RESUMEN

BACKGROUND: KCNJ10 and CAPN1 variants cause "spinocerebellar" ataxia in dogs, but their association with generalized myokymia and neuromyotonia remains unclear. OBJECTIVE: To investigate the association between KCNJ10 and CAPN1 and myokymia or neuromyotonia, with or without concurrent spinocerebellar ataxia. ANIMALS: Thirty-three client-owned dogs with spinocerebellar ataxia, myokymia neuromytonia, or a combination of these signs. METHODS: Genetic analysis of a cohort of dogs clinically diagnosed with spinocerebellar ataxia, myokymia or neuromyotonia. KCNJ10 c.627C>G and CAPN1 c.344G>A variants and the coding sequence of KCNA1, KCNA2, KCNA6, KCNJ10 and HINT1 were sequenced using DNA extracted from blood samples. RESULTS: Twenty-four Jack Russell terriers, 1 Jack Russell terrier cross, 1 Dachshund and 1 mixed breed with spinocerebellar ataxia were biallelic (homozygous) for the KCNJ10 c.627C>G variant. Twenty-one of those dogs had myokymia, neuromyotonia, or both. One Parson Russell terrier with spinocerebellar ataxia alone was biallelic for the CAPN1 c.344G>A variant. Neither variant was found in 1 Jack Russell terrier with ataxia alone, nor in 3 Jack Russell terriers and 1 Yorkshire terrier with myokymia and neuromyotonia alone. No other causal variants were found in the coding sequence of the investigated candidate genes in these latter 5 dogs. CONCLUSION: The KCNJ10 c.627C>G variant, or rarely the CAPN1 c.344G>A variant, was confirmed to be the causal variant of spinocerebellar ataxia. We also report the presence of the KCNJ10 c.627C>G variant in the Dachshund breed. In dogs with myokymia and neuromyotonia alone the reported gene variants were not found. Other genetic or immune-mediated causes should be investigated to explain the clinical signs of these cases.

Asunto(s)

Enfermedades de los Perros , Síndrome de Isaacs , Miocimia , Ataxias Espinocerebelosas , Humanos , Perros , Animales , Miocimia/genética , Miocimia/veterinaria , Síndrome de Isaacs/genética , Síndrome de Isaacs/veterinaria , Ataxias Espinocerebelosas/veterinaria , Ataxia/veterinaria , Cruzamiento , Proteínas del Tejido Nervioso , Canal de Potasio Kv1.6 , Enfermedades de los Perros/genética

RESUMEN

OBJECTIVE: Mutations in the genes encoding neuronal ion channels are a common cause of Mendelian neurological diseases. We sought to identify novel de novo sequence variants in cases with early infantile epileptic phenotypes and neurodevelopmental anomalies. METHODS: Following clinical diagnosis, we performed whole exome sequencing of the index cases and their parents. Identified channel variants were expressed in Xenopus oocytes and their functional properties assessed using two-electrode voltage clamp. RESULTS: We identified novel de novo variants in KCNA6 in four unrelated individuals variably affected with neurodevelopmental disorders and seizures with onset in the first year of life. Three of the four identified mutations affect the pore-lining S6 α-helix of KV 1.6. A prominent finding of functional characterization in Xenopus oocytes was that the channel variants showed only minor effects on channel activation but slowed channel closure and shifted the voltage dependence of deactivation in a hyperpolarizing direction. Channels with a mutation affecting the S6 helix display dominant effects on channel deactivation when co-expressed with wild-type KV 1.6 or KV 1.1 subunits. SIGNIFICANCE: This is the first report of de novo nonsynonymous variants in KCNA6 associated with neurological or any clinical features. Channel variants showed a consistent effect on channel deactivation, slowing the rate of channel closure following normal activation. This specific gain-of-function feature is likely to underlie the neurological phenotype in our patients. Our data highlight KCNA6 as a novel channelopathy gene associated with early infantile epileptic phenotypes and neurodevelopmental anomalies.

