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1.
Int J Mol Sci ; 20(18)2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31514419

RESUMO

This paper reports on the discovery of a novel three-membrane channel unit exhibiting very steep voltage dependence and strong cooperative behavior. It was reconstituted into planar phospholipid membranes formed by the monolayer method and studied under voltage-clamp conditions. The behavior of the novel channel-former, isolated from Escherichia coli, is consistent with a linearly organized three-channel unit displaying steep voltage-gating (a minimum of 14 charges in the voltage sensor) that rivals that of channels in mammalian excitable membranes. The channels also display strong cooperativity in that closure of the first channel permits the second to close and closure of the second channel permits closure of the third. All three have virtually the same conductance and selectivity, and yet the first and third close at positive potentials whereas the second closes at negative potentials. Thus, is it likely that the second channel-former is oriented in the membrane in a direction opposite to that of the other two. This novel structure is named "triplin." The extraordinary behavior of triplin indicates that it must have important and as yet undefined physiological roles.


Assuntos
Eletricidade , Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Ativação do Canal Iônico , Cinética , Modelos Biológicos , Porinas/metabolismo
2.
Invest Ophthalmol Vis Sci ; 60(12): 3821-3829, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31529078

RESUMO

Purpose: Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation. Methods: We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule. Results: cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46. Conclusions: These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.


Assuntos
Conexina 43/metabolismo , Conexinas/metabolismo , AMP Cíclico/metabolismo , Cristalino/metabolismo , Sistemas do Segundo Mensageiro/fisiologia , Corantes Fluorescentes , Junções Comunicantes/fisiologia , Células HeLa , Humanos , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp , Permeabilidade , Transfecção
3.
Int J Mol Sci ; 20(18)2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31540178

RESUMO

Ion channels contribute fundamental properties to cell membranes. Although highly diverse in conductivity, structure, location, and function, many of them can be regulated by common mechanisms, such as voltage or (de-)phosphorylation. Primarily considering ion channels involved in the nociceptive system, this review covers more novel and less known features. Accordingly, we outline noncanonical operation of voltage-gated sodium, potassium, transient receptor potential (TRP), and hyperpolarization-activated cyclic nucleotide (HCN)-gated channels. Noncanonical features discussed include properties as a memory for prior voltage and chemical exposure, alternative ion conduction pathways, cluster formation, and silent subunits. Complementary to this main focus, the intention is also to transfer knowledge between fields, which become inevitably more separate due to their size.


Assuntos
Canais Iônicos/metabolismo , Dor/etiologia , Dor/metabolismo , Animais , Suscetibilidade a Doenças , Descoberta de Drogas , Humanos , Ativação do Canal Iônico , Canais Iônicos/química , Canais Iônicos/genética , Dor/tratamento farmacológico
4.
Int J Nanomedicine ; 14: 5595-5609, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31413565

RESUMO

Background: Platinum nanoparticles (PtNPs) have been considered a nontoxic nanomaterial and been clinically used in cancer chemotherapy. PtNPs can also be vehicle exhausts and environmental pollutants. These situations increase the possibility of human exposure to PtNPs. However, the potential biotoxicities of PtNPs including that on cardiac electrophysiology have been poorly understood. Methods: Ion channel currents of cardiomyocytes were recorded by patch clamp. Heart rhythm was monitored by electrocardiogram recording. Morphology and characteristics of PtNPs were examined by transmission electron microscopy, dynamic light scattering and electrophoretic light scattering analyses. Results: In cultured neonatal mice ventricular cardiomyocytes, PtNPs with diameters 5 nm (PtNP-5) and 70 nm (PtNP-70) concentration-dependently (10-9 - 10-5 g/mL) depolarized the resting potentials, suppressed the depolarization of action potentials and delayed the repolarization of action potentials. At the ion channel level, PtNPs decreased the current densities of INa, IK1 and Ito channels, but did not affect the channel activity kinetics. In vivo, PtNP-5 and PtNP-70 dose-dependently (3-10 mg/kg, i.v.) decreased the heart rate and induced complete atrioventricular conduction block (AVB) at higher doses. Both PtNP-5 and PtNP-70 (10-9 - 10-5 g/mL) did not significantly increase the generation of ROS and leak of lactate dehydrogenase (LDH) from cardiomyocytes within 5 mins after exposure except that only very high PtNP-5 (10-5 g/mL) slightly increased LDH leak. The internalization of PtNP-5 and PtNP-70 did not occur within 5 mins but occurred 1 hr after exposure. Conclusion: PtNP-5 and PtNP-70 have similar acute toxic effects on cardiac electrophysiology and can induce threatening cardiac conduction block. These acute electrophysiological toxicities of PtNPs are most likely caused by a nanoscale interference of PtNPs on ion channels at the extracellular side, rather than by oxidative damage or other slower biological processes.


