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1.
J Gen Physiol ; 156(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38832889

RESUMO

Voltage-gated ion channels are responsible for the electrical excitability of neurons and cardiomyocytes. Thus, they are obvious targets for pharmaceuticals aimed to modulate excitability. Compounds activating voltage-gated potassium (KV) channels are expected to reduce excitability. To search for new KV-channel activators, we performed a high-throughput screen of 10,000 compounds on a specially designed Shaker KV channel. Here, we report on a large family of channel-activating compounds with a carboxyl (COOH) group as the common motif. The most potent COOH activators are lipophilic (4 < LogP <7) and are suggested to bind at the interface between the lipid bilayer and the channel's positively charged voltage sensor. The negatively charged form of the COOH-group compounds is suggested to open the channel by electrostatically pulling the voltage sensor to an activated state. Several of the COOH-group compounds also activate the therapeutically important KV7.2/7.3 channel and can thus potentially be developed into antiseizure drugs. The COOH-group compounds identified in this study are suggested to act via the same site and mechanism of action as previously studied COOH-group compounds, such as polyunsaturated fatty acids and resin acids, but distinct from sites for several other types of potassium channel-activating compounds.


Assuntos
Ativação do Canal Iônico , Animais , Ativação do Canal Iônico/efeitos dos fármacos , Superfamília Shaker de Canais de Potássio/metabolismo , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ2/agonistas , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/efeitos dos fármacos , Canal de Potássio KCNQ3/metabolismo , Humanos , Xenopus laevis
2.
eNeuro ; 11(7)2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38886063

RESUMO

Persistent activity in excitatory pyramidal cells (PYRs) is a putative mechanism for maintaining memory traces during working memory. We have recently demonstrated persistent interruption of firing in fast-spiking parvalbumin-expressing interneurons (PV-INs), a phenomenon that could serve as a substrate for persistent activity in PYRs through disinhibition lasting hundreds of milliseconds. Here, we find that hippocampal CA1 PV-INs exhibit type 2 excitability, like striatal and neocortical PV-INs. Modeling and mathematical analysis showed that the slowly inactivating potassium current KV1 contributes to type 2 excitability, enables the multiple firing regimes observed experimentally in PV-INs, and provides a mechanism for robust persistent interruption of firing. Using a fast/slow separation of times scales approach with the KV1 inactivation variable as a bifurcation parameter shows that the initial inhibitory stimulus stops repetitive firing by moving the membrane potential trajectory onto a coexisting stable fixed point corresponding to a nonspiking quiescent state. As KV1 inactivation decays, the trajectory follows the branch of stable fixed points until it crosses a subcritical Hopf bifurcation (HB) and then spirals out into repetitive firing. In a model describing entorhinal cortical PV-INs without KV1, interruption of firing could be achieved by taking advantage of the bistability inherent in type 2 excitability based on a subcritical HB, but the interruption was not robust to noise. Persistent interruption of firing is therefore broadly applicable to PV-INs in different brain regions but is only made robust to noise in the presence of a slow variable, KV1 inactivation.


Assuntos
Interneurônios , Modelos Neurológicos , Parvalbuminas , Parvalbuminas/metabolismo , Interneurônios/fisiologia , Interneurônios/metabolismo , Animais , Potenciais de Ação/fisiologia , Região CA1 Hipocampal/fisiologia , Região CA1 Hipocampal/metabolismo , Inibição Neural/fisiologia , Células Piramidais/fisiologia , Células Piramidais/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Córtex Entorrinal/fisiologia , Córtex Entorrinal/metabolismo , Masculino
3.
Laryngoscope ; 134(3): 1363-1371, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-37551886