Asunto(s)

Epilepsia , Trastornos del Neurodesarrollo , Humanos , Epilepsia/genética , Mutación/genética , Convulsiones/genética , Canal de Potasio Kv1.6/genética

RESUMEN

BACKGROUND: Glyphosate is the most commonly used herbicide in the world and is purported to have a variety of health effects, including endocrine disruption and an elevated risk of several types of cancer. Blood DNA methylation has been shown to be associated with many other environmental exposures, but to our knowledge, no studies to date have examined the association between blood DNA methylation and glyphosate exposure. OBJECTIVE: We conducted an epigenome-wide association study to identify DNA methylation loci associated with urinary glyphosate and its metabolite aminomethylphosphonic acid (AMPA) levels. Secondary goals were to determine the association of epigenetic age acceleration with glyphosate and AMPA and develop blood DNA methylation indices to predict urinary glyphosate and AMPA levels. METHODS: For 392 postmenopausal women, white blood cell DNA methylation was measured using the Illumina Infinium MethylationEPIC BeadChip array. Glyphosate and AMPA were measured in two urine samples per participant using liquid chromatography-tandem mass spectrometry. Methylation differences at the probe and regional level associated with glyphosate and AMPA levels were assessed using a resampling-based approach. Probes and regions that had an false discovery rate q<0.1 in ≥90% of 1,000 subsamples of the study population were considered differentially methylated. Differentially methylated sites from the probe-specific analysis were combined into a methylation index. Epigenetic age acceleration from three epigenetic clocks and an epigenetic measure of pace of aging were examined for associations with glyphosate and AMPA. RESULTS: We identified 24 CpG sites whose methylation level was associated with urinary glyphosate concentration and two associated with AMPA. Four regions, within the promoters of the MSH4, KCNA6, ABAT, and NDUFAF2/ERCC8 genes, were associated with glyphosate levels, along with an association between ESR1 promoter hypomethylation and AMPA. The methylation index accurately predicted glyphosate levels in an internal validation cohort. AMPA, but not glyphosate, was associated with greater epigenetic age acceleration. DISCUSSION: Glyphosate and AMPA exposure were associated with DNA methylation differences that could promote the development of cancer and other diseases. Further studies are warranted to replicate our results, determine the functional impact of glyphosate- and AMPA-associated differential DNA methylation, and further explore whether DNA methylation could serve as a biomarker of glyphosate exposure. https://doi.org/10.1289/EHP10174.

Asunto(s)

Metilación de ADN , Posmenopausia , Estudios Transversales , Enzimas Reparadoras del ADN , Femenino , Glicina/análogos & derivados , Humanos , Canal de Potasio Kv1.6 , Factores de Transcripción , Glifosato

RESUMEN

BACKGROUND: Genetic variants have been found to influence red blood cell (RBC) susceptibility to hemolytic stress and affect transfusion outcomes and the severity of blood diseases. Males have a higher susceptibility to hemolysis than females, but little is known about the genetic mechanism contributing to the difference. RESULTS: To investigate the sex differences in RBC susceptibility to hemolysis, we conducted a sex-stratified genome-wide association study and a genome-wide gene-by-sex interaction scan in a multi-ethnic dataset with 12,231 blood donors who have in vitro osmotic hemolysis measurements during routine blood storage. The estimated SNP-based heritability for osmotic hemolysis was found to be significantly higher in males than in females (0.46 vs. 0.41). We identified SNPs associated with sex-specific susceptibility to osmotic hemolysis in five loci (SPTA1, KCNA6, SLC4A1, SUMO1P1, and PAX8) that impact RBC function and hemolysis. CONCLUSION: Our study established a best practice to identify sex-specific genetic modifiers for sexually dimorphic traits in datasets with mixed ancestries, providing evidence of different genetic regulations of RBC susceptibility to hemolysis between sexes. These and other variants may help explain observed sex differences in the severity of hemolytic diseases, such as sickle cell and malaria, as well as the viability of red cell storage and recovery.

