RESUMEN
The transmembrane protein TMEM206 was recently identified as the molecular basis of the extracellular proton-activated Cl- channel (PAC), which plays an essential role in neuronal death in ischemia-reperfusion. The PAC channel is activated by extracellular acid, but the proton-sensitive mechanism remains unclear, although different acid-sensitive pockets have been suggested based on the cryo-EM structure of the human PAC (hPAC) channel. In the present study, we firstly identified two acidic amino acid residues that removed the pH-dependent activation of the hPAC channel by neutralization all the conservative negative charged residues located in the extracellular domain of the hPAC channel and some positively charged residues at the hotspot combined with two-electrode voltage-clamp (TEVC) recording in the Xenopus oocytes system. Double-mutant cycle analysis and double cysteine mutant of these two residues proved that these two residues cooperatively form a proton-sensitive site. In addition, we found that chloral hydrate activates the hPAC channel depending on the normal pH sensitivity of the hPAC channel. Furthermore, the PAC channel knock-out (KO) male mice (C57BL/6J) resist chloral hydrate-induced sedation and hypnosis. Our study provides a molecular basis for understanding the proton-dependent activation mechanism of the hPAC channel and a novel drug target of chloral hydrate.SIGNIFICANCE STATEMENT Proton-activated Cl- channel (PAC) channels are widely distributed in the nervous system and play a vital pathophysiological role in ischemia and endosomal acidification. The main discovery of this paper is that we identified the proton activation mechanism of the human proton-activated chloride channel (hPAC). Intriguingly, we also found that anesthetic chloral hydrate can activate the hPAC channel in a pH-dependent manner. We found that the chloral hydrate activates the hPAC channel and needs the integrity of the pH-sensitive site. In addition, the PAC channel knock-out (KO) mice are resistant to chloral hydrate-induced anesthesia. The study on PAC channels' pH activation mechanism enables us to better understand PAC's biophysical mechanism and provides a novel target of chloral hydrate.
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Hidrato de Cloral , Canales de Cloruro , Ratones , Animales , Masculino , Humanos , Hidrato de Cloral/farmacología , Canales de Cloruro/genética , Canales de Cloruro/metabolismo , Protones , Cloruros/metabolismo , Ratones Endogámicos C57BLRESUMEN
The Slack channel (KCNT1, Slo2.2) is a sodium-activated and chloride-activated potassium channel that regulates heart rate and maintains the normal excitability of the nervous system. Despite intense interest in the sodium gating mechanism, a comprehensive investigation to identify the sodium-sensitive and chloride-sensitive sites has been missing. In the present study, we identified two potential sodium-binding sites in the C-terminal domain of the rat Slack channel by conducting electrophysical recordings and systematic mutagenesis of cytosolic acidic residues in the rat Slack channel C terminus. In particular, by taking advantage of the M335A mutant, which results in the opening of the Slack channel in the absence of cytosolic sodium, we found that among the 92 screened negatively charged amino acids, E373 mutants could completely remove sodium sensitivity of the Slack channel. In contrast, several other mutants showed dramatic decreases in sodium sensitivity but did not abolish it altogether. Furthermore, molecular dynamics (MD) simulations performed at the hundreds of nanoseconds timescale revealed one or two sodium ions at the E373 position or an acidic pocket composed of several negatively charged residues. Moreover, the MD simulations predicted possible chloride interaction sites. By screening predicted positively charged residues, we identified R379 as a chloride interaction site. Thus, we conclude that the E373 site and the D863/E865 pocket are two potential sodium-sensitive sites, while R379 is a chloride interaction site in the Slack channel.SIGNIFICANCE STATEMENT The research presented here identified two distinct sodium and one chloride interaction sites located in the intracellular C-terminal domain of the Slack (Slo2.2, KCNT1) channel. Identification of the sites responsible for the sodium and chloride activation of the Slack channel sets its gating property apart from other potassium channels in the BK channel family. This finding sets the stage for future functional and pharmacological studies of this channel.