RESUMO

OBJECTIVE: Fragile X Syndrome (FXS) is a hereditary form of autism spectrum disorder. It is caused by a trinucleotide repeat expansion in the Fmr1 gene, leading to a loss of Fragile X Protein (FMRP) expression. The loss of FMRP causes auditory hypersensitivity: FXS patients display hyperacusis and the Fmr1- knock-out (KO) mouse model for FXS exhibits auditory seizures. FMRP is strongly expressed in the cochlear nucleus and other auditory brainstem nuclei. We hypothesize that the Fmr1-KO mouse has altered gene expression in the cochlear nucleus that may contribute to auditory hypersensitivity. METHODS: RNA was isolated from cochlear nuclei of Fmr1-KO and WT mice. Using next-generation sequencing (RNA-seq), the transcriptomes of Fmr1-KO mice and WT mice (n = 3 each) were compared and analyzed using gene ontology programs. RESULTS: We identified 270 unique, differentially expressed genes between Fmr1-KO and WT cochlear nuclei. Upregulated genes (67%) are enriched in those encoding secreted molecules. Downregulated genes (33%) are enriched in neuronal function, including synaptic pathways, some of which are ideal candidate genes that may contribute to hyperacusis. CONCLUSION: The loss of FMRP can affect the expression of genes in the cochlear nucleus that are important for neuronal signaling. One of these, Kcnab2, which encodes a subunit of the Shaker voltage-gated potassium channel, is expressed at an abnormally low level in the Fmr1-KO cochlear nucleus. Kcnab2 and other differentially expressed genes may represent pathways for the development of hyperacusis. Future studies will be aimed at investigating the effects of these altered genes on hyperacusis. LEVEL OF EVIDENCE: N/A Laryngoscope, 134:1363-1371, 2024.


Assuntos
Transtorno do Espectro Autista , Núcleo Coclear , Síndrome do Cromossomo X Frágil , Humanos , Camundongos , Animais , Núcleo Coclear/metabolismo , Hiperacusia/genética , Transcriptoma , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/metabolismo , Camundongos Knockout , Modelos Animais de Doenças , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo
4.
JCI Insight ; 7(3)2022 02 08.
Artigo em Inglês | MEDLINE | ID: mdl-35132967

RESUMO

There is a high prevalence of ventricular arrhythmias related to sudden cardiac death in patients with chronic kidney disease (CKD). To explored the possible mechanism of CKD-related ventricular arrhythmias, a CKD rat model was created, and indoxyl sulfate (IS) was further used in vivo and in vitro. This project used the following methods: patch clamp, electrocardiogram, and some molecular biology experimental techniques. IS was found to be significantly elevated in the serum of CKD rats. Interestingly, the expression levels of the fast transient outward potassium current-related (Ito,f-related) proteins (Kv4.2, Kv4.3, and KChIP2) in the heart of CKD rats and rats treated with IS decreased. IS dose-dependently reduced Ito,f density, accompanied by the decreases in Kv4.2, Kv4.3, and KChIP2 proteins in vitro. IS also prolonged the action potential duration and QT interval, and paroxysmal ventricular tachycardia could be induced by IS. In-depth studies have shown that ROS/p38MAPK, ROS-p44/42 MAPK, and NF-κB signaling pathways play key roles in the reduction of Ito,f density and Ito,f-related proteins caused by IS. These data suggest that IS reduces Ito,f-related proteins and Ito,f density by activating ROS/MAPK and NF-κB signaling pathways, and the action potential duration and QT interval are subsequently prolonged, which contributes to increasing the susceptibility to arrhythmia in CKD.