Asunto(s)

Conservación de la Sangre , Eritrocitos , Hemólisis , Presión Osmótica , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Canal de Potasio Kv1.6/genética , Masculino , Ósmosis , Factores Sexuales

RESUMEN

The potassium channel Kv1.6 has recently been implicated as a major modulatory channel subunit expressed in primary nociceptors. Furthermore, its expression at juxtaparanodes of myelinated primary afferents is induced following traumatic nerve injury as part of an endogenous mechanism to reduce hyperexcitability and pain-related hypersensitivity. In this study, we compared two mouse models of constitutive Kv1.6 knock-out (KO) achieved by different methods: traditional gene trap via homologous recombination and CRISPR-mediated excision. Both Kv1.6 KO mouse lines exhibited an unexpected reduction in sensitivity to noxious heat stimuli, to differing extents: the Kv1.6 mice produced via gene trap had a far more significant hyposensitivity. These mice (Kcna6lacZ ) expressed the bacterial reporter enzyme LacZ in place of Kv1.6 as a result of the gene trap mechanism, and we found that their central primary afferent presynaptic terminals developed a striking neurodegenerative phenotype involving accumulation of lipid species, development of "meganeurites," and impaired transmission to dorsal horn wide dynamic range neurons. The anatomic defects were absent in CRISPR-mediated Kv1.6 KO mice (Kcna6-/-) but were present in a third mouse model expressing exogenous LacZ in nociceptors under the control of a Nav1.8-promoted Cre recombinase. LacZ reporter enzymes are thus intrinsically neurotoxic to sensory neurons and may induce pathologic defects in transgenic mice, which has confounding implications for the interpretation of gene KOs using lacZ Nonetheless, in Kcna6-/- mice not affected by LacZ, we demonstrated a significant role for Kv1.6 regulating acute noxious thermal sensitivity, and both mechanical and thermal pain-related hypersensitivity after nerve injury.SIGNIFICANCE STATEMENT In recent decades, the expansion of technologies to experimentally manipulate the rodent genome has contributed significantly to the field of neuroscience. While introduction of enzymatic or fluorescent reporter proteins to label neuronal populations is now commonplace, often potential toxicity effects are not fully considered. We show a role of Kv1.6 in acute and neuropathic pain states through analysis of two mouse models lacking Kv1.6 potassium channels: one with additional expression of LacZ and one without. We show that LacZ reporter enzymes induce unintended defects in sensory neurons, with an impact on behavioral data outcomes. To summarize we highlight the importance of Kv1.6 in recovery of normal sensory function following nerve injury, and careful interpretation of data from LacZ reporter models.

Asunto(s)

Técnicas de Inactivación de Genes/efectos adversos , Genes Reporteros , Canal de Potasio Kv1.6/genética , Operón Lac , Neuralgia/metabolismo , Nociceptores/metabolismo , Animales , Sistemas CRISPR-Cas , Femenino , Técnicas de Inactivación de Genes/métodos , Integrasas/metabolismo , Canal de Potasio Kv1.6/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Nociceptores/patología , Sinapsis/metabolismo , Sinapsis/patología

RESUMEN

Temporal coding precision of bushy cells in the ventral cochlear nucleus (VCN), critical for sound localization and communication, depends on the generation of rapid and temporally precise action potentials (APs). Voltage-gated potassium (Kv) channels are critically involved in this. The bushy cells in rat VCN express Kv1.1, 1.2, 1.3, 1.6, 3.1, 4.2, and 4.3 subunits. The Kv1.1 subunit contributes to the generation of a temporally precise single AP. However, the understanding of the functions of other Kv subunits expressed in the bushy cells is limited. Here, we investigated the functional diversity of Kv subunits concerning their contributions to temporal coding. We characterized the electrophysiological properties of the Kv channels with different subunits using whole cell patch-clamp recording and pharmacological methods. The neuronal firing pattern changed from single to multiple APs only when the Kv1.1 subunit was blocked. The Kv subunits, including the Kv1.1, 1.2, 1.6, or 3.1, were involved in enhancing temporal coding by lowering membrane excitability, shortening AP latencies, reducing jitter, and regulating AP kinetics. Meanwhile, all the Kv subunits contributed to rapid repolarization and sharpening peaks by narrowing half-width and accelerating fall rate, and the Kv1.1 subunit also affected the depolarization of AP. The Kv1.1, 1.2, and 1.6 subunits endowed bushy cells with a rapid time constant and a low input resistance of membrane for enhancing spike timing precision. The present results indicate that the Kv channels differentially affect intrinsic membrane properties to optimize the generation of rapid and reliable APs for temporal coding.NEW & NOTEWORTHY This study investigates the roles of Kv channels in effecting precision using electrophysiological and pharmacological methods in bushy cells. Different Kv channels have varying electrophysiological characteristics, which contribute to the interplay between changes in the membrane properties and regulation of neuronal excitability which then improve temporal coding. We conclude that the Kv channels are specialized to promote the precise and rapid coding of acoustic input by optimizing the generation of reliable APs.