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Canales de potasio activados por Sodio , Animales , Ratas , Cloruros/metabolismo , Canales de potasio activados por Sodio/metabolismo , Sodio/metabolismoRESUMEN
Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. In the present study, we find that the global Slack KO male mice exhibit anxious behaviors, whereas the Slack Y777H male mice manifest anxiolytic behaviors. The expression of Slack channels is rich in basolateral amygdala (BLA) glutamatergic neurons and downregulated in chronic corticosterone-treated mice. In addition, electrophysiological data show enhanced excitability of BLA glutamatergic neurons in the Slack KO mice and decreased excitability of these neurons in the Slack Y777H mice. Furthermore, the Slack channel deletion in BLA glutamatergic neurons is sufficient to result in enhanced avoidance behaviors, whereas Kcnt1 gene expression in the BLA or BLA-ventral hippocampus (vHPC) glutamatergic projections reverses anxious behaviors of the Slack KO mice. Our study identifies the role of the Slack channel in controlling anxious behaviors by decreasing the excitability of BLA-vHPC glutamatergic projections, providing a potential target for anxiolytic therapies.SIGNIFICANCE STATEMENT Anxiety disorders are a series of mental disorders characterized by anxiety and fear, but the molecular basis of these disorders remains unclear. Here, we examined the behaviors of loss- and gain-of-function of Slack channel mice in elevated plus maze and open field tests and found the anxiolytic role of the Slack channel. By altering the Slack channel expression in the specific neuronal circuit, we demonstrated that the Slack channel played its anxiolytic role by decreasing the excitability of BLA-vHPC glutamatergic projections. Our data reveal the role of the Slack channel in the regulation of anxiety, which may provide a potential molecular target for anxiolytic therapies.
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Ansiedad , Complejo Nuclear Basolateral , Proteínas del Tejido Nervioso , Canales de potasio activados por Sodio , Animales , Ansiedad/metabolismo , Complejo Nuclear Basolateral/metabolismo , Hipocampo/metabolismo , Masculino , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/metabolismo , Canales de potasio activados por Sodio/metabolismoRESUMEN
Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.
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Antiarrítmicos , Péptidos y Proteínas de Señalización Intercelular , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Neurotoxinas , Péptidos , Venenos de Araña , Animales , Antiarrítmicos/química , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Lípidos , Ratones , Neurotoxinas/química , Neurotoxinas/farmacología , Péptidos/química , Péptidos/farmacología , Venenos de Araña/química , Venenos de Araña/farmacología , Venenos de Araña/uso terapéuticoRESUMEN
The cardiac mechanosensitive BK (Slo1) channels are gated by Ca2+, voltage, and membrane stretch. The neuropeptide GsMTx4 is a selective inhibitor of mechanosensitive (MS) channels. It has been reported to suppress stretch-induced cardiac fibrillation in the heart, but the mechanism underlying the specificity and even the targeting channel(s) in the heart remain elusive. Here, we report that GsMTx4 inhibits a stretch-activated BK channel (SAKcaC) in the heart through a modulation specific to mechano-gating. We show that membrane stretching increases while GsMTx4 decreases the open probability (Po) of SAKcaC. These effects were mostly abolished by the deletion of the STREX axis-regulated (STREX) exon located between RCK1 and RCK2 domains in BK channels. Single-channel kinetics analysis revealed that membrane stretch activates SAKcaC by prolonging the open-time duration (τO) and shortening the closed-time constant (τC). In contrast, GsMTx4 reversed the effects of membrane stretch, suggesting that GsMTx4 inhibits SAKcaC activity by interfering with mechano-gating of the channel. Moreover, GsMTx4 exerted stronger efficacy on SAKcaC under membrane-hyperpolarized/resting conditions. Molecular dynamics simulation study revealed that GsMTx4 appeared to have the ability to penetrate deeply within the bilayer, thus generating strong membrane deformation under the hyperpolarizing/resting conditions. Immunostaining results indicate that BK variants containing STREX are also expressed in mouse ventricular cardiomyocytes. Our results provide common mechanisms of peptide actions on MS channels and may give clues to therapeutic suppression of cardiac arrhythmias caused by excitatory currents through MS channels under hyper-mechanical stress in the heart.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Venenos de Araña/metabolismo , Animales , Membrana Celular/metabolismo , Pollos , Embrión no Mamífero/metabolismo , Cinética , Canales de Potasio de Gran Conductancia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Gran Conductancia Activados por el Calcio/genética , Ratones , Simulación de Dinámica Molecular , Miocitos Cardíacos/clasificación , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Dominios ProteicosRESUMEN