Assuntos
Regulação da Expressão Gênica , Indicã/farmacologia , Miócitos Cardíacos/metabolismo , NF-kappa B/genética , Insuficiência Renal Crônica/complicações , Superfamília Shaker de Canais de Potássio/metabolismo , Taquicardia Ventricular/genética , Animais , Células Cultivadas , Modelos Animais de Doenças , Eletrocardiografia , Masculino , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Miócitos Cardíacos/patologia , NF-kappa B/biossíntese , Técnicas de Patch-Clamp , RNA/genética , Ratos , Ratos Wistar , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/metabolismo , Transdução de Sinais , Taquicardia Ventricular/tratamento farmacológico , Taquicardia Ventricular/etiologia
5.
J Neurophysiol ; 127(1): 116-129, 2022 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-34817286

RESUMO

Diverse physiological phenotypes in a neuronal population can broaden the range of computational capabilities within a brain region. The avian cochlear nucleus angularis (NA) contains a heterogeneous population of neurons whose variation in intrinsic properties results in electrophysiological phenotypes with a range of sensitivities to temporally modulated input. The low-threshold potassium conductance (GKLT) is a key feature of neurons involved in fine temporal structure coding for sound localization, but a role for these channels in intensity or spectrotemporal coding has not been established. To determine whether GKLT affects the phenotypical variation and temporal properties of NA neurons, we applied dendrotoxin-I (DTX), a potent antagonist of Kv1-type potassium channels, to chick brain stem slices in vitro during whole cell patch-clamp recordings. We found a cell-type specific subset of NA neurons that was sensitive to DTX: single-spiking NA neurons were most profoundly affected, as well as a subset of tonic-firing neurons. Both tonic I (phasic onset bursting) and tonic II (delayed firing) neurons showed DTX sensitivity in their firing rate and phenotypical firing pattern. Tonic III neurons were unaffected. Spike time reliability and fluctuation sensitivity measured in DTX-sensitive NA neurons was also reduced with DTX. Finally, DTX reduced spike threshold adaptation in these neurons, suggesting that GKLT contributes to the temporal properties that allow coding of rapid changes in the inputs to NA neurons. These results suggest that variation in Kv1 channel expression may be a key factor in functional diversity in the avian cochlear nucleus.NEW & NOTEWORTHY The dendrotoxin-sensitive voltage-gated potassium conductance typically associated with neuronal coincidence detection in the timing pathway for sound localization is demonstrated to affect spiking patterns and temporal input sensitivity in the intensity pathway in the avian auditory brain stem. The Kv1-family channels appear to be present in a subset of cochlear nucleus angularis neurons, regulate spike threshold dynamics underlying high-pass membrane filtering, and contribute to intrinsic firing diversity.


Assuntos
Potenciais de Ação/fisiologia , Núcleo Coclear/fisiologia , Neurônios/fisiologia , Bloqueadores dos Canais de Potássio/farmacologia , Superfamília Shaker de Canais de Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Galinhas , Núcleo Coclear/efeitos dos fármacos , Núcleo Coclear/metabolismo , Venenos Elapídicos/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Superfamília Shaker de Canais de Potássio/efeitos dos fármacos
6.
PLoS One ; 16(12): e0261087, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34932577

RESUMO

Age-related changes in ion channel expression are likely to affect neuronal signaling. Here, we examine how age affects Kv4/Shal and Kv1/Shaker K+ channel protein levels in Drosophila. We show that Kv4/Shal protein levels decline sharply from 3 days to 10 days, then more gradually from 10 to 40 days after eclosion. In contrast, Kv1/Shaker protein exhibits a transient increase at 10 days that then stabilizes and eventually declines at 40 days. We present data that begin to show a relationship between reactive oxygen species (ROS), Kv4/Shal, and locomotor performance. We show that Kv4/Shal levels are negatively affected by ROS, and that over-expression of Catalase or RNAi knock-down of the ROS-generating enzyme, Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Oxidase (NOX), can attenuate the loss of Kv4/Shal protein. Finally, we compare levels of Kv4.2 and Kv4.3 in the hippocampus, olfactory bulb, cerebellum, and motor cortex of mice aged 6 weeks and 1 year. While there was no global decline in Kv4.2/4.3 that parallels what we report in Drosophila, we did find that Kv4.2/4.3 are differentially affected in various brain regions; this survey of changes may help inform mammalian studies that examine neuronal function with age.