Assuntos
Frequência Cardíaca/efeitos dos fármacos , Canais Iônicos/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Miócitos Cardíacos/metabolismo , Platina/toxicidade , Testes de Toxicidade Aguda , Animais , Animais Recém-Nascidos , Células Cultivadas , Eletrocardiografia , Endocitose/efeitos dos fármacos , Sistema de Condução Cardíaco/efeitos dos fármacos , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Nanopartículas Metálicas/administração & dosagem , Nanopartículas Metálicas/ultraestrutura , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Estresse Oxidativo/efeitos dos fármacos , Platina/administração & dosagem
5.
Nat Commun ; 10(1): 3584, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31395867

RESUMO

In voltage-gated potassium channels (VGKC), voltage sensors (VSD) endow voltage-sensitivity to pore domains (PDs) through a not fully understood mechanism. Shaker-like VGKC show domain-swapped configuration: VSD of one subunit is covalently connected to its PD by the protein backbone (far connection) and non-covalently to the PD of the next subunit (near connection). VSD-to-PD coupling is not fully explained by far connection only, therefore an additional mechanistic component may be based on near connection. Using tandem dimers of Shaker channels we show functional data distinguishing VSD-to-PD far from near connections. Near connections influence both voltage-dependence of C-type inactivation at the selectivity filter and overall PD open probability. We speculate a conserved residue in S5 (S412 in Shaker), within van der Waals distance from next subunit S4 residues is key for the noncanonical VSD-to-PD coupling. Natural mutations of S412-homologous residues in brain and heart VGKC are related to neurological and cardiac diseases.


Assuntos
Ativação do Canal Iônico/fisiologia , Multimerização Proteica/fisiologia , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Feminino , Potenciais da Membrana/fisiologia , Mutagênese , Oócitos , Domínios Proteicos/genética , Superfamília Shaker de Canais de Potássio/genética , Xenopus laevis
6.
Nat Commun ; 10(1): 3769, 2019 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-31434906

RESUMO

The calcium-activated chloride channel (CaCC) TMEM16A plays crucial roles in regulating neuronal excitability, smooth muscle contraction, fluid secretion and gut motility. While opening of TMEM16A requires binding of intracellular Ca2+, prolonged Ca2+-dependent activation results in channel desensitization or rundown, the mechanism of which is unclear. Here we show that phosphatidylinositol (4,5)-bisphosphate (PIP2) regulates TMEM16A channel activation and desensitization via binding to a putative binding site at the cytosolic interface of transmembrane segments (TMs) 3-5. We further demonstrate that the ion-conducting pore of TMEM16A is constituted of two functionally distinct modules: a Ca2+-binding module formed by TMs 6-8 and a PIP2-binding regulatory module formed by TMs 3-5, which mediate channel activation and desensitization, respectively. PIP2 dissociation from the regulatory module results in ion-conducting pore collapse and subsequent channel desensitization. Our findings thus provide key insights into the mechanistic understanding of TMEM16 channel gating and lipid-dependent regulation.