All vertebrate inwardly rectifying potassium (Kir) channels are activated by phosphatidylinositol 4,5-bisphosphate (PIP2) (Logothetis, D. E., Petrou, V. I., Zhang, M., Mahajan, R., Meng, X. Y., Adney, S. K., Cui, M., and Baki, L. (2015) Annu. Rev. Physiol. 77, 81-104; Fürst, O., Mondou, B., and D'Avanzo, N. (2014) Front. Physiol. 4, 404-404). Structural components of a PIP2-binding site are conserved in vertebrate Kir channels but not in distantly related animals such as sponges and sea anemones. To expand our understanding of the structure-function relationships of PIP2 regulation of Kir channels, we studied AqKir, which was cloned from the marine sponge Amphimedon queenslandica, an animal that represents the phylogenetically oldest metazoans. A requirement for PIP2 in the maintenance of AqKir activity was examined in intact oocytes by activation of a co-expressed voltage-sensing phosphatase, application of wortmannin (at micromolar concentrations), and activation of a co-expressed muscarinic acetylcholine receptor. All three mechanisms to reduce the availability of PIP2 resulted in inhibition of AqKir current. However, time-dependent rundown of AqKir currents in inside-out patches could not be re-activated by direct application to the inside membrane surface of water-soluble dioctanoyl PIP2, and the current was incompletely re-activated by the more hydrophobic arachidonyl stearyl PIP2. When we introduced mutations to AqKir to restore two positive charges within the vertebrate PIP2-binding site, both forms of PIP2 strongly re-activated the mutant sponge channels in inside-out patches. Molecular dynamics simulations validate the additional hydrogen bonding potential of the sponge channel mutants. Thus, nature's mutations conferred a high affinity activation of vertebrate Kir channels by PIP2, and this is a more recent evolutionary development than the structures that explain ion channel selectivity and inward rectification.
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Mutación , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/genética , Vertebrados/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Pollos , Evolución Molecular , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Fosfatidilinositol 4,5-Difosfato/química , Poríferos/genética , Poríferos/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Alineación de Secuencia , Vertebrados/clasificación , Vertebrados/metabolismoRESUMEN
Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca(2+)). Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca(2+)-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP2 regulation of this channel's activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca(2+) and PIP2 in regulating BK channel activity. In the absence of Ca(2+), RCK1 structural elements limit channel activation through a decrease in the channel's PIP2 apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca(2+) coordination site (Asp(367) or its flanking basic residues in the KDRDD loop) to the PIP2-interacting residues (Lys(392) and Arg(393)) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca(2+), the RCK1-inhibitory influence of channel-PIP2 interactions and channel activity is relieved by Ca(2+) engaging Asp(367). Our results demonstrate that, along with Ca(2+) and voltage, PIP2 is a third factor critical to the integral control of BK channel activity.
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Calcio/metabolismo , Canal de Potasio Kv.1.1/química , Canal de Potasio Kv.1.1/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Regulación Alostérica , Secuencia de Aminoácidos , Animales , Sitios de Unión , Membrana Celular/metabolismo , Citosol/metabolismo , Activación del Canal Iónico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Ratones , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutagénesis , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Voltage-gated K(+) (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP(2)) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP(2) depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP(2). Gating current measurements revealed that PIP(2) constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP(2) controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.