Assuntos
Potenciais de Ação , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neurônios/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Canais de Potássio Shal/metabolismo , Fatores Etários , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Masculino , Neurônios/citologia , Superfamília Shaker de Canais de Potássio/genética , Canais de Potássio Shal/genética
7.
Plant Physiol ; 187(4): 2092-2109, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34618033

RESUMO

Potassium (K+) channels serve a wide range of functions in plants from mineral nutrition and osmotic balance to turgor generation for cell expansion and guard cell aperture control. Plant K+ channels are members of the superfamily of voltage-dependent K+ channels, or Kv channels, that include the Shaker channels first identified in fruit flies (Drosophila melanogaster). Kv channels have been studied in depth over the past half century and are the best-known of the voltage-dependent channels in plants. Like the Kv channels of animals, the plant Kv channels are regulated over timescales of milliseconds by conformational mechanisms that are commonly referred to as gating. Many aspects of gating are now well established, but these channels still hold some secrets, especially when it comes to the control of gating. How this control is achieved is especially important, as it holds substantial prospects for solutions to plant breeding with improved growth and water use efficiencies. Resolution of the structure for the KAT1 K+ channel, the first channel from plants to be crystallized, shows that many previous assumptions about how the channels function need now to be revisited. Here, I strip the plant Kv channels bare to understand how they work, how they are gated by voltage and, in some cases, by K+ itself, and how the gating of these channels can be regulated by the binding with other protein partners. Each of these features of plant Kv channels has important implications for plant physiology.


Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Fenômenos Fisiológicos Vegetais/efeitos dos fármacos , Plantas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo
8.
J Plant Physiol ; 266: 153529, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34583134

RESUMO

Potassium is a major cationic nutrient involved in numerous physiological processes in plants. The uptake of K+ is mediated by K+ channels and transporters, and the Shaker K+ channel gene family plays an essential role in K+ uptake and stress resistance in plants. However, little is known regarding this family in soybean. In this study, 14 members of the Shaker K+ channel gene family were identified in soybean and were classified into five groups. Protein domain analysis revealed that Shaker K+ channel gene members have an ion transport domain (ion trans), a cyclic nucleotide-binding domain, ankyrin repeat domains, and a dimerization domain in the potassium ion channel. Quantitative real-time polymerase chain reaction analysis indicated that the expression of eight genes (notably GmAKT1) in soybean leaves and roots was significantly increased in response to salt and drought stress. Furthermore, the overexpression of GmAKT1 in Arabidopsis enhanced root length, K+ concentration, and fresh/dry weight ratio compared with wild-type plants subjected to salt and drought stress; this suggests that GmAKT1 improves the tolerance of soybean to abiotic stress. Our results provide important insight into the characterization of Shaker K+ channel gene family members in soybean and highlight the function of GmAKT1 in soybean plants under salt and drought stress.


Assuntos
Arabidopsis/fisiologia , Glycine max/genética , Proteínas de Plantas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Arabidopsis/genética , Secas , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/fisiologia , Superfamília Shaker de Canais de Potássio/genética , Cloreto de Sódio , Estresse Fisiológico
9.
Biochem Pharmacol ; 190: 114646, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34090876

RESUMO

Chimeric potassium channels KcsA-Kv1, which are among the most intensively studied hybrid membrane proteins to date, were constructed by replacing a part of the pore domain of bacterial potassium channel KcsA (K channel of streptomyces A) with corresponding regions of the mammalian voltage-gated potassium channels belonging to the Kv1 subfamily. In this way, the pore blocker binding site of Kv1 channels was transferred to KcsA, opening up possibility to use the obtained hybrids as receptors of Kv1-channel pore blockers of different origin. In this review the recent progress in KcsA-Kv1 channel design and applications is discussed with a focus on the development of new assays for studying interactions of pore blockers with the channels. A summary of experimental data is presented demonstrating that hybrid channels reproduce the blocker-binding profiles of parental Kv1 channels. It is overviewed how the KcsA-Kv1 chimeras are used to get new insight into the structure of potassium channels, to determine molecular basis for high affinity and selectivity of binding of peptide blockers to Kv1 channels, as well as to identify new peptide ligands.


