , Receptores AMPA/efectos de los fármacos , Receptores AMPA/metabolismo , Receptores de GABA-A/química , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Receptores de Glutamato/química , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Receptores de Glicina/química , Receptores de Glicina/efectos de los fármacos , Receptores de Glicina/metabolismo , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/efectos de los fármacos , Receptores de Ácido Kaínico/metabolismo , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Receptores Purinérgicos P2X/química , Receptores Purinérgicos P2X/efectos de los fármacos , Receptores Purinérgicos P2X/metabolismo , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/efectos de los fármacos , Receptores de Serotonina 5-HT3/metabolismo

RESUMEN

Rational construction of interfaces based on multicomponent responsive systems in which molecular transport is mediated by structures of nanoscale dimensions has become a very fertile research area in biomimetic supramolecular chemistry. Herein, we describe the creation of hybrid mesostructured interfaces with reversible gate-like transport properties that can be controlled by chemical inputs, such as protons or calcium ions. This was accomplished by taking advantage of the surface-initiated polymerization of 2-(methacryloyloxy)ethyl phosphate (MEP) monomer units into and onto mesoporous silica thin films. In this way, phosphate-bearing polymer brushes were used as "gatekeepers" located not only on the outer surface of mesoporous thin films but also in the inner environment of the porous scaffold. Pore-confined PMEP brushes respond to the external triggering chemical signals not only by altering their physicochemical properties but also by switching the transport properties of the mesoporous film. The ion-gate response/operation was based on the protonation and/or chelation of phosphate monomer units in which the polymer brush works as an off-on switch in response to the presence of protons or Ca(2+) ions. The hybrid meso-architectured interface and their functional features were studied by a combination of experimental techniques including ellipso-porosimetry, cyclic voltammetry, X-ray reflectivity, grazing incidence small-angle X-ray scattering, X-ray photoelectron spectroscopy, and in situ atomic force microscopy. In this context, we believe that the integration of stimuli-responsive polymer brushes into nanoscopic supramolecular architectures would provide new routes toward multifunctional biomimetic nanosystems displaying transport properties similar to those encountered in biological ligand-gated ion channels.

Asunto(s)

Materiales Biomiméticos/química , Canales Iónicos Activados por Ligandos/química , Membranas Artificiales , Polímeros/química , Biomimética/métodos , Calcio , Nanoestructuras , Fosfatos/química , Porosidad , Protones

RESUMEN

It is well known that ethanol modulates the function of the Cys loop ligand-gated ion channels, which include the inhibitory glycine receptors (GlyRs). Previous studies have consistently shown that transmembrane and extracellular sites are essential for ethanol actions in GlyRs. In addition, recent evidence has shown that the ethanol modulation of GlyRs is also affected by G protein activation through Gßγ subunits. However, more specific roles of G protein α subunits on ethanol actions are unknown. Here, we show that the allosteric effect of ethanol on the human α(1) GlyR is selectively enhanced by the expression of Gα(s) Q-L. For example, constitutively active Gα(s), but not Gα(q) or Gα(i), was able to displace the alcohol sensitivity of GlyRs toward low millimolar concentrations (17 ± 4 versus 48 ± 5% at 100 mM). Experiments under conditions that increased cAMP and protein kinase A (PKA)-mediated signaling, on the contrary, did not produce the same enhancement in sensitivity, suggesting that the Gα(s) Q-L effect was not dependent on cAMP/PKA-dependent signaling. On the other hand, the effect of Gα(s) Q-L was blocked by a Gßγ scavenger (9 ± 3% of control). Furthermore, two mutant receptors previously shown to have impaired interactions with Gßγ were not affected by Gα(s) Q-L, suggesting that Gßγ is needed for enhancing ethanol sensitivity. These results support the conclusion that activated Gα(s) can facilitate the Gßγ interaction with GlyRs in presence of ethanol, independent of increases in cAMP signaling. Thus, these data indicate that the activated form of Gα(s) is able to positively influence the effect of ethanol on a type of inhibitory receptor important for motor control, pain, and respiration.

Asunto(s)

Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gs/metabolismo , Proteínas de Unión al GTP/metabolismo , Receptores de Glicina/metabolismo , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Técnica del Anticuerpo Fluorescente , Células HEK293 , Humanos , Canales Iónicos Activados por Ligandos , Técnicas de Placa-Clamp , Fosforilación , Receptores de Glicina/genética , Transducción de Señal

RESUMEN

Ligand-gated ion channel kinetics were studied in mammalian transfected cells encoding adult mouse muscle acetylcholine (ACh) receptors. We measured macroscopic and single-channel currents using the outside-out and cell-attached patch-clamp configurations. Cultured cells were exposed to moderate intensity inhomogeneous static magnetic fields up to 180 mT and measurements were performed for temperatures ranging from 5 to 50 °C. We found no significant changes in ACh-elicited macroscopic or single-channel currents. We observed the expected dependence in current decay constants with temperature, but negligible magnetic field influence on the channel's kinetics.

Asunto(s)

Campos Magnéticos , Receptores Nicotínicos/metabolismo , Animales , Células Cultivadas , Canales Iónicos Activados por Ligandos/metabolismo , Ratones , Músculo Esquelético/citología , Técnicas de Placa-Clamp , Temperatura

RESUMEN

In silico simulation based on Markov chains is a powerful way to describe and predict the activity of many transport proteins including ion channels. However, modeling and simulation using realistic models of voltage- or ligand-gated ion channels exposed to a wide range of experimental conditions require building complex kinetic schemes and solving complicated differential equations. To circumvent these problems, we developed IonChannelLab a software tool that includes a user-friendly Graphical User Interface and a simulation library. This program supports channels with Ohmic or Goldman-Hodgkin-Katz behavior and can simulate the time-course of ionic and gating currents, single channel behavior and steady-state conditions. The program allows the simulation of experiments where voltage, ligand and ionic concentration are varied independently or simultaneously.

Asunto(s)

Simulación por Computador , Canales Iónicos/metabolismo , Modelos Moleculares , Programas Informáticos , Activación del Canal Iónico , Cinética , Canales Iónicos Activados por Ligandos/metabolismo , Cadenas de Markov , Interfaz Usuario-Computador