Asunto(s)
Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Modelos Moleculares , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Cristalografía por Rayos X , Cinética , Simulación de Dinámica Molecular , Mutagénesis , Oocitos/metabolismo , Técnicas de Placa-Clamp , Fosfolípidos/metabolismo , Subunidades de Proteína/metabolismo , XenopusRESUMEN
BACKGROUND: Cholesterol modulates inwardly rectifying potassium (Kir) channels. RESULTS: A two-way molecular cytosolic switch controls channel modulation by cholesterol and PI(4,5)P(2). CONCLUSION: Cholesterol and PI(4,5)P(2) induce a common gating pathway of Kir2.1 despite their opposite impact on channel function. SIGNIFICANCE: These findings provide insights into structure-function relationship of ion channels and contribute to understanding of the mechanisms underlying their regulation by lipids. Inwardly rectifying potassium (Kir) channels play an important role in setting the resting membrane potential and modulating membrane excitability. An emerging feature of several Kir channels is that they are regulated by cholesterol. However, the mechanism by which cholesterol affects channel function is unclear. Here we show that mutations of two distant Kir2.1 cytosolic residues, Leu-222 and Asn-251, form a two-way molecular switch that controls channel modulation by cholesterol and affects critical hydrogen bonding. Notably, these two residues are linked by a residue chain that continues from Asn-251 to connect adjacent subunits. Furthermore, our data indicate that the same switch also regulates the sensitivity of the channels to phosphatidylinositol 4,5-bisphosphate, a phosphoinositide that is required for activation of Kir channels. Thus, although cholesterol and phosphatidylinositol 4,5-bisphosphate do not interact with the same region of Kir2.1, these different modulators induce a common gating pathway of the channel.
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Colesterol/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células HEK293 , Humanos , Datos de Secuencia Molecular , Mutación , Oocitos/metabolismo , Canales de Potasio de Rectificación Interna/genética , XenopusRESUMEN
Background: The histaminergic neurons in the hypothalamic tuberomammillary nucleus (TMN) have been suggested to play a vital role in maintaining a rising state. But the neuronal types of the TMN are in debate and the role of GABAergic neurons remains unclear. Methods: In the present study, we examined the role of TMN GABAergic neurons in general anesthesia using chemogenetics and optogenetics strategies to regulate the activity of TMN GABAergic neurons. Results: The results indicated that either chemogenetic or optogenetic activation of TMN GABAergic neurons in mice decreased the effect of sevoflurane and propofol anesthesia. In contrast, inhibition of the TMN GABAergic neurons facilitates the sevoflurane anesthesia effect. Conclusion: Our results suggest that the activity of TMN GABAergic neurons produces an anti-anesthesia effect in loss of consciousness and analgesia.
RESUMEN
A cDNA encoding a potassium channel of the two-pore domain family (K(2P), KCNK) of leak channels was cloned from the marine sponge Amphimedon queenslandica. Phylogenetic analysis indicated that AquK(2P) cannot be placed into any of the established functional groups of mammalian K(2P) channels. We used the Xenopus oocyte expression system, a two-electrode voltage clamp and inside-out patch clamp electrophysiology to determine the physiological properties of AquK(2P). In whole cells, non-inactivating, voltage-independent, outwardly rectifying K(+) currents were generated by external application of micromolar concentrations of arachidonic acid (AA; EC(50) â¼30 µmol l(-1)), when applied in an alkaline solution (≥pH 8.0). Prior activation of channels facilitated the pH-regulated, AA-dependent activation of AquK(2P) but external pH changes alone did not activate the channels. Unlike certain mammalian fatty-acid-activated K(2P) channels, the sponge K(2P) channel was not activated by temperature and was insensitive to osmotically induced membrane distortion. In inside-out patch recordings, alkalinization of the internal pH (pK(a) 8.18) activated the AquK(2P) channels independently of AA and also facilitated activation by internally applied AA. The gating of the sponge K(2P) channel suggests that voltage-independent outward rectification and sensitivity to pH and AA are ancient and fundamental properties of animal K(2P) channels. In addition, the membrane potential of some poriferan cells may be dynamically regulated by pH and AA.