Assuntos
Proteínas de Bactérias/química , Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Bioengenharia , Bloqueadores dos Canais de Potássio , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes , Venenos de Escorpião/química , Superfamília Shaker de Canais de Potássio/química
10.
PLoS One ; 16(3): e0248688, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33755670

RESUMO

Voltage-gated potassium (Kv) channels regulate the membrane potential and conductance of excitable cells to control the firing rate and waveform of action potentials. Even though Kv channels have been intensely studied for over 70 year, surprisingly little is known about how specific channels expressed in various neurons and their functional properties impact neuronal network activity and behavior in vivo. Although many in vivo genetic manipulations of ion channels have been tried, interpretation of these results is complicated by powerful homeostatic plasticity mechanisms that act to maintain function following perturbations in excitability. To better understand how Kv channels shape network function and behavior, we have developed a novel optogenetic technology to acutely regulate Kv channel expression with light by fusing the light-sensitive LOV domain of Vaucheria frigida Aureochrome 1 to the N-terminus of the Kv1 subunit protein to make an Opto-Kv1 channel. Recording of Opto-Kv1 channels expressed in Xenopus oocytes, mammalian cells, and neurons show that blue light strongly induces the current expression of Opto-Kv1 channels in all systems tested. We also find that an Opto-Kv1 construct containing a dominant-negative pore mutation (Opto-Kv1(V400D)) can be used to down-regulate Kv1 currents in a blue light-dependent manner. Finally, to determine whether Opto-Kv1 channels can elicit light-dependent behavioral effect in vivo, we targeted Opto-Kv1 (V400D) expression to Kv1.3-expressing mitral cells of the olfactory bulb in mice. Exposure of the bulb to blue light for 2-3 hours produced a significant increase in sensitivity to novel odors after initial habituation to a similar odor, comparable to behavioral changes seen in Kv1.3 knockout animals. In summary, we have developed novel photoactivatable Kv channels that provide new ways to interrogate neural circuits in vivo and to examine the roles of normal and disease-causing mutant Kv channels in brain function and behavior.


Assuntos
Neurônios , Superfamília Shaker de Canais de Potássio/metabolismo , Potenciais de Ação , Animais , Células HEK293 , Humanos , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/metabolismo , Oócitos/citologia , Oócitos/metabolismo , Xenopus laevis
11.
Brain ; 144(6): 1711-1726, 2021 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-33693512

RESUMO

Patients with neuropathic pain often experience innocuous cooling as excruciating pain. The cell and molecular basis of this cold allodynia is little understood. We used in vivo calcium imaging of sensory ganglia to investigate how the activity of peripheral cold-sensing neurons was altered in three mouse models of neuropathic pain: oxaliplatin-induced neuropathy, partial sciatic nerve ligation, and ciguatera poisoning. In control mice, cold-sensing neurons were few in number and small in size. In neuropathic animals with cold allodynia, a set of normally silent large diameter neurons became sensitive to cooling. Many of these silent cold-sensing neurons responded to noxious mechanical stimuli and expressed the nociceptor markers Nav1.8 and CGRPα. Ablating neurons expressing Nav1.8 resulted in diminished cold allodynia. The silent cold-sensing neurons could also be activated by cooling in control mice through blockade of Kv1 voltage-gated potassium channels. Thus, silent cold-sensing neurons are unmasked in diverse neuropathic pain states and cold allodynia results from peripheral sensitization caused by altered nociceptor excitability.


Assuntos
Temperatura Baixa/efeitos adversos , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Neurônios/metabolismo , Nociceptores/metabolismo , Animais , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Sensação Térmica/fisiologia
12.
J Gen Physiol ; 153(4)2021 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-33683319

RESUMO

Voltage-gated potassium (KV) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker KV channel increased resin-acid-induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker KV channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid-binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1-S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.


