RESUMO
The Shaker family of voltage-gated K+ channels has been thought of as an animal-specific ion channel family that diversified in concert with nervous systems. It comprises four functionally independent gene subfamilies (Kv1-4) that encode diverse neuronal K+ currents. Comparison of animal genomes predicts that only the Kv1 subfamily was present in the animal common ancestor. Here, we show that some choanoflagellates, the closest protozoan sister lineage to animals, also have Shaker family K+ channels. Choanoflagellate Shaker family channels are surprisingly most closely related to the animal Kv2-4 subfamilies which were believed to have evolved only after the divergence of ctenophores and sponges from cnidarians and bilaterians. Structural modeling predicts that the choanoflagellate channels share a T1 Zn2+ binding site with Kv2-4 channels that is absent in Kv1 channels. We functionally expressed three Shakers from Salpingoeca helianthica (SheliKvT1.1-3) in Xenopus oocytes. SheliKvT1.1-3 function only in two heteromultimeric combinations (SheliKvT1.1/1.2 and SheliKvT1.1/1.3) and encode fast N-type inactivating K+ channels with distinct voltage dependence that are most similar to the widespread animal Kv1-encoded A-type Shakers. Structural modeling of the T1 assembly domain supports a preference for heteromeric assembly in a 2:2 stoichiometry. These results push the origin of the Shaker family back into a common ancestor of metazoans and choanoflagellates. They also suggest that the animal common ancestor had at least two distinct molecular lineages of Shaker channels, a Kv1 subfamily lineage predicted from comparison of animal genomes and a Kv2-4 lineage predicted from comparison of animals and choanoflagellates.
Assuntos
Coanoflagelados , Evolução Molecular , Superfamília Shaker de Canais de Potássio , Animais , Coanoflagelados/genética , Coanoflagelados/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Filogenia , Sequência de AminoácidosRESUMO
Here we explore the evolutionary origins of fast N-type ball-and-chain inactivation in Shaker (Kv1) K+ channels by functionally characterizing Shaker channels from the ctenophore (comb jelly) Mnemiopsis leidyi. Ctenophores are the sister lineage to other animals and Mnemiopsis has >40 Shaker-like K+ channels, but they have not been functionally characterized. We identified three Mnemiopsis channels (MlShak3-5) with N-type inactivation ball-like sequences at their N termini and functionally expressed them in Xenopus oocytes. Two of the channels, MlShak4 and MlShak5, showed rapid inactivation similar to cnidarian and bilaterian Shakers with rapid N-type inactivation, whereas MlShak3 inactivated â¼100-fold more slowly. Fast inactivation in MlShak4 and MlShak5 required the putative N-terminal inactivation ball sequences. Furthermore, the rate of fast inactivation in these channels depended on the number of inactivation balls/channel, but the rate of recovery from inactivation did not. These findings closely match the mechanism of N-type inactivation first described for Drosophila Shaker in which 1) inactivation balls on the N termini of each subunit can independently block the pore, and 2) only one inactivation ball occupies the pore binding site at a time. These findings suggest classical N-type activation evolved in Shaker channels at the very base of the animal phylogeny in a common ancestor of ctenophores, cnidarians, and bilaterians and that fast-inactivating Shakers are therefore a fundamental type of animal K+ channel. Interestingly, we find evidence from functional co-expression experiments and molecular dynamics that MlShak4 and MlShak5 do not co-assemble, suggesting that Mnemiopsis has at least two functionally independent N-type Shaker channels.