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Álcalis/farmacología , Organismos Acuáticos/fisiología , Ácidos Grasos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Poríferos/fisiología , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Secuencia de Aminoácidos , Animales , Organismos Acuáticos/efectos de los fármacos , Ácido Araquidónico/farmacología , Concentración de Iones de Hidrógeno/efectos de los fármacos , Datos de Secuencia Molecular , Ósmosis/efectos de los fármacos , Filogenia , Poríferos/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/química , Homología de Secuencia de Aminoácido , Temperatura , Xenopus laevisRESUMEN
The role of the Slack (also known as Slo2.2, KNa1.1, or KCNT1) channel in pain-sensing is still in debate on which kind of pain it regulates. In the present study, we found that the Slack-/- mice exhibited decreased mechanical pain threshold but normal heat and cold pain sensitivity. Subsequently, X-gal staining, in situ hybridization, and immunofluorescence staining revealed high expression of the Slack channel in Isolectin B4 positive (IB4+) neurons in the dorsal root ganglion (DRG) and somatostatin-positive (SOM+) neurons in the spinal cord. Patch-clamp recordings indicated the firing frequency was increased in both small neurons in DRG and spinal SOM+ neurons in the Slack-/- mice whereas no obvious slow afterhyperpolarization was observed in both WT mice and Slack-/- mice. Furthermore, we found Kcnt1 gene expression in spinal SOM+ neurons in Slack-/- mice partially relieved the mechanical pain hypersensitivity of Slack-/- mice and decreased AP firing rates of the spinal SOM+ neurons. Finally, deletion of the Slack channel in spinal SOM+ neurons is sufficient to result in mechanical pain hypersensitivity in mice. In summary, our results suggest the important role of the Slack channel in the regulation of mechanical pain-sensing both in small neurons in DRG and SOM+ neurons in the spinal dorsal horn.
RESUMEN
Slo2 Na(+)-activated potassium channels are widely expressed in neurons and other cells, such as kidney, heart, and skeletal muscle. Although their important physiological roles continue to be appreciated, molecular determinants responsible for sensing intracellular Na(+) remain unknown. Here we report identification of an Na(+) regulatory site, similar to an Na(+) coordination motif described in Kir channels, localized in the RCK2 domain of Slo2.2 channels. Molecular simulations of the homology-modeled Slo2.2 RCK2 domain provided structural insights into the organization of this Na(+) coordination site. Furthermore, free energy calculations reproduced the experimentally derived monovalent cation selectivity. Our results suggest that Slo2.2 and Kir channels share a similar mechanism to coordinate Na(+). The localization of an Na(+) sensor within the RCK2 domain of Slo2.2 further supports the role of RCK (regulators of conductance of K(+)) domains of Slo channels in coupling ion sensing to channel gating.
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Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canal de Potasio Kv1.6/metabolismo , Sodio/metabolismo , Secuencia de Aminoácidos , Aminoácidos/genética , Animales , Fenómenos Biofísicos/efectos de los fármacos , Fenómenos Biofísicos/genética , Fenómenos Biofísicos/fisiología , Cloruros/farmacología , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica/métodos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/genética , Canal de Potasio Kv1.6/genética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microinyecciones/métodos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación/genética , Oocitos , Técnicas de Placa-Clamp , Sodio/farmacología , Xenopus laevisRESUMEN
The Slo3 gene encodes a high conductance potassium channel, which is activated by both voltage and intracellular alkalinization. Slo3 is specifically expressed in mammalian sperm cells, where it gives rise to pH-dependent outwardly rectifying K(+) currents. Sperm Slo3 is the main current responsible for the capacitation-induced hyperpolarization, which is required for the ensuing acrosome reaction, an exocytotic process essential for fertilization. Here we show that in intact spermatozoa and in a heterologous expression system, the activation of Slo3 currents is regulated by phosphatidylinositol 4,5-bisphosphate (PIP(2)). Depletion of endogenous PIP(2) in inside-out macropatches from Xenopus oocytes inhibited heterologously expressed Slo3 currents. Whole-cell recordings of sperm Slo3 currents or of Slo3 channels co-expressed in Xenopus oocytes with epidermal growth factor receptor, demonstrated that stimulation by epidermal growth factor (EGF) could inhibit channel activity in a PIP(2)-dependent manner. High concentrations of PIP(2) in the patch pipette not only resulted in a strong increase in sperm Slo3 current density but also prevented the EGF-induced inhibition of this current. Mutation of positively charged residues involved in channel-PIP(2) interactions enhanced the EGF-induced inhibition of Slo3 currents. Overall, our results suggest that PIP(2) is an important regulator for Slo3 activation and that receptor-mediated hydrolysis of PIP(2) leads to inhibition of Slo3 currents both in native and heterologous expression systems.