Assuntos
Canais de Potássio , Superfamília Shaker de Canais de Potássio , Sítios de Ligação , Fenômenos Biofísicos , Simulação por Computador , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo
13.
Elife ; 102021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33620313

RESUMO

In Shaker K+ channels, the S4-S5 linker couples the voltage sensor (VSD) and pore domain (PD). Another coupling mechanism is revealed using two W434F-containing channels: L361R:W434F and L366H:W434F. In L361R:W434F, W434F affects the L361R VSD seen as a shallower charge-voltage (Q-V) curve that crosses the conductance-voltage (G-V) curve. In L366H:W434F, L366H relieves the W434F effect converting a non-conductive channel in a conductive one. We report a chain of residues connecting the VSD (S4) to the selectivity filter (SF) in the PD of an adjacent subunit as the molecular basis for voltage sensor selectivity filter gate (VS-SF) coupling. Single alanine substitutions in this region (L409A, S411A, S412A, or F433A) are enough to disrupt the VS-SF coupling, shown by the absence of Q-V and G-V crossing in L361R:W434F mutant and by the lack of ionic conduction in the L366H:W434F mutant. This residue chain defines a new coupling between the VSD and the PD in voltage-gated channels.


Assuntos
Proteínas de Drosophila/genética , Superfamília Shaker de Canais de Potássio/genética , Xenopus laevis/fisiologia , Animais , Proteínas de Drosophila/metabolismo , Feminino , Superfamília Shaker de Canais de Potássio/metabolismo
14.
Cardiovasc Toxicol ; 21(4): 322-335, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33389602

RESUMO

Consumption of illicit pharmaceutical products containing sibutramine has been reported to cause cardiovascular toxicity problems. This study aimed to demonstrate the toxicity profile of sibutramine, and thereby provide important implications for the development of more effective strategies in both clinical approaches and drug design studies. Action potentials (APs) were determined from freshly isolated ventricular cardiomyocytes with whole-cell configuration of current clamp as online. The maximum amplitude of APs (MAPs), the resting membrane potential (RMP), and AP duration from the repolarization phases were calculated from original records. The voltage-dependent K+-channel currents (IK) were recorded in the presence of external Cd2+ and both inward and outward parts of the current were calculated, while their expression levels were determined with qPCR. The levels of intracellular free Ca2+ and H+ (pHi) as well as reactive oxygen species (ROS) were measured using either a ratiometric micro-spectrofluorometer or confocal microscope. The mechanical activity of isolated hearts was observed with Langendorff-perfusion system. Acute sibutramine applications (10-8-10-5 M) induced significant alterations in both MAPs and RMP as well as the repolarization phases of APs and IK in a concentration-dependent manner. Sibutramine (10 µM) induced Ca2+-release from the sarcoplasmic reticulum under either electrical or caffeine stimulation, whereas it depressed left ventricular developed pressure with a marked decrease in the end-diastolic pressure. pHi inhibition by sibutramine supports the observed negative alterations in contractility. Changes in mRNA levels of different IK subunits are consistent with the acute inhibition of the repolarizing IK, affecting AP parameters, and provoke the cardiotoxicity.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Fármacos Antiobesidade/toxicidade , Ciclobutanos/toxicidade , Cardiopatias/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Cálcio/metabolismo , Cardiotoxicidade , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/fisiopatologia , Concentração de Íons de Hidrogênio , Preparação de Coração Isolado , Masculino , Miócitos Cardíacos/metabolismo , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Fatores de Tempo , Função Ventricular Esquerda/efeitos dos fármacos
16.
Gene ; 768: 145311, 2021 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-33220344