Asunto(s)

Potenciales de Acción/fisiología , Núcleo Coclear/fisiología , Neuronas/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Femenino , Canal de Potasio Kv.1.1/antagonistas & inhibidores , Canal de Potasio Kv.1.1/fisiología , Canal de Potasio Kv.1.2/antagonistas & inhibidores , Canal de Potasio Kv.1.2/fisiología , Canal de Potasio Kv1.6/antagonistas & inhibidores , Canal de Potasio Kv1.6/fisiología , Masculino , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley

RESUMEN

BACKGROUND: The Val122Ile mutation in Transthyretin (TTR) gene causes a rare, difficult to diagnose hereditary form of cardiac amyloidosis. This mutation is most common in the United States and mainly present in people of African descent. The carriers have an increased risk of congestive heart failure, peripheral edema, and several other noncardiac phenotypes such as carpal tunnel syndrome, and arthroplasty which are top reasons for ambulatory/outpatient surgeries (OSs) in the country. METHODS: We conducted first-ever epigenome-wide association study using the Illumina's EPIC array, in Val122Ile carriers of African descent for heart disease and multiple OSs-an early disease indicator. Differential methylation across genome wide cytosine-phosphate guanine (CpG) sites was tested between carriers with and without heart disease and OS. Significant CpG sites were investigated for cis-mQTLs loci, followed by gene ontology and protein-protein interaction network. We also investigated the significant CpG sites in a secondary cohort of carriers for replication. RESULTS: Five differentially methylated sites (P≤2.1×10-8) in genes-FAM129B, SKI, WDR27, GLS, and an intergenic site near RP11-550A5.2, and one differentially methylated region containing KCNA6 and GALNT3 (P=1.1×10-12) were associated with heart disease. For OS, we observe 4 sites-2 sites in UBE2E3 and SEC14L5, and other 2 in intergenic regions (P≤1.8×10-7) and 3 regions overlapping SH3D21, EVA1B, LTB4R2, and CIDEB (P≤3.9×10-7). Functional protein-interaction module analysis identified ABCA1 (P=0.001) for heart disease. Six cis-mQTLs were associated with one of the significant CpG sites (FAM129B; P=4.1×10-24). We replicated 2 CpG sites (cg18546846 and cg06641417; P<0.05) in an external cohort of biopsy-confirmed cases of TTR (transthyretin) amyloidosis. The genes identified are involved in transport and clearance of amyloid deposits (GLS, ABCA1, FAM129B); cardiac fibrosis (SKI); and muscle tissue regulation (SKI, FAM129B). CONCLUSIONS: These findings highlight the link between a complex amyloid circuit and diverse symptoms of Val122Ile.