Assuntos
Ctenóforos , Ativação do Canal Iônico , Superfamília Shaker de Canais de Potássio , Animais , Ctenóforos/metabolismo , Ctenóforos/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/química , Sequência de Aminoácidos , Filogenia , Oócitos/metabolismoRESUMO
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/metabolismoRESUMO
Shaker potassium channels have been an essential model for studying inactivation of ion channels and shaped our earliest understanding of N-type vs. C-type mechanisms. In early work describing C-type inactivation, López-Barneo and colleagues systematically characterized numerous mutations of Shaker residue T449, demonstrating that this position was a key determinant of C-type inactivation rate. In most of the closely related mammalian Kv1 channels, however, a persistent enigma has been that residue identity at this position has relatively modest effects on the rate of inactivation in response to long depolarizations. In this study, we report alternative ways to measure or elicit conformational changes in the outer pore associated with C-type inactivation. Using a strategically substituted cysteine in the outer pore, we demonstrate that mutation of Kv1.2 V381 (equivalent to Shaker T449) or W366 (Shaker W434) markedly increases susceptibility to modification by extracellularly applied MTSET. Moreover, due to the cooperative nature of C-type inactivation, Kv1.2 assembly in heteromeric channels markedly inhibits MTSET modification of this substituted cysteine in neighboring subunits. The identity of Kv1.2 residue V381 also markedly influences function in conditions that bias channels toward C-type inactivation, namely when Na+ is substituted for K+ as the permeant ion or when channels are blocked by an N-type inactivation particle (such as Kvß1.2). Overall, our findings illustrate that in mammalian Kv1 channels, the identity of the T449-equivalent residue can strongly influence function in certain experimental conditions, even while having modest effects on apparent inactivation during long depolarizations. These findings contribute to reconciling differences in experimental outcomes in many Kv1 channels vs. Shaker.
Assuntos
Ativação do Canal Iônico , Canal de Potássio Kv1.2 , Animais , Canal de Potássio Kv1.2/metabolismo , Canal de Potássio Kv1.2/química , Canal de Potássio Kv1.2/genética , Mutação , Superfamília Shaker de Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/química , Superfamília Shaker de Canais de Potássio/genética , HumanosRESUMO
Voltage-gated K+ channels have distinct gates that regulate ion flux: the activation gate (A-gate) formed by the bundle crossing of the S6 transmembrane helices and the slow inactivation gate in the selectivity filter. These two gates are bidirectionally coupled. If coupling involves the rearrangement of the S6 transmembrane segment, then we predict state-dependent changes in the accessibility of S6 residues from the water-filled cavity of the channel with gating. To test this, we engineered cysteines, one at a time, at S6 positions A471, L472, and P473 in a T449A Shaker-IR background and determined the accessibility of these cysteines to cysteine-modifying reagents MTSET and MTSEA applied to the cytosolic surface of inside-out patches. We found that neither reagent modified either of the cysteines in the closed or the open state of the channels. On the contrary, A471C and P473C, but not L472C, were modified by MTSEA, but not by MTSET, if applied to inactivated channels with open A-gate (OI state). Our results, combined with earlier studies reporting reduced accessibility of residues I470C and V474C in the inactivated state, strongly suggest that the coupling between the A-gate and the slow inactivation gate is mediated by rearrangements in the S6 segment. The S6 rearrangements are consistent with a rigid rod-like rotation of S6 around its longitudinal axis upon inactivation. S6 rotation and changes in its environment are concomitant events in slow inactivation of Shaker KV channels.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana , Superfamília Shaker de Canais de Potássio , Superfamília Shaker de Canais de Potássio/genética , Metanossulfonato de Etila , Cisteína/genética , Cisteína/química , Potássio/metabolismoRESUMO
The malignancy with the greatest global mortality rate is lung cancer. Lung adenocarcinoma (LUAD) is the most common subtype. The evidence demonstrated that voltage-gated potassium channel subunit beta-2 (KCNAB2) significantly participated in the initiation of colorectal cancer and its progression. However, the biological function of KCNAB2 in LUAD and its effect on the tumor immune microenvironment are still unknown. In this study, we found that the expression of KCNAB2 in tissues of patients with LUAD was markedly downregulated, and its downregulation was linked to accelerated cancer growth and poor clinical outcomes. In addition, low KCNAB2 expression was correlated with a deficiency in immune infiltration. The mechanism behind this issue might be that KCNAB2 influenced the immunological process such that the directed migration of immune cells was affected. Furthermore, overexpression of KCNAB2 in cell lines promoted the expression of CCL2, CCL3, CCL4, CCL18, CXCL9, CXCL10, and CXCL12, which are necessary for the recruitment of immune cells. In conclusion, KCNAB2 may play a key function in immune infiltration and can be exploited as a predictive biomarker for evaluating prognosis and a possible immunotherapeutic target.