RESUMO

The Shaker K+ channel family plays a vital role in potassium absorption and stress resistance in plants. However little information on the genes family is available about sweetpotato. In the present study, eleven sweetpotato Shaker K+ channel genes were identified and classified into five groups based on phylogenetic relationships, conserved motifs, and gene structure analyses. Based on synteny analysis, four duplicated gene pairs were identified, derived from both ancient and recent duplication, whereas only one resulted from tandem duplication events. Different expression pattern of Shaker K+ channel genes in roots of Xu32 and NZ1 resulted in different K+ deficiency tolerances, suggesting there is different mechanism of K+ uptake in sweetpotato cultivars with different K+-tolerance levels. Quantitative real-time PCR analysis revealed that the shaker K+ channel genes responded to drought and high salt stresses. Higher K+ influx under normal condition and lower K+ efflux under K+ deficiency stress were observed in IbAKT1 overexpressing transgenic roots than in adventitious roots, which indicated that IbAKT1 may play an important role in the regulation of K+ deficiency tolerance in sweetpotato. This is the first genome-wide analysis of Shaker K+ channel genes and the first functional analysis of IbAKT1 in sweetpotato. Our results provide valuable information on the gene structure, evolution, expression and functions of the Shaker K+ channel gene family in sweetpotato.


Assuntos
Perfilação da Expressão Gênica/métodos , Ipomoea batatas/crescimento & desenvolvimento , Superfamília Shaker de Canais de Potássio/genética , Sequenciamento Completo do Genoma/métodos , Mapeamento Cromossômico , Secas , Evolução Molecular , Duplicação Gênica , Regulação da Expressão Gênica de Plantas , Sequenciamento de Nucleotídeos em Larga Escala , Ipomoea batatas/genética , Ipomoea batatas/metabolismo , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Estresse Salino , Superfamília Shaker de Canais de Potássio/metabolismo , Sintenia
17.
Eur J Clin Invest ; 51(3): e13454, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33216974

RESUMO

BACKGROUND: Atherosclerosis (AS) is a chronic progressive inflammatory condition with a leading prevalence worldwide. Endothelial dysfunction leads to low-density lipoprotein trafficking into subendothelial space and the subsequent form of oxidized LDL (ox-LDL) within intimal layer, perpetuating the vicious cycle of endothelial dysfunction. K+ exerts beneficial effects in vascular wall by reducing LDL oxidization, vascular smooth muscle cells (VSMCs) proliferation, and free radical generation. K+ also modulates vascular tone through a regulatory effect on cell membrane potential. MATERIALS AND METHODS: The most relevant papers on the association between 'potassium channels' and 'atherosclerosis' were selected among those deposited on PubMed from 1990 to 2020. RESULTS: Here, we provide a short narrative review that elaborates on the role of K+ in atherosclerosis. This review also update the current knowledge about potential pharmacological agents targeting K+ channels with a special focus on pleiotropic activities of agents such as statins, sulfonylureas and dihydropyridines. CONCLUSION: In this review, the mechanism of different K+ channels on vascular endothelium will be summarized, mainly focusing on their pathophysiological role in atherosclerosis and potential therapeutic application.


Assuntos
Aterosclerose/metabolismo , Endotélio Vascular/fisiopatologia , Potenciais da Membrana , Potássio/metabolismo , Aterosclerose/fisiopatologia , Proliferação de Células , Radicais Livres/metabolismo , Humanos , Canais KATP/metabolismo , Lipoproteínas LDL/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Canais de Potássio Cálcio-Ativados/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo
18.
Elife ; 92020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33185190