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Factor de Crecimiento Epidérmico/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Fosfatos de Fosfatidilinositol/farmacología , Espermatozoides/metabolismo , Reacción Acrosómica , Animales , Regulación de la Expresión Génica , Concentración de Iones de Hidrógeno , Hidrólisis , Masculino , Ratones , Oocitos/metabolismo , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato , Fosfatidilinositoles/química , Canales de Potasio/química , Xenopus laevisRESUMEN
Kir3 channels control heart rate and neuronal excitability through GTP-binding (G) protein and phosphoinositide signaling pathways. These channels were the first characterized effectors of the ßγ subunits of G proteins. Because we currently lack structures of complexes between G proteins and Kir3 channels, their interactions leading to modulation of channel function are not well understood. The recent crystal structure of a chimera between the cytosolic domain of a mammalian Kir3.1 and the transmembrane region of a prokaryotic KirBac1.3 (Kir3.1 chimera) has provided invaluable structural insight. However, it was not known whether this chimera could form functional K(+) channels. Here, we achieved the functional reconstitution of purified Kir3.1 chimera in planar lipid bilayers. The chimera behaved like a bona fide Kir channel displaying an absolute requirement for PIP(2) and Mg(2+)-dependent inward rectification. The channel could also be blocked by external tertiapin Q. The three-dimensional reconstruction of the chimera by single particle electron microscopy revealed a structure consistent with the crystal structure. Channel activity could be stimulated by ethanol and activated G proteins. Remarkably, the presence of both activated Gα and Gßγ subunits was required for gating of the channel. These results confirm the Kir3.1 chimera as a valid structural and functional model of Kir3 channels.
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Proteínas Bacterianas/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Activación del Canal Iónico , Membrana Dobles de Lípidos/química , Proteínas Recombinantes de Fusión/química , Animales , Proteínas Bacterianas/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Células HEK293 , Humanos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Xenopus laevisRESUMEN
BACKGROUND AND PURPOSE: The Slo3 potassium (KCa 5.1) channel, which is specifically expressed in the testis and sperm, is essential for mammalian male fertilization. The sequence divergence of the bovine, mouse and human Slo3 α-subunit revealed a rapid evolution rate across different species. The rat Slo3 (rSlo3) channel has not been cloned and characterized previously. EXPERIMENTAL APPROACH: We used molecular cloning, electrophysiology (inside-out patches and outside-out patches) and mutagenesis to investigate the biophysical properties and pharmacological characteristics of the rSlo3 channel. KEY RESULTS: The rat Slo3 channel (rSlo3) is gated by voltage and cytosolic pH rather than intracellular calcium. The characteristics of voltage-dependent, pH-sensitivity and activation kinetics of the rSlo3 channel differ from the characteristics of other Slo3 orthologues. In terms of pharmacology, the 4-AP blockade of the rSlo3 channel also shows properties distinct from its blockade of the mSlo3 channel. Iberiotoxin and progesterone weakly inhibit the rSlo3 channel. Finally, we found that propofol, one of the widely used general anaesthetics, blocks the rSlo3 channel from both intracellular and extracellular sides, whereas ketamine only blocks the rSlo3 channel at the extracellular side. CONCLUSION AND IMPLICATIONS: Our findings suggest that the rSlo3 channel possesses unique biophysical and pharmacological properties. Our results provide new insights into the diversities of the Slo3 family of channels, which are valuable for estimating the effects of the use of these drugs to improve sperm quality.