Assuntos
Adenocarcinoma de Pulmão , Superfamília Shaker de Canais de Potássio , Humanos , Adenocarcinoma de Pulmão/imunologia , Adenocarcinoma de Pulmão/patologia , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Superfamília Shaker de Canais de Potássio/genética , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , PrognósticoRESUMO
Ion channel complexes typically consist of both pore-forming subunits and auxiliary subunits that do not directly conduct current but can regulate trafficking or alter channel properties. Isolating the role of these auxiliary subunits in neurons has proved difficult due to a lack of specific pharmacological agents and the potential for developmental compensation in constitutive knockout models. Here, we use cell-type-specific viral-mediated CRISPR/Cas9 mutagenesis to target the potassium channel auxiliary subunit Kvß2 (Kcnab2) in dopamine neurons in the adult mouse brain. We find that mutagenesis of Kcnab2 reduces surface expression of Kv1.2, the primary Kv1 pore-forming subunit expressed in dopamine neurons, and shifts the voltage dependence of inactivation of potassium channel currents toward more hyperpolarized potentials. Loss of Kcnab2 broadens the action potential waveform in spontaneously firing dopamine neurons recorded in slice, reduces the afterhyperpolarization amplitude, and increases spike timing irregularity and excitability, all of which is consistent with a reduction in potassium channel current. Similar effects were observed with mutagenesis of the pore-forming subunit Kv1.2 (Kcna2). These results identify Kv1 currents as important contributors to dopamine neuron firing and demonstrate a role for Kvß2 subunits in regulating the trafficking and gating properties of these ion channels. Furthermore, they demonstrate the utility of CRISPR-mediated mutagenesis in the study of previously difficult to isolate ion channel subunits.NEW & NOTEWORTHY Here, we utilize CRISPR/Cas9-mediated mutagenesis in dopamine neurons in mice to target the gene encoding Kvß2, an auxiliary subunit that forms a part of Kv1 channel complexes. We find that the absence of Kvß2 alters action potential properties by reducing surface expression of pore-forming subunits and shifting the voltage dependence of channel inactivation. This work establishes a new function for Kvß2 subunits and Kv1 complexes in regulating dopamine neuron activity.
Assuntos
Neurônios Dopaminérgicos , Canais de Potássio , Animais , Neurônios Dopaminérgicos/metabolismo , Camundongos , Canais de Potássio/metabolismo , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
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/genéticaRESUMO
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 FisiológicoRESUMO
Temperature-dependent regulation of ion channel activity is critical for a variety of physiological processes ranging from immune response to perception of noxious stimuli. Our understanding of the structural mechanisms that underlie temperature sensing remains limited, in part due to the difficulty of combining high-resolution structural analysis with temperature stimulus. Here, we use NMR to compare the temperature-dependent behavior of Shaker potassium channel voltage sensor domain (WT-VSD) to its engineered temperature sensitive (TS-VSD) variant. Further insight into the molecular basis for temperature-dependent behavior is obtained by analyzing the experimental results together with molecular dynamics simulations. Our studies reveal that the overall secondary structure of the engineered TS-VSD is identical to the wild-type channels except for local changes in backbone torsion angles near the site of substitution (V369S and F370S). Remarkably however, these structural differences result in increased hydration of the voltage-sensing arginines and the S4-S5 linker helix in the TS-VSD at higher temperatures, in contrast to the WT-VSD. These findings highlight how subtle differences in the primary structure can result in large-scale changes in solvation and thereby confer increased temperature-dependent activity beyond that predicted by linear summation of solvation energies of individual substituents.
Assuntos
Engenharia de Proteínas , Superfamília Shaker de Canais de Potássio/química , Escherichia coli , Temperatura Alta , Simulação de Dinâmica Molecular , Mutação , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
[Figure: see text].