Assuntos
Anoctamina-1/efeitos dos fármacos , Anoctamina-1/metabolismo , Cálcio/metabolismo , Agonistas dos Canais de Cloreto/metabolismo , Canais de Cloreto/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/farmacologia , Sítios de Ligação , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Modelos Moleculares
7.
Nat Commun ; 10(1): 3703, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420535

RESUMO

A family of plant nuclear ion channels, including DMI1 (Does not Make Infections 1) and its homologs CASTOR and POLLUX, are required for the establishment of legume-microbe symbioses by generating nuclear and perinuclear Ca2+ spiking. Here we show that CASTOR from Lotus japonicus is a highly selective Ca2+ channel whose activation requires cytosolic/nucleosolic Ca2+, contrary to the previous suggestion of it being a K+ channel. Structurally, the cytosolic/nucleosolic ligand-binding soluble region of CASTOR contains two tandem RCK (Regulator of Conductance for K+) domains, and four subunits assemble into the gating ring architecture, similar to that of large conductance, Ca2+-gated K+ (BK) channels despite the lack of sequence similarity. Multiple ion binding sites are clustered at two locations within each subunit, and three of them are identified to be Ca2+ sites. Our in vitro and in vivo assays also demonstrate the importance of these gating-ring Ca2+ binding sites to the physiological function of CASTOR as well as DMI1.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Lotus , Micorrizas , Rhizobium , Simbiose , Ativação do Canal Iônico , Membrana Nuclear/metabolismo , Proteínas de Plantas/metabolismo , Domínios Proteicos
8.
Nat Commun ; 10(1): 3740, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431622

RESUMO

The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca2+. The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca2+-bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca2+ alone, and with both ADPR and Ca2+, resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Estrutura Quaternária de Proteína , Canais de Cátion TRPM/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Células HEK293 , Humanos , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Células Sf9 , Spodoptera , Canais de Cátion TRPM/química , Peixe-Zebra , Proteínas de Peixe-Zebra/química
9.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31331039

RESUMO

Psychiatric disorders are mental, behavioral or emotional disorders. These conditions are prevalent, one in four adults suffer from any type of psychiatric disorders world-wide. It has always been observed that psychiatric disorders have a genetic component, however, new methods to sequence full genomes of large cohorts have identified with high precision genetic risk loci for these conditions. Psychiatric disorders include, but are not limited to, bipolar disorder, schizophrenia, autism spectrum disorder, anxiety disorders, major depressive disorder, and attention-deficit and hyperactivity disorder. Several risk loci for psychiatric disorders fall within genes that encode for voltage-gated calcium channels (CaVs). Calcium entering through CaVs is crucial for multiple neuronal processes. In this review, we will summarize recent findings that link CaVs and their auxiliary subunits to psychiatric disorders. First, we will provide a general overview of CaVs structure, classification, function, expression and pharmacology. Next, we will summarize tools to study risk loci associated with psychiatric disorders. We will examine functional studies of risk variations in CaV genes when available. Finally, we will review pharmacological evidence of the use of CaV modulators to treat psychiatric disorders. Our review will be of interest for those studying pathophysiological aspects of CaVs.


Assuntos
Canais de Cálcio/genética , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Transtornos Mentais/genética , Animais , Biomarcadores , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Ensaios Clínicos como Assunto , Regulação da Expressão Gênica , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Transtornos Mentais/diagnóstico , Transtornos Mentais/tratamento farmacológico , Transtornos Mentais/metabolismo , Terapia de Alvo Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Resultado do Tratamento
10.
J Pharmacol Exp Ther ; 370(3): 350-359, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31201216