Asunto(s)

Amiloidosis/diagnóstico , Negro o Afroamericano/genética , Epigenómica , Prealbúmina/genética , Transportador 1 de Casete de Unión a ATP/genética , Amiloidosis/genética , Metilación de ADN , Redes Reguladoras de Genes/genética , Estudio de Asociación del Genoma Completo , Cardiopatías/genética , Cardiopatías/patología , Cardiopatías/cirugía , Humanos , Canal de Potasio Kv1.6/genética , Fosfoproteínas/genética , Polimorfismo de Nucleótido Simple , Sitios de Carácter Cuantitativo , Enzimas Ubiquitina-Conjugadoras/genética

RESUMEN

Spinocerebellar ataxia type 3 (SCA3) is the second-most common CAG repeat disease, caused by a glutamine-encoding expansion in the ATXN3 protein. SCA3 is characterized by spinocerebellar degeneration leading to progressive motor incoordination and early death. Previous studies suggest that potassium channel dysfunction underlies early abnormalities in cerebellar cortical Purkinje neuron firing in SCA3. However, cerebellar cortical degeneration is often modest both in the human disease and mouse models of SCA3, raising uncertainty about the role of cerebellar dysfunction in SCA3. Here, we address this question by investigating Purkinje neuron excitability in SCA3. In early-stage SCA3 mice, we confirm a previously identified increase in excitability of cerebellar Purkinje neurons and associate this excitability with reduced transcripts of two voltage-gated potassium (KV) channels, Kcna6 and Kcnc3, as well as motor impairment. Intracerebroventricular delivery of antisense oligonucleotides (ASO) to reduce mutant ATXN3 restores normal excitability to SCA3 Purkinje neurons and rescues transcript levels of Kcna6 and Kcnc3. Interestingly, while an even broader range of KV channel transcripts shows reduced levels in late-stage SCA3 mice, cerebellar Purkinje neuron physiology was not further altered despite continued worsening of motor impairment. These results suggest the progressive motor phenotype observed in SCA3 may not reflect ongoing changes in the cerebellar cortex but instead dysfunction of other neuronal structures within and beyond the cerebellum. Nevertheless, the early rescue of both KV channel expression and neuronal excitability by ASO treatment suggests that cerebellar cortical dysfunction contributes meaningfully to motor dysfunction in SCA3.

Asunto(s)

Ataxina-3/genética , Enfermedad de Machado-Joseph/tratamiento farmacológico , Enfermedad de Machado-Joseph/genética , Oligonucleótidos Antisentido/uso terapéutico , Células de Purkinje/patología , Proteínas Represoras/genética , Animales , Conducta Animal , Humanos , Inyecciones Intraventriculares , Canal de Potasio Kv1.6/efectos de los fármacos , Canal de Potasio Kv1.6/genética , Enfermedad de Machado-Joseph/psicología , Ratones , Ratones Transgénicos , Técnicas de Placa-Clamp , Fenotipo , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio Shaw/efectos de los fármacos , Canales de Potasio Shaw/genética , Resultado del Tratamiento

RESUMEN

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.

Asunto(s)

Venenos de Moluscos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Animales , Caracol Conus , Activación del Canal Iónico , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.4/antagonistas & inhibidores , Canal de Potasio Kv1.4/genética , Canal de Potasio Kv1.4/metabolismo , Canal de Potasio Kv1.5/antagonistas & inhibidores , Canal de Potasio Kv1.5/genética , Canal de Potasio Kv1.5/metabolismo , Canal de Potasio Kv1.6/antagonistas & inhibidores , Canal de Potasio Kv1.6/genética , Canal de Potasio Kv1.6/metabolismo , Potenciales de la Membrana , Oocitos , Canales de Potasio de la Superfamilia Shaker/genética , Canales de Potasio de la Superfamilia Shaker/metabolismo , Xenopus laevis

RESUMEN

RCK domains regulate the activity of K+ channels and transporters in eukaryotic and prokaryotic organisms by responding to ions or nucleotides. The mechanisms of RCK activation by Ca2+ in the eukaryotic BK and bacterial MthK K+ channels are well understood. However, the molecular details of activation in nucleotide-dependent RCK domains are not clear. Through a functional and structural analysis of the mechanism of ATP activation in KtrA, a RCK domain from the B. subtilis KtrAB cation channel, we have found that activation by nucleotide requires binding of cations to an intra-dimer interface site in the RCK dimer. In particular, divalent cations are coordinated by the γ-phosphates of bound-ATP, tethering the two subunits and stabilizing the active state conformation. Strikingly, the binding site residues are highly conserved in many different nucleotide-dependent RCK domains, indicating that divalent cations are a general cofactor in the regulatory mechanism of many nucleotide-dependent RCK domains.