Assuntos
Circulação Coronária , Canal de Potássio Kv1.3/metabolismo , Músculo Liso Vascular/metabolismo , Oxigênio/metabolismo , Superfamília Shaker de Canais de Potássio/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Canal de Potássio Kv1.3/genética , Ácido Láctico , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/citologia , Músculo Liso Vascular/fisiologia , Contração Miocárdica , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Miócitos de Músculo Liso/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Vasoconstrição , VasodilataçãoRESUMO
Physiological functioning and homeostasis of the brain rely on finely tuned synaptic transmission, which involves nanoscale alignment between presynaptic neurotransmitter-release machinery and postsynaptic receptors. However, the molecular identity and physiological significance of transsynaptic nanoalignment remain incompletely understood. Here, we report that epilepsy gene products, a secreted protein LGI1 and its receptor ADAM22, govern transsynaptic nanoalignment to prevent epilepsy. We found that LGI1-ADAM22 instructs PSD-95 family membrane-associated guanylate kinases (MAGUKs) to organize transsynaptic protein networks, including NMDA/AMPA receptors, Kv1 channels, and LRRTM4-Neurexin adhesion molecules. Adam22ΔC5/ΔC5 knock-in mice devoid of the ADAM22-MAGUK interaction display lethal epilepsy of hippocampal origin, representing the mouse model for ADAM22-related epileptic encephalopathy. This model shows less-condensed PSD-95 nanodomains, disordered transsynaptic nanoalignment, and decreased excitatory synaptic transmission in the hippocampus. Strikingly, without ADAM22 binding, PSD-95 cannot potentiate AMPA receptor-mediated synaptic transmission. Furthermore, forced coexpression of ADAM22 and PSD-95 reconstitutes nano-condensates in nonneuronal cells. Collectively, this study reveals LGI1-ADAM22-MAGUK as an essential component of transsynaptic nanoarchitecture for precise synaptic transmission and epilepsy prevention.
Assuntos
Proteínas ADAM/genética , Epilepsia/genética , Guanilato Quinases/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Transmissão Sináptica/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Proteínas de Ligação ao Cálcio/genética , Modelos Animais de Doenças , Epilepsia/patologia , Epilepsia/prevenção & controle , Técnicas de Introdução de Genes , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Proteínas de Membrana/genética , Camundongos , Moléculas de Adesão de Célula Nervosa/genética , Receptores de AMPA/genética , Receptores de N-Metil-D-Aspartato/genética , Superfamília Shaker de Canais de Potássio/genéticaRESUMO
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 fármacosRESUMO
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 , SinteniaRESUMO
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-HíbridoRESUMO
Prion diseases are fatal and transmissible neurodegenerative disorders caused by the misfolding and aggregation of prion protein. Although recent studies have implicated epigenetic variation in common neurodegenerative disorders, no study has yet explored their role in human prion diseases. Here we profiled genome-wide blood DNA methylation in the most common human prion disease, sporadic Creutzfeldt-Jakob disease (sCJD). Our case-control study (n = 219), when accounting for differences in cell type composition between individuals, identified 38 probes at genome-wide significance (p < 1.24 × 10-7). Nine of these sites were taken forward in a replication study, performed in an independent case-control (n = 186) cohort using pyrosequencing. Sites in or close to FKBP5, AIM2 (2 probes), UHRF1, KCNAB2 successfully replicated. The blood-based DNA methylation signal was tissue- and disease-specific, in that the replicated probe signals were unchanged in case-control studies using sCJD frontal-cortex (n = 84), blood samples from patients with Alzheimer's disease, and from inherited and acquired prion diseases. Machine learning algorithms using blood DNA methylation array profiles accurately distinguished sCJD patients and controls. Finally, we identified sites whose methylation levels associated with prolonged survival in sCJD patients. Altogether, this study has identified a peripheral DNA methylation signature of sCJD with a variety of potential biomarker applications.