RESUMO

Glucose-stimulated insulin secretion from pancreatic ß-cells is controlled by ATP-regulated potassium (KATP) channels composed of Kir6.2 and sulfonylurea receptor 1 (SUR1) subunits. The KATP channel-opener diazoxide is FDA-approved for treating hyperinsulinism and hypoglycemia but suffers from off-target effects on vascular KATP channels and other ion channels. The development of more specific openers would provide critically needed tool compounds for probing the therapeutic potential of Kir6.2/SUR1 activation. Here, we characterize a novel scaffold activator of Kir6.2/SUR1 that our group recently discovered in a high-throughput screen. Optimization efforts with medicinal chemistry identified key structural elements that are essential for VU0071063-dependent opening of Kir6.2/SUR1. VU0071063 has no effects on heterologously expressed Kir6.1/SUR2B channels or ductus arteriole tone, indicating it does not open vascular KATP channels. VU0071063 induces hyperpolarization of ß-cell membrane potential and inhibits insulin secretion more potently than diazoxide. VU0071063 exhibits metabolic and pharmacokinetic properties that are favorable for an in vivo probe and is brain penetrant. Administration of VU0071063 inhibits glucose-stimulated insulin secretion and glucose-lowering in mice. Taken together, these studies indicate that VU0071063 is a more potent and specific opener of Kir6.2/SUR1 than diazoxide and should be useful as an in vitro and in vivo tool compound for investigating the therapeutic potential of Kir6.2/SUR1 expressed in the pancreas and brain.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Receptores Sulfonilureia/metabolismo , Xantinas/farmacologia , Xantinas/farmacocinética , Animais , Canal Arterial/efeitos dos fármacos , Canal Arterial/fisiologia , Glucose/farmacologia , Células HEK293 , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Camundongos , Relação Estrutura-Atividade , Vasodilatação/efeitos dos fármacos , Xantinas/química
11.
Int J Mol Sci ; 20(11)2019 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-31167355

RESUMO

KcsA is a tetrameric potassium channel formed out of four identical monomeric subunits used as a standard model for selective potassium transport and pH-dependent gating. Large conformational changes are reported for tetramer and monomer upon gating, and the response of the monomer being controversial with the two major studies partially contradicting each other. KcsA was analyzed as functional tetramers embedded in liposomes and as monomer subunits with confocal Raman microscopy under physiological conditions for the active and the closed channel state, using 532 nm excitation to avoid introducing conformational changes during the measurement. Channel function was confirmed using liposome flux assay. While the classic fingerprint region below 1800 rel. cm-1 in the Raman spectrum of the tetramer was unaffected, the CH-stretching region between 2800 and 3200 rel. cm-1 was found to be strongly affected by the conformation. No pH-dependency was observed in the Raman spectra of the monomer subunits, which closely resembled the Raman spectrum of the tetramer in its active conformation, indicating that the open conformation of the monomer and not the closed conformation as postulated may equal the relaxed state of the molecule.


Assuntos
Proteínas de Bactérias/química , Concentração de Íons de Hidrogênio , Canais de Potássio/química , Conformação Proteica , Multimerização Proteica , Análise Espectral Raman , Proteínas de Bactérias/metabolismo , Ativação do Canal Iônico , Lipossomos , Modelos Moleculares , Canais de Potássio/metabolismo , Ligação Proteica , Relação Estrutura-Atividade
12.
Biophys Chem ; 252: 106196, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31203196

RESUMO

Potassium channels are pore-forming membrane proteins that open and close in response to changes in a chemical or electrical potential, thereby regulating the flow of potassium ions across biological membranes. Two regions of the same channels are acting in tandem and enable ion flow through the channel pore. I refer to this coupled action as a "gate linker". To closely examine the role of the gate linker in the channel function, I mutated the amino acids in the cDNA of this region, and used from knowen mutaion, either alone or together with the amino acids of adjacent regions. I have emphasized the importance of the linker between these two gates - mutations in this region may cause conformational changes that play a fundamental role in mediating the coupling between the voltage sensor, activation gate and selectivity filter elements of Kv channels. I observe that free energy considerations show the significance of the coupling between the activation and inactivation gates. Moreover, a symmetry between the coupling and sensor spring strength leads to the destruction of ion conductivity. I present a thermodynamic framework for the possible study of multiple channel blocks. The arising physical perspective of the gating process gives rise to new research avenues of the coupling mode of potassium channels and may assist in explaining the centrality of the "gate linker" to the channel function.