Asunto(s)

Proteínas Bacterianas/química , Proteínas de Transporte de Catión/química , Nucleótidos/química , Conformación Proteica , Adenosina Trifosfato/química , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Sitios de Unión/genética , Calcio/metabolismo , Proteínas de Transporte de Catión/genética , Proteínas de Transporte de Catión/ultraestructura , Cationes/química , Cristalografía por Rayos X , Canal de Potasio Kv1.6/química , Canal de Potasio Kv1.6/ultraestructura , Nucleótidos/genética , Potasio/química , Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio/ultraestructura , Dominios Proteicos/genética , Estructura Terciaria de Proteína , Proteínas Ribosómicas

RESUMEN

Supplementation of 100% oxygen is a very common intervention in intensive care units (ICU) and critical care centers for patients with dysfunctional lung and lung disorders. Although there is advantage in delivering sufficient levels of oxygen, hyperoxia is reported to be directly associated with increasing in-hospital deaths. Our previous studies reported ventricular and electrical remodeling in hyperoxia treated mouse hearts, and in this article, for the first time, we are investigating the effects of hyperoxia on atrial electrophysiology using whole-cell patch-clamp electrophysiology experiments along with assessment of Kv1.5, Kv4.2, and KChIP2 transcripts and protein profiles using real-time quantitative RT-PCR and Western blotting. Our data showed that induction of hyperoxia for 3 days in mice showed larger outward potassium currents with shorter action potential durations (APD). This increase in current densities is due to significant increase in ultrarapid delayed rectifier outward K+ currents (IKur ) and rapidly activating, rapidly inactivating transient outward K+ current (Ito ) densities. We also observed a significant increase in both transcripts and protein levels of Kv1.5 and KChIP2 in hyperoxia treated atrial cardiomyocytes, whereas no significant change was observed in Kv4.2 transcripts or protein. The data presented here further support our previous findings that hyperoxia induces not only ventricular remodeling, but also atrial electrical remodeling.

Asunto(s)

Proteínas de Interacción con los Canales Kv/genética , Canal de Potasio Kv1.6/genética , Enfermedades Pulmonares/terapia , Oxígeno/efectos adversos , Canales de Potasio Shal/genética , Potenciales de Acción/efectos de los fármacos , Animales , Regulación de la Expresión Génica , Atrios Cardíacos/fisiopatología , Mortalidad Hospitalaria , Humanos , Hiperoxia/etiología , Hiperoxia/fisiopatología , Unidades de Cuidados Intensivos , Pulmón/metabolismo , Pulmón/fisiopatología , Enfermedades Pulmonares/complicaciones , Enfermedades Pulmonares/mortalidad , Enfermedades Pulmonares/fisiopatología , Ratones , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Técnicas de Placa-Clamp , Potasio/metabolismo

RESUMEN

Potassium voltage-gated Kv1.6 channel, which is distributed primarily in neurons of central and peripheral nervous systems, is of significant physiological importance. To date, several high-affinity Kv1.6-channel blockers are known, but the lack of selective ones among them hampers the studies of tissue localization and functioning of Kv1.6 channels. Here we present an approach to advanced understanding of interactions of peptide toxin blockers with a Kv1.6 pore. It combines molecular modeling studies and an application of a new bioengineering system based on a KcsA-Kv1.6 hybrid channel for the quantitative fluorescent analysis of blocker-channel interactions. Using this system we demonstrate that peptide toxins agitoxin 2, kaliotoxin1 and OSK1 have similar high affinity to the extracellular vestibule of the K+-conducting pore of Kv1.6, hetlaxin is a low-affinity ligand, whereas margatoxin and scyllatoxin do not bind to Kv1.6 pore. Binding of toxins to Kv1.6 pore has considerable inverse dependence on the ionic strength. Model structures of KcsA-Kv1.6 and Kv1.6 complexes with agitoxin 2, kaliotoxin 1 and OSK1 were obtained using homology modeling and molecular dynamics simulation. Interaction interfaces, which are formed by 15-19 toxin residues and 10 channel residues, are described and compared. Specific sites of Kv1.6 pore recognition are identified for targeting of peptide blockers. Analysis of interactions between agitoxin 2 derivatives with point mutations (S7K, S11G, L19S, R31G) and KcsA-Kv1.6 confirms reliability of the calculated complex structure.

Asunto(s)

Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Modelos Moleculares , Bloqueadores de los Canales de Potasio/metabolismo , Canales de Potasio/metabolismo , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Canales de Potasio de la Superfamilia Shaker/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Relación Dosis-Respuesta a Droga , Humanos , Canal de Potasio Kv1.6 , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/química , Canales de Potasio/genética , Unión Proteica/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Canales de Potasio de la Superfamilia Shaker/química , Canales de Potasio de la Superfamilia Shaker/genética

RESUMEN

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides µ-SIIIA and µ-PIIIA, thus, are not specific for NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.

Asunto(s)

Conotoxinas/química , Canal de Potasio Kv.1.1/antagonistas & inhibidores , Canal de Potasio Kv.1.2/antagonistas & inhibidores , Canal de Potasio Kv1.4/antagonistas & inhibidores , Canal de Potasio Kv1.6/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/química , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Electrofisiología , Células HEK293 , Humanos , Neuronas/metabolismo , Péptidos/química , Dominios Proteicos

RESUMEN

Scorpion toxins that block potassium channels and antimicrobial plant defensins share a common structural CSαß-motif. These toxins contain a toxin signature (K-C4-X-N) in their amino acid sequence, and based on in silico analysis of 18 plant defensin sequences, we noted the presence of a toxin signature (K-C5-R-G) in the amino acid sequence of the Arabidopsis thaliana defensin AtPDF2.3. We found that recombinant (r)AtPDF2.3 blocks Kv1.2 and Kv1.6 potassium channels, akin to the interaction between scorpion toxins and potassium channels. Moreover, rAtPDF2.3[G36N], a variant with a KCXN toxin signature (K-C5-R-N), is more potent in blocking Kv1.2 and Kv1.6 channels than rAtPDF2.3, whereas rAtPDF2.3[K33A], devoid of the toxin signature, is characterized by reduced Kv channel blocking activity. These findings highlight the importance of the KCXN scorpion toxin signature in the plant defensin sequence for blocking potassium channels. In addition, we found that rAtPDF2.3 inhibits the growth of Saccharomyces cerevisiae and that pathways regulating potassium transport and/or homeostasis confer tolerance of this yeast to rAtPDF2.3, indicating a role for potassium homeostasis in the fungal defence response towards rAtPDF2.3. Nevertheless, no differences in antifungal potency were observed between the rAtPDF2.3 variants, suggesting that antifungal activity and Kv channel inhibitory function are not linked.

Asunto(s)

Antifúngicos/farmacología , Proteínas de Arabidopsis/farmacología , Proteínas de Homeodominio/farmacología , Canal de Potasio Kv.1.2/antagonistas & inhibidores , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de la Superfamilia Shaker/antagonistas & inhibidores , Antifúngicos/química , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Canal de Potasio Kv.1.2/genética , Canal de Potasio Kv.1.2/metabolismo , Canal de Potasio Kv1.6 , Bloqueadores de los Canales de Potasio/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Canales de Potasio de la Superfamilia Shaker/genética , Canales de Potasio de la Superfamilia Shaker/metabolismo

RESUMEN

Cholinergic regulation of arterial luminal diameter involves intricate network of intercellular communication between the endothelial and smooth muscle cells that is highly dependent on the molecular mediators released by the endothelium. Albeit the well-recognized contribution of nitric oxide (NO) towards vasodilation, the identity of compensatory mechanisms that maintain vasomotor tone when NO synthesis is deranged remain largely unknown in the ophthalmic artery. This is the first study to identify the vasodilatory signalling mechanisms of the ophthalmic artery employing wild type mice. Acetylcholine (ACh)-induced vasodilation was only partially attenuated when NO synthesis was inhibited. Intriguingly, the combined blocking of cytochrome P450 oxygenase (CYP450) and lipoxygenase (LOX), as well as CYP450 and gap junctions, abolished vasodilation; demonstrating that the key compensatory mechanisms comprise arachidonic acid metabolites which, work in concert with gap junctions for downstream signal transmission. Furthermore, the voltage-gated potassium ion channel, Kv1.6, was functionally relevant in mediating vasodilation. Its localization was found exclusively in the smooth muscle. In conclusion, ACh-induced vasodilation of mouse ophthalmic artery is mediated in part by NO and predominantly via arachidonic acid metabolites, with active involvement of gap junctions. Particularly, the Kv1.6 channel represents an attractive therapeutic target in ophthalmopathologies when NO synthesis is compromised.

Asunto(s)

Arteria Oftálmica/fisiología , Acetilcolina/farmacología , Animales , Factores Biológicos/metabolismo , Colinérgicos/farmacología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/metabolismo , Epoprostenol/farmacología , Canal de Potasio Kv1.6/metabolismo , Ratones , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Arteria Oftálmica/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Vasodilatación/efectos de los fármacos

RESUMEN

Clinical use of non-steroidal anti-inflammatory drugs (NSAIDs) is well known to cause gastrointestinal ulcer formation via several mechanisms that include inhibiting epithelial cell migration and mucosal restitution. The drug-affected signaling pathways that contribute to inhibition of migration by NSAIDs are poorly understood, though previous studies have shown that NSAIDs depolarize membrane potential and suppress expression of calpain proteases and voltage-gated potassium (Kv) channel subunits. Kv channels play significant roles in cell migration and are targets of NSAID activity in white blood cells, but the specific functional effects of NSAID-induced changes in Kv channel expression, particularly on cell migration, are unknown in intestinal epithelial cells. Accordingly, we investigated the effects of NSAIDs on expression of Kv1.3, 1.4, and 1.6 in vitro and/or in vivo and evaluated the functional significance of loss of Kv subunit expression. Indomethacin or NS-398 reduced total and plasma membrane protein expression of Kv1.3 in cultured intestinal epithelial cells (IEC-6). Additionally, depolarization of membrane potential with margatoxin (MgTx), 40mM K(+), or silencing of Kv channel expression with siRNA significantly reduced IEC-6 cell migration and disrupted calpain activity. Furthermore, in rat small intestinal epithelia, indomethacin and NS-398 had significant, yet distinct, effects on gene and protein expression of Kv1.3, 1.4, or 1.6, suggesting that these may be clinically relevant targets. Our results show that inhibition of epithelial cell migration by NSAIDs is associated with decreased expression of Kv channel subunits, and provide a mechanism through which NSAIDs inhibit cell migration and may contribute to NSAID-induced gastrointestinal (GI) toxicity.

Asunto(s)

Antiinflamatorios no Esteroideos/farmacología , Calpaína/antagonistas & inhibidores , Movimiento Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Animales , Calpaína/metabolismo , Línea Celular , Movimiento Celular/fisiología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.4/antagonistas & inhibidores , Canal de Potasio Kv1.4/metabolismo , Canal de Potasio Kv1.6/antagonistas & inhibidores , Canal de Potasio Kv1.6/metabolismo , Potenciales de la Membrana/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Ratas , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología

RESUMEN

The functions of voltage-gated potassium (Kv) channels in neurons have been well defined, whereas their roles in glial cells are not fully understood. Kv1.1, Kv1.3 and Kv1.6 are endogenously expressed in C6 astrocytoma cells, but their trafficking and subcellular localization have not been well studied. In C6 cells, Kv1.1 was localized to the cell surface, Kv1.3 was predominantly localized in the cis-Golgi, and Kv1.6 was enriched in the endoplasmic reticulum. Disruption of the Golgi stacks with brefeldin A treatment redirected Kv1.3 to the endoplasmic reticulum, further confirming that Kv