Assuntos
Encéfalo/patologia , Síndrome de Creutzfeldt-Jakob/genética , Síndrome de Creutzfeldt-Jakob/metabolismo , Metilação de DNA/fisiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Encéfalo/metabolismo , Estudos de Casos e Controles , Síndrome de Creutzfeldt-Jakob/patologia , Feminino , Predisposição Genética para Doença/genética , Humanos , Masculino , Pessoa de Meia-Idade , Doenças Priônicas/metabolismo , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismoRESUMO
Recently, Conorfamide-Sr3 (CNF-Sr3) was isolated from the venom of Conus spurius and was demonstrated to have an inhibitory concentration-dependent effect on the Shaker K+ channel. The voltage-gated potassium channels play critical functions on cellular signaling, from the regeneration of action potentials in neurons to the regulation of insulin secretion in pancreatic cells, among others. In mammals, there are at least 40 genes encoding voltage-gated K+ channels and the process of expression of some of them may include alternative splicing. Given the enormous variety of these channels and the proven use of conotoxins as tools to distinguish different ligand- and voltage-gated ion channels, in this work, we explored the possible effect of CNF-Sr3 on four human voltage-gated K+ channel subtypes homologous to the Shaker channel. CNF-Sr3 showed a 10 times higher affinity for the Kv1.6 subtype with respect to Kv1.3 (IC50 = 2.7 and 24 µM, respectively) and no significant effect on Kv1.4 and Kv1.5 at 10 µM. Thus, CNF-Sr3 might become a novel molecular probe to study diverse aspects of human Kv1.3 and Kv1.6 channels.
Assuntos
Venenos de Moluscos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Superfamília Shaker de Canais de Potássio/antagonistas & inibidores , Animais , Caramujo Conus , Ativação do Canal Iônico , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/genética , Canal de Potássio Kv1.4/metabolismo , Canal de Potássio Kv1.5/antagonistas & inibidores , Canal de Potássio Kv1.5/genética , Canal de Potássio Kv1.5/metabolismo , Canal de Potássio Kv1.6/antagonistas & inibidores , Canal de Potássio Kv1.6/genética , Canal de Potássio Kv1.6/metabolismo , Potenciais da Membrana , Oócitos , Superfamília Shaker de Canais de Potássio/genética , Superfamília Shaker de Canais de Potássio/metabolismo , Xenopus laevisRESUMO
The existing mosquito pesticide repertoire faces great challenges to sustainability, and new classes of pesticides are vitally needed to address established and emerging mosquito-borne infectious diseases. RNA interference- (RNAi-) based pesticides are emerging as a promising new biorational mosquito control strategy. In this investigation, we describe characterization of an interfering RNA pesticide (IRP) corresponding to the mosquito Shaker (Sh) gene, which encodes an evolutionarily conserved voltage-gated potassium channel subunit. Delivery of the IRP to Aedes aegypti adult mosquitoes in the form of siRNA that was injected or provided as an attractive toxic sugar bait (ATSB) led to Sh gene silencing that resulted in severe neural and behavioral defects and high levels of adult mortality. Likewise, when provided to A. aegypti larvae in the form of short hairpin RNA (shRNA) expressed in Saccharomyces cerevisiae (baker's yeast) that had been formulated into a dried inactivated yeast tablet, the yeast IRP induced neural defects and larval death. Although the Sh IRP lacks a known target site in humans or other non-target organisms, conservation of the target site in the Sh genes of multiple mosquito species suggested that it may function as a biorational broad-range mosquito insecticide. In support of this, the Sh IRP induced both adult and larval mortality in treated Aedes albopictus, Anopheles gambiae, and Culex quinquefasciatus mosquitoes, but was not toxic to non-target arthropods. These studies indicated that IRPs targeting Sh could one day be used in integrated biorational mosquito control programs for the prevention of multiple mosquito-borne illnesses. The results of this investigation also suggest that the species-specificity of ATSB technology, a new paradigm for vector control, could be enhanced through the use of RNAi-based pesticides.