Assuntos
Canais de Potássio/metabolismo , Termodinâmica , Transistores Eletrônicos , Ativação do Canal Iônico , Canais de Potássio/química , Conformação Proteica
13.
Nat Commun ; 10(1): 2008, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043612

RESUMO

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Ativação do Canal Iônico/fisiologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
14.
Cell Mol Neurobiol ; 39(6): 809-822, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31037516

RESUMO

We established a model of Alzheimer's disease in vitro by exposing primary hippocampal neurons of neonatal Wistar rats to the ß-Amyloid peptide fragment 25-35, Aß25-35. We then observed the effects of genistein, a type of soybean isoflavone, on Aß25-35-incubated hippocampal neuron viability, and the electrophysiological properties of voltage-gated sodium channels (NaV) and potassium channels (KV) in the hippocampal neurons. Aß25-35 exposure reduced the viability of hippocampal neurons, decreased the peak amplitude of voltage-activated sodium channel currents (INa), and significantly reduced INa at different membrane potentials. Moreover, Aß25-35 shifted the activation curve toward depolarization, shifted the inactivation curve toward hyperpolarization, and increased the time constant of recovery from inactivation. Aß25-35 exposure significantly shifted the inactivation curve of transient outward K+ currents (IA) toward hyperpolarization and increased its time constant of recovery from inactivation. In addition, Aß25-35 significantly decreased the peak density of outward-delayed rectifier potassium channel currents (IDR) and significantly reduced IDR value at different membrane potentials. We found that genistein partially reversed the decrease in hippocampal neuron viability, and the alterations in electrophysiological properties of NaV and KV induced by Aß25-35. Our results suggest that genistein could inhibit Aß25-35-induced neuronal damage with changes in the electrophysiological properties of NaV and KV.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Genisteína/farmacologia , Neurônios/metabolismo , Neurônios/patologia , Fragmentos de Peptídeos/toxicidade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/patologia , Ativação do Canal Iônico/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Ratos Wistar
15.
Cell Tissue Res ; 378(1): 59-66, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31041505

RESUMO

Several studies have been reported in various domains from induction methods to utilities of somatic cell pluripotent reprogramming. However, one of the major struggles facing the research field of induced pluripotent stem cell (iPSC)-derived target cells is the lack of consistency in observations. This could be due to variety of reasons including varied culture periods post-differentiation. The cardiomyocytes (CMs) derived from iPSCs are commonly studied and proposed to be utilized in the comprehensive in vitro proarrhythmia initiative for drug safety screening. As the influence of varied culture periods on the electrophysiological properties of iPSC-CMs is not clearly known, using whole-cell patch clamp technique, we compared two groups of differentiated ventricular-like iPSC-CMs that are cultured for 10 to 15 days (D10-15) and more than 30 days (≥ D30) both under current and voltage clamps. The prolonged culture imparts increased excitability with high-frequency spontaneous action potentials, robust increase in the magnitude of peak Na+ current density, relatively shallow inactivation kinetics of Na+ channels, faster recovery from inactivation, and augmented Ca2+ current density. Quantitative real-time PCR studies of α-subunit transcripts showed enhanced mRNA expression of SCN1A, SCN5A Na+ channel subtypes, and CACNA1C, CACNA1G, and CACNA1I Ca2+ channel subtypes, in ≥ D30 group. Conclusively, the prolonged culture of differentiated iPSC-CMs affects the excitability, single-cell electrophysiological properties, and ion channel expressions. Therefore, following standard periods of culture across research studies while utilizing ventricular-like iPSC-CMs for in vitro health/disease modeling to study cellular functional mechanisms or test high-throughput drugs' efficacy and toxicity becomes crucial.