RESUMO

The palmitoyl acyltransferase (PAT) ZDHHC14 is highly expressed in the hippocampus and is the only PAT predicted to bind Type-I PDZ domain-containing proteins. However, ZDHHC14's neuronal roles are unknown. Here, we identify the PDZ domain-containing Membrane-associated Guanylate Kinase (MaGUK) PSD93 as a direct ZDHHC14 interactor and substrate. PSD93, but not other MaGUKs, localizes to the axon initial segment (AIS). Using lentiviral-mediated shRNA knockdown in rat hippocampal neurons, we find that ZDHHC14 controls palmitoylation and AIS clustering of PSD93 and also of Kv1 potassium channels, which directly bind PSD93. Neurodevelopmental expression of ZDHHC14 mirrors that of PSD93 and Kv1 channels and, consistent with ZDHHC14's importance for Kv1 channel clustering, loss of ZDHHC14 decreases outward currents and increases action potential firing in hippocampal neurons. To our knowledge, these findings identify the first neuronal roles and substrates for ZDHHC14 and reveal a previously unappreciated role for palmitoylation in control of neuronal excitability.


Assuntos
Aciltransferases/metabolismo , Axônios/enzimologia , Superfamília Shaker de Canais de Potássio/metabolismo , Aciltransferases/genética , Animais , Fenômenos Eletrofisiológicos , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Hipocampo/citologia , Humanos , Camundongos , Ligação Proteica , Superfamília Shaker de Canais de Potássio/genética , Técnicas do Sistema de Duplo-Híbrido
19.
Mar Drugs ; 18(11)2020 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-33114777

RESUMO

κ-Conotoxin-PVIIA (κ-PVIIA) is a potassium-channel blocking peptide from the venom of the fish-hunting snail, Conus purpurascens, which is essential for quick prey's excitotoxic immobilization. Binding of one κ-PVIIA to Shaker K-channels occludes the K+-conduction pore without additional conformational effects. Because this 27-residue toxin is +4-charged at neutral pH, we asked if electrostatic interactions play a role in binding. With Voltage-Clamp electrophysiology, we tested how ionic strength (IS) affects κ-PVIIA blockade to Shaker. When IS varied from ~0.06 to ~0.16 M, the dissociation constant for open and closed channels increased by ~5- and ~16-fold, respectively. While the association rates decreased equally, by ~4-fold, in open and closed channels, the dissociation rates increased 4-5-fold in closed channels but was IS-insensitive in open channels. To explain this differential IS-dependency, we propose that the bound κ-PVIIA wobbles, so that in open channels the intracellular environment, via ion-conduction pore, buffers the imposed IS-changes in the toxin-channel interface. A Brønsted-Bjerrum analysis on the rates predicts that if, instead of fish, the snail preyed on organisms with seawater-like lymph ionic composition, a severely harmless toxin, with >100-fold diminished affinity, would result. Thus, considerations of the native ionic environment are essential for conotoxins evaluation as pharmacological leads.


Assuntos
Conotoxinas/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Animais , Conotoxinas/química , Oócitos , Concentração Osmolar , Bloqueadores dos Canais de Potássio/farmacologia , Ligação Proteica , Superfamília Shaker de Canais de Potássio/química , Xenopus laevis
20.
Proc Natl Acad Sci U S A ; 117(43): 27016-27021, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33051293

RESUMO

The opening and closing of voltage-gated ion channels are regulated by voltage sensors coupled to a gate that controls the ion flux across the cellular membrane. Modulation of any part of gating constitutes an entry point for pharmacologically regulating channel function. Here, we report on the discovery of a large family of warfarin-like compounds that open the two voltage-gated type 1 potassium (KV1) channels KV1.5 and Shaker, but not the related KV2-, KV4-, or KV7-type channels. These negatively charged compounds bind in the open state to positively charged arginines and lysines between the intracellular ends of the voltage-sensor domains and the pore domain. This mechanism of action resembles that of endogenous channel-opening lipids and opens up an avenue for the development of ion-channel modulators.


Assuntos
Ativação do Canal Iônico , Canal de Potássio Kv1.5/agonistas , Superfamília Shaker de Canais de Potássio/agonistas , Animais , Ensaios de Triagem em Larga Escala , Canal de Potássio Kv1.5/metabolismo , Simulação de Acoplamento Molecular , Técnicas de Patch-Clamp , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevis
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