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Canales de Potasio de Gran Conductancia Activados por el Calcio , Canales de Potasio con Entrada de Voltaje , Animales , Biofisica , Bovinos , Clonación Molecular , Masculino , Ratones , Canales de Potasio con Entrada de Voltaje/genética , Ratas , EspermatozoidesRESUMEN
We previously reported that SAK(CA), a stretch-activated, large-conductance, calcium- and voltage-activated potassium (BK(Ca)) channel is present in chick embryonic heart. Here, we cloned SAK(CA) and identified that Stress-Axis Regulated Exon (STREX) is responsible for the stretch sensitivity. Single patch-clamp recordings from CHO cells transfected with the cloned SAK(CA) showed stretch sensitivity, whereas deletion of the STREX insert diminished the stretch sensitivity of the channel. Sequence analysis revealed that the ERA (672-674) sequence of the STREX is indispensable for channel stretch sensitivity and single amino acid substitution from Ala674 to Thr674 completely eliminated the stretch sensitivity. Co-expression of chick STREX-EGFP and SAK(CA) in CHO cells, induced a strong GFP signal in the cell membrane and inhibited the stretch sensitivity significantly. These results suggest that SAK(CA) senses membrane tension through an interaction between STREX and submembranous components.
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Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Mecanotransducción Celular , Secuencia de Aminoácidos , Animales , Células CHO , Embrión de Pollo , Cricetinae , Cricetulus , Biblioteca de Genes , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Estrés MecánicoRESUMEN
Oxidative stress has been considered as one of pathogenesis of brain damage led by epilepsy. Reducing oxidative stress can ameliorate brain damage during seizures. However, expression levels of important antioxidative enzymes such as thioredoxin-1 (TRX1), thioredoxin-like 1 protein (TXNL1) and thioredoxin reductase 1 (TXNRD1) during seizures have not been investigated. In this study, we examined protein and mRNA expression levels of TRX1, TXNL1 and TXNRD1 in different brain regions in PTZ induced seizure model mice. We found that protein expression levels of TRX1, TXNL1 and TXNRD1 are simultaneously up-regulated by 2- or 3-fold in the cortex of both acute and chronic seizure model mice. But there is no unified expression pattern change of these enzymes in the hippocampus, cerebellum and diencephalon in the seizure model mice. Less extent up-regulation of mRNA expression of these enzymes were also observed in the cortex of seizure mice. These data suggest that antioxidative enzymes may provide a protective effect against oxidative stress in the cortex during seizures.
Asunto(s)
Excitación Neurológica/metabolismo , Convulsiones/metabolismo , Tiorredoxina Reductasa 1/metabolismo , Tiorredoxinas/metabolismo , Animales , Modelos Animales de Enfermedad , Excitación Neurológica/genética , Masculino , Ratones , Convulsiones/genética , Tiorredoxina Reductasa 1/genética , Tiorredoxinas/genéticaRESUMEN
General anesthesia is an unconscious state induced by anesthetics for surgery. The molecular targets and cellular mechanisms of general anesthetics in the mammalian nervous system have been investigated during past decades. In recent years, K+ channels have been identified as important targets of both volatile and intravenous anesthetics. This review covers achievements that have been made both on the regulatory effect of general anesthetics on the activity of K+ channels and their underlying mechanisms. Advances in research on the modulation of K+ channels by general anesthetics are summarized and categorized according to four large K+ channel families based on their amino-acid sequence homology. In addition, research achievements on the roles of K+ channels in general anesthesia in vivo, especially with regard to studies using mice with K+ channel knockout, are particularly emphasized.
Asunto(s)
Anestésicos Generales/farmacología , Canales de Potasio/metabolismo , Anestésicos Generales/uso terapéutico , Animales , HumanosRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0191749.].