Assuntos
Canais de Cálcio/metabolismo , Ventrículos do Coração/citologia , Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Canais de Potássio/metabolismo , Potenciais de Ação , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Ativação do Canal Iônico , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo
16.
Cell Mol Life Sci ; 76(22): 4539-4550, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31055644

RESUMO

The initiation of action potentials (APs) by membrane depolarization occurs after a brief vulnerability period, during which excitation can be abolished by the reversal of the stimulus polarity. This vulnerability period is determined by the time needed for gating of voltage-gated sodium channels (VGSC). We compared nerve excitation by ultra-short uni- and bipolar stimuli to define the time frame of bipolar cancellation and of AP initiation. Propagating APs in isolated frog sciatic nerve were elicited by cathodic pulses (200 ns-300 µs), followed by an anodic (canceling) pulse of the same duration after a 0-200-µs delay. We found that the earliest and the latest boundaries for opening the critical number of VGSC needed to initiate AP are, respectively, between 11 and 20 µs and between 100 and 200 µs after the onset of depolarization. Stronger depolarization accelerated AP initiation, apparently due to faster VGSC opening, but not beyond the 11-µs limit. Bipolar cancellation was augmented by reducing pulse duration, shortening the delay between pulses, decreasing the amplitude of the cathodic pulse, and increasing the amplitude of the anodic one. Some of these characteristics contrasted the bipolar cancellation of cell membrane electroporation (Pakhomov et al. in Bioelectrochemistry 122:123-133, 2018; Gianulis et al. in Bioelectrochemistry 119:10-19, 2017), suggesting different mechanisms. The ratio of nerve excitation thresholds for a unipolar cathodic pulse and a symmetrical bipolar pulse increased as a power function as the pulse duration decreased, in remarkable agreement with the predictions of SENN model of nerve excitation (Reilly and Diamant in Health Phys 83(3):356-365, 2002).


Assuntos
Potenciais de Ação/fisiologia , Ativação do Canal Iônico/fisiologia , Sistema Nervoso/metabolismo , Canais de Sódio/metabolismo , Animais , Anuros/metabolismo , Anuros/fisiologia , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Permeabilidade da Membrana Celular/fisiologia , Eletroporação/métodos , Feminino
17.
EBioMedicine ; 43: 641-649, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31078519

RESUMO

Epilepsy remains refractory to medical treatment in ~30% of patients despite decades of new drug development. Neurosurgery to remove or disconnect the seizure focus is often curative but frequently contraindicated by risks of irreversible impairment to brain function. Novel therapies are therefore required that better balance seizure suppression against the risks of side effects. Among experimental gene therapies, chemogenetics has the major advantage that the action on the epileptogenic zone can be modulated on demand. Two broad approaches are to use a designer G-protein-coupled receptor or a modified ligand gated ion channel, targeted to specific neurons in the epileptogenic zone using viral vectors and cell-type selective promoters. The receptor can be activated on demand by either an exogenous compound or by pathological levels of extracellular glutamate that occur in epileptogenic tissue. We review the principal designer receptor technologies and their modes of action. We compare the drawbacks and benefits of each designer receptor with particular focus on the drug activators and the potential for clinical translation in epilepsy.


Assuntos
Desenho de Drogas , Epilepsia/etiologia , Epilepsia/terapia , Receptores Acoplados a Proteínas-G/genética , Animais , Epilepsia/metabolismo , Humanos , Ativação do Canal Iônico , Ligantes , Terapia de Alvo Molecular , Relação Quantitativa Estrutura-Atividade , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/metabolismo
18.
Eur J Pharmacol ; 855: 50-55, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31063774

RESUMO

Previous studies indicated that one of the action targets of carvedilol is the voltage-gated potassium (Kv) channel, which has a fundamental role in the control of electrical properties in excitable cells. It is not clear whether this compound exerts any actions specifically on delayed rectifier Kv2.1 channels. The present study is conducted on Kv2.1 currents heterologously expressed in HEK293 cells to characterize the block by carvedilol in detail, identifying molecular determinants and providing biophysical insights of the block. Macroscopic Kv2.1 currents obtained by whole-cell recording were substantially inhibited after addition of carvedilol with an IC50 value of 5.1 µM. This drug also led to a largely hyperpolarizing shift (30 mV) of the inactivation curve, which may contribute to the blocking action due to more inactivation of Kv2.1 currents occurred in depolarization potentials. Mutations at Y380 (a putative TEA binding site) and K356 (a K+ binding site) in the outer vestibule of Kv2.1 channels significantly eliminated the inhibitory effects of carvedilol and prevented the leftward shift of inactivation. Moreover, mutations at above positions modulated the effects of carvedilol on the deactivation but not activation kinetics of Kv2.1 channels. Collected data indicate that carvedilol can exert a blocking effect on the closed-state of Kv2.1 channels, and specific residues within the S5-P and P-S6 linkers in the outer vestibule may play crucial roles in carvedilol-induced blocking for Kv2.1 channels.


