RESUMEN
Vertebrate neurons are highly dynamic cells that undergo several alterations in their functioning and physiologies in adaptation to various external stimuli. In particular, how these neurons respond to physical exercise has long been an area of active research. Studies of the vertebrate locomotor system's adaptability suggest multiple mechanisms are involved in the regulation of neuronal activity and properties during exercise. In this brief review, we highlight recent results and insights from the field with a focus on the following mechanisms: (a) alterations in neuronal excitability during acute exercise; (b) alterations in neuronal excitability after chronic exercise; (c) exercise-induced changes in neuronal membrane properties via modulation of ion channel activity; (d) exercise-enhanced dendritic plasticity; and (e) exercise-induced alterations in neuronal gene expression and protein synthesis. Our hope is to update the community with a cellular and molecular understanding of the recent mechanisms underlying the adaptability of the vertebrate locomotor system in response to both acute and chronic physical exercise.
Asunto(s)
Neuronas , Condicionamiento Físico Animal , Animales , Neuronas/fisiología , Vertebrados , Condicionamiento Físico Animal/fisiologíaRESUMEN
SignificanceCholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.
Asunto(s)
Colesterol , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Membrana Celular/metabolismo , Colesterol/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Potasio/metabolismo , Transducción de SeñalRESUMEN
Selective ion channel modulators play a critical role in physiology in defining the contribution of specific ion channels to physiological function and as proof of concept for novel therapeutic strategies. Antibodies are valuable research tools that have broad uses including defining the expression and localization of ion channels in native tissue, and capturing ion channel proteins for subsequent analyses. In this review, we detail how renewable and recombinant antibodies can be used to control ion channel function. We describe the different forms of renewable and recombinant antibodies that have been used and the mechanisms by which they modulate ion channel function. We highlight the use of recombinant antibodies that are expressed intracellularly (intrabodies) as genetically encoded tools to control ion channel function. We also offer perspectives of avenues of future research that may be opened by the application of emerging technologies for engineering recombinant antibodies for enhanced utility in ion channel research. Overall, this review provides insights that may help stimulate and guide interested researchers to develop and incorporate renewable and recombinant antibodies as valuable tools to control ion channel function.
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Anticuerpos , Canales IónicosRESUMEN
Transient receptor potential vanilloid (TRPV) channels are part of the TRP channel superfamily and named after the first identified member TRPV1, that is sensitive to the vanillylamide capsaicin. Their overall structure is similar to the structure of voltage gated potassium channels (Kv) built up as homotetramers from subunits with six transmembrane helices (S1-S6). Six TRPV channel subtypes (TRPV1-6) are known, that can be subdivided into the thermoTRPV (TRPV1-4) and the Ca2+-selective TRPV channels (TRPV5, TRPV6). Contrary to Kv channels, TRPV channels are not primary voltage gated. All six channels have distinct properties and react to several endogenous ligands as well as different gating stimuli such as heat, pH, mechanical stress, or osmotic changes. Their physiological functions are highly diverse and subtype as well as tissue specific. In many tissues they serve as sensors for different pain stimuli (heat, pressure, pH) and contribute to the homeostasis of electrolytes, the maintenance of barrier functions and the development of macrophages. Due to their fundamental role in manifold physiological and pathophysiological processes, TRPV channels are promising targets for drug development. However, drugs targeting specific TRPV channels, that are suitable for drug therapy, are rare. Moreover, selective and potent compounds for further research at TRPV channels are often lacking. In this review different aspects of the structure, the different gating stimuli, the expression pattern, the physiological and pathophysiological roles as well as the modulating mechanisms of synthetic, natural and endogenous ligands are summarized.
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Analgésicos/farmacología , Antineoplásicos/farmacología , Factores Inmunológicos/farmacología , Moduladores del Transporte de Membrana/farmacología , Canales Catiónicos TRPV/metabolismo , Analgésicos/química , Analgésicos/clasificación , Antineoplásicos/química , Antineoplásicos/clasificación , Sitios de Unión , Encéfalo/citología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/clasificación , Activación del Canal Iónico/efectos de los fármacos , Ligandos , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Moduladores del Transporte de Membrana/química , Moduladores del Transporte de Membrana/clasificación , Modelos Moleculares , Especificidad de Órganos , Unión Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Bazo/citología , Bazo/efectos de los fármacos , Bazo/metabolismo , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/clasificaciónRESUMEN
Modifications of the composition or organization of the cancer cell membrane seem to be a promising targeted therapy. This approach can significantly enhance drug uptake or intensify the response of cancer cells to chemotherapeutics. There are several methods enabling lipid bilayer modifications, e.g., pharmacological, physical, and mechanical. It is crucial to keep in mind the significance of drug resistance phenomenon, ion channel and specific receptor impact, and lipid bilayer organization in planning the cell membrane-targeted treatment. In this review, strategies based on cell membrane modulation or reorganization are presented as an alternative tool for future therapeutic protocols.
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Membrana Celular , Sistemas de Liberación de Medicamentos , Neoplasias , Membrana Celular/metabolismo , Membrana Celular/patología , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
KCNQ1, also known as Kv7.1, is a voltage-dependent K+ channel that regulates gastric acid secretion, salt and glucose homeostasis, and heart rhythm. Its functional properties are regulated in a tissue-specific manner through co-assembly with beta subunits KCNE1-5. In non-excitable cells, KCNQ1 forms a complex with KCNE3, which suppresses channel closure at negative membrane voltages that otherwise would close it. Pore opening is regulated by the signaling lipid PIP2. Using cryoelectron microscopy (cryo-EM), we show that KCNE3 tucks its single-membrane-spanning helix against KCNQ1, at a location that appears to lock the voltage sensor in its depolarized conformation. Without PIP2, the pore remains closed. Upon addition, PIP2 occupies a site on KCNQ1 within the inner membrane leaflet, which triggers a large conformational change that leads to dilation of the pore's gate. It is likely that this mechanism of PIP2 activation is conserved among Kv7 channels.
Asunto(s)
Canal de Potasio KCNQ1/metabolismo , Canal de Potasio KCNQ1/ultraestructura , Microscopía por Crioelectrón , Humanos , Activación del Canal Iónico/fisiología , Canal de Potasio KCNQ1/química , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Canales de Potasio con Entrada de Voltaje/ultraestructuraRESUMEN
Phosphatidylinositol 4,5-bisphosphate (PIP2) in the plasma membrane regulates the function of many ion channels, including M-type (potassium voltage-gated channel subfamily Q member (KCNQ), Kv7) K+ channels; however, the molecular mechanisms involved remain unclear. To this end, we here focused on the KCNQ3 subtype that has the highest apparent affinity for PIP2 and performed extensive mutagenesis in regions suggested to be involved in PIP2 interactions among the KCNQ family. Using perforated patch-clamp recordings of heterologously transfected tissue culture cells, total internal reflection fluorescence microscopy, and the zebrafish (Danio rerio) voltage-sensitive phosphatase to deplete PIP2 as a probe, we found that PIP2 regulates KCNQ3 channels through four different domains: 1) the A-B helix linker that we previously identified as important for both KCNQ2 and KCNQ3, 2) the junction between S6 and the A helix, 3) the S2-S3 linker, and 4) the S4-S5 linker. We also found that the apparent strength of PIP2 interactions within any of these domains was not coupled to the voltage dependence of channel activation. Extensive homology modeling and docking simulations with the WT or mutant KCNQ3 channels and PIP2 were consistent with the experimental data. Our results indicate that PIP2 modulates KCNQ3 channel function by interacting synergistically with a minimum of four cytoplasmic domains.
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Citoplasma/metabolismo , Canal de Potasio KCNQ3/química , Canal de Potasio KCNQ3/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetulus , Humanos , Canal de Potasio KCNQ3/genética , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios ProteicosRESUMEN
KEY POINTS: The AMPA-type ionotropic glutamate receptors (AMPARs) mediate the majority of excitatory synaptic transmission and their function impacts learning, cognition and behaviour. The gating of AMPARs occurs in milliseconds, precisely controlled by a variety of auxiliary subunits that are expressed differentially in the brain, but the difference in mechanisms underlying AMPAR gating modulation by auxiliary subunits remains elusive and is investigated. The elements of the AMPAR that are functionally recruited by auxiliary subunits, stargazin and cornichon 3, are located not only in the extracellular domains but also in the lipid-accessible surface of the AMPAR. We reveal that the two auxiliary subunits require a shared surface on the transmembrane domain of the AMPAR for their function, but the gating is influenced by this surface in opposing directions for each auxiliary subunit. Our results provide new insights into the mechanistic difference of AMPAR modulation by auxiliary subunits and a conceptual framework for functional engineering of the complex. ABSTRACT: During excitatory synaptic transmission, various structurally unrelated transmembrane auxiliary subunits control the function of AMPA receptors (AMPARs), but the underlying mechanisms remain unclear. We identified lipid-exposed residues in the transmembrane domain (TMD) of the GluA2 subunit of AMPARs that are critical for the function of AMPAR auxiliary subunits, stargazin (Stg) and cornichon 3 (CNIH3). These residues are essential for stabilizing the AMPAR-CNIH3 complex in detergents and overlap with the contacts made between GluA2 TMD and Stg in the cryoEM structures. Mutating these residues had opposite effects on gating modulation and complex stability when Stg- and CNIH3-bound AMPARs were compared. Specifically, in detergent the GluA2-A793F formed an unstable complex with CNIIH3 but in the membrane the GluA2-A793F-CNIH3 complex expressed a gain of function. In contrast, the GluA2-A793F-Stg complex was stable, but had diminished gating modulation. GluA2-C528L destabilized the AMPAR-CNIH3 complex but stabilized the AMPAR-Stg complex, with overall loss of function in gating modulation. Furthermore, loss-of-function mutations in this TMD region cancelled the effects of a gain-of-function Stg carrying mutation in its extracellular loop, demonstrating that both the extracellular and the TMD elements contribute independently to gating modulation. The elements of AMPAR functionally recruited by auxiliary subunits are, therefore, located not only in the extracellular domains but also in the lipid accessible surface of the AMPAR. The TMD surface we defined is a potential target for auxiliary subunit-specific compounds, because engineering of this hotspot induces opposing functional outcomes by Stg and CNIH3. The collection of mutant-phenotype mapping provides a framework for engineering AMPAR gating using auxiliary subunits.
Asunto(s)
Canales de Calcio/fisiología , Receptores AMPA/fisiología , Canales de Calcio/genética , Línea Celular , Membrana Celular , Humanos , Activación del Canal Iónico , Mutación , Dominios Proteicos , Receptores AMPA/genéticaRESUMEN
The membrane dipole potential (Ψd) constitutes one of three electrical potentials generated by cell membranes. Ψd arises from the unfavorable parallel alignment of phospholipid and water dipoles, and varies in magnitude both longitudinally and laterally across the bilayer according to membrane composition and phospholipid packing density. In this work, we propose that dynamic counter-balancing between Ψd and the transmembrane potential (ΔΨm) governs the conformational state transitions of voltage-gated ion channels. Ψd consists of 1) static outer, and dynamic inner leaflet components (Ψd(extra) and Ψd(intra), respectively); and 2) a transmembrane component (ΔΨd(inner-outer)), ariing from differences in intra- and extracellular leaflet composition. Ψd(intra), which transitions between high and low energy states (Ψd(intra, high) and Ψd(intra, low)) as a function of channel conformation, is transduced by the pore domain. ΔΨd(inner-outer) is transduced by the voltage-sensing (VS) domain in summation with ΔΨm. Potentiation of voltage-gated ion channels is of interest for the treatment of cardiac, neuronal, and other disorders arising from inherited/acquired ion channel dysfunction. Potentiators are widely believed to alter the rates and voltage-dependencies of channel gating transitions by binding to pockets in the membrane-facing and other regions of ion channel targets. Here, we propose that potentiators alter Ψd(intra) and/or Ψd(extra), thereby increasing or decreasing the energy barriers governing channel gating transitions. We used quantum mechanical and molecular dynamics (MD) simulations to predict the overall Ψd-modulating effects of a series of published positive hERG potentiators partitioned into model DOPC bilayers. Our findings suggest a strong correlation between the magnitude of Ψd-lowering and positive hERG potentiation across the series.
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Cationes/metabolismo , Membrana Celular/fisiología , Activación del Canal Iónico/fisiología , Canales Iónicos/metabolismo , Potenciales de la Membrana/fisiología , Sitios de Unión/fisiología , Fenómenos Biofísicos/fisiología , Humanos , Membrana Dobles de Lípidos/metabolismo , Simulación de Dinámica Molecular , Unión Proteica/fisiología , Regulador Transcripcional ERG/metabolismoRESUMEN
Ca2+ release-activated Ca2+ (CRAC) channels play an essential role in the immune system. The pore-forming subunit, Orai1, is an important pharmacological target. Here, we summarize the recent discoveries on the structure-function relationship of Orai1, as well as its interaction with the native channel opener STIM1 and chemical modulator 2-aminoethoxydiphenyl borate (2-APB). We first introduce the critical structural elements of Orai1, which include a Ca2+ accumulating region, ion selectivity filter, hydrophobic centre, basic region, extended transmembrane Orai1 N-terminal (ETON) region, transmembrane (TM) regions 2 and 3, P245 bend, 263 SHK265 hinge linker and L273-L276 hydrophobic patch. We then hypothesize the possible mechanisms by which STIM1 triggers the conformational transitions of TM regions and exquisitely shapes the ion conduction pathway during generation of the CRAC current (Icrac ) with high Ca2+ selectivity. Finally, we propose mechanisms by which 2-APB modulates Icrac . On the STIM1-activated Orai1 channel, a low dose of 2-APB acts directly, dilating its extremely narrow pore diameter from 3.8 to 4.6 Å, increasing its unitary channel conductance, and potentiating the Icrac . Further elucidation of the structure of the opened CRAC channel and a better understanding of structure-function relationship will benefit the future development of novel immune modulators.
Asunto(s)
Compuestos de Boro/farmacología , Proteínas de Neoplasias/fisiología , Proteína ORAI1/química , Proteína ORAI1/fisiología , Molécula de Interacción Estromal 1/fisiología , Humanos , Activación del Canal Iónico/efectos de los fármacosRESUMEN
The endogenous neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) is secreted by both neuronal and non-neuronal cells in the brain and spinal cord, in response to pathological conditions such as stroke, seizures, chronic inflammatory and neuropathic pain. PACAP has been shown to exert various neuromodulatory and neuroprotective effects. However, direct influence of PACAP on the function of intrinsically excitable ion channels that are critical to both hyperexcitation as well as cell death, remain largely unexplored. The major dendritic K(+) channel Kv4.2 is a critical regulator of neuronal excitability, back-propagating action potentials in the dendrites, and modulation of synaptic inputs. We identified, cloned and characterized the downstream signaling originating from the activation of three PACAP receptor (PAC1) isoforms that are expressed in rodent hippocampal neurons that also exhibit abundant expression of Kv4.2 protein. Activation of PAC1 by PACAP leads to phosphorylation of Kv4.2 and downregulation of channel currents, which can be attenuated by inhibition of either PKA or ERK1/2 activity. Mechanistically, this dynamic downregulation of Kv4.2 function is a consequence of reduction in the density of surface channels, without any influence on the voltage-dependence of channel activation. Interestingly, PKA-induced effects on Kv4.2 were mediated by ERK1/2 phosphorylation of the channel at two critical residues, but not by direct channel phosphorylation by PKA, suggesting a convergent phosphomodulatory signaling cascade. Altogether, our findings suggest a novel GPCR-channel signaling crosstalk between PACAP/PAC1 and Kv4.2 channel in a manner that could lead to neuronal hyperexcitability.
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Dendritas/efectos de los fármacos , Neuronas/citología , Neurotransmisores/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Canales de Potasio Shal/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Humanos , Masculino , Ratones , Mutagénesis/genética , Mutación/genética , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/genética , Ratas , Ratas Sprague-Dawley , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
Ion channels facilitate the passive movement of ions down an electrochemical gradient and across lipid bilayers in cells. This phenomenon is essential for life and underlies many critical homeostatic processes in cells. Ion channels are diverse and differ with respect to how they open and close (gating) and to their ionic conductance/selectivity (permeation). Fundamental understanding of ion channel structure-function mechanisms, their physiological roles, how their dysfunction leads to disease, their utility as biosensors, and development of novel molecules to modulate their activity are important and active research frontiers. In this review, we focus on ion channel engineering approaches that have been applied to investigate these aspects of ion channel function, with a major emphasis on voltage-gated ion channels.
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Canales Iónicos/fisiología , Transporte Iónico , Ingeniería de Proteínas , Animales , HumanosRESUMEN
The large-conductance voltage- and Ca(2+)-activated K(+) channel (BKCa) is an important regulator of membrane excitability in a wide variety of cells and tissues. In myometrial smooth muscle, activation of BKCa plays essential roles in buffering contractility to maintain uterine quiescence during pregnancy and in the transition to a more contractile state at the onset of labor. Multiple mechanisms of modulation have been described to alter BKCa channel activity, expression, and cellular localization. In the myometrium, BKCa is regulated by alternative splicing, protein targeting to the plasma membrane, compartmentation in membrane microdomains, and posttranslational modifications. In addition, interaction with auxiliary proteins (i.e., ß1- and ß2-subunits), association with G-protein coupled receptor signaling pathways, such as those activated by adrenergic and oxytocin receptors, and hormonal regulation provide further mechanisms of variable modulation of BKCa channel function in myometrial smooth muscle. Here, we provide an overview of these mechanisms of BKCa channel modulation and provide a context for them in relation to myometrial function.
RESUMEN
Professional deep-water divers exposed to high pressure (HP) above 1.1 MPa suffer from High Pressure Neurological Syndrome (HPNS), which is associated with CNS hyperexcitability. We have previously reported that HP augments N-methyl-D-aspartate receptor (NMDAR) synaptic responses, increases neuronal excitability, and potentially causes irreversible neuronal damage. We now report that HP (10.1 MPa) differentially affects eight specific NMDAR subtypes. GluN1(1a or 1b) was co-expressed with one of the four GluN2(A-D) subunits in Xenopus laevis oocytes. HP increased ionic currents (measured by two electrode voltage clamps) of one subtype, reduced the current in four others, and did not affect the current in the remaining three. 3D theoretical modeling was aimed at revealing specific receptor domains involved with HP selectivity. In light of the information on the CNS spatial distribution of the different NMDAR subtypes, we conclude that the NMDAR's diverse responses to HP may lead to selective HP effects on different brain regions. These discoveries call for further and more specific investigation of deleterious HP effects and suggest the need for a re-evaluation of deep-diving safety guidelines.