Assuntos
Carvedilol/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Shab/antagonistas & inibidores , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio Shab/metabolismo
19.
Eur J Pharmacol ; 855: 160-166, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31063775

RESUMO

P2Y1 receptors play an essential role in inhibitory neuromuscular transmission in the gastrointestinal tract. The signalling pathway involves the opening of small conductance calcium activated potassium-channels (Kca2 family) that results in smooth muscle hyperpolarization and relaxation. Inorganic polyphosphates and dinucleotidic polyphosphates are putative neurotransmitters that potentially act on P2Y1 receptors. A pharmacological approach using both orthosteric (MRS2500) and allosteric (BPTU) blockers of the P2Y1 receptor and openers (CyPPA) and blockers (apamin) of Kca2 channels was used to pharmacologically characterise the effect of these neurotransmitters. Organ bath and microelectrodes were used to evaluate the effect of P1,P4-Di (adenosine-5') tetraphosphate ammonium salt (Ap4A), inorganic polyphosphates (PolyP) and CyPPA on spontaneous contractions and membrane potential of mouse colonic smooth muscle cells. PolyP neither modified contractions nor membrane potential. In contrast, Ap4A caused a concentration-dependent inhibition of spontaneous contractions reaching a maximum effect at 100 µM Ap4A response was antagonised by MRS2500 (1 µM), BPTU (3 µM) and apamin (1 µM). CyPPA (10 µM) inhibited spontaneous contractions and this response was antagonised by apamin but it was not affected by MRS2500 or BPTU. Both CyPPA and Ap4A caused smooth muscle hyperpolarization that was blocked by apamin and MRS2500 respectively. We conclude that Ap4A but not PolyP activates P2Y1 receptors causing smooth muscle hyperpolarization and relaxation. Ap4A signalling causes activation of Kca2 channels through activation of P2Y1 receptors. In contrast, CyPPA acts directly on Kca2 channels. Further studies are needed to evaluate if dinucleotidic polyphosphates are released from inhibitory motor neurons.


Assuntos
Colo/efeitos dos fármacos , Fosfatos de Dinucleosídeos/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/fisiologia , Receptores Purinérgicos P2Y1/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Colo/fisiologia , Nucleotídeos de Desoxiadenina/farmacologia , Feminino , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Músculo Liso/citologia , Músculo Liso/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Transdução de Sinais/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/metabolismo
20.
Molecules ; 24(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091684

RESUMO

Venom from mammals, amphibians, snakes, arachnids, sea anemones and insects provides diverse sources of peptides with different potential medical applications. Several of these peptides have already been converted into drugs and some are still in the clinical phase. Diabetes type 2 is one of the diseases with the highest mortality rate worldwide, requiring specific attention. Diverse drugs are available (e.g., Sulfonylureas) for effective treatment, but with several adverse secondary effects, most of them related to the low specificity of these compounds to the target. In this context, the search for specific and high-affinity compounds for the management of this metabolic disease is growing. Toxins isolated from animal venom have high specificity and affinity for different molecular targets, of which the most important are ion channels. This review will present an overview about the electrical activity of the ion channels present in pancreatic ß cells that are involved in the insulin secretion process, in addition to the diversity of peptides that can interact and modulate the electrical activity of pancreatic ß cells. The importance of prospecting bioactive peptides for therapeutic use is also reinforced.


Assuntos
Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Peptídeos/farmacologia , Peçonhas/química , Animais , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Desenvolvimento de Medicamentos , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Peptídeos/química , Pesquisa Médica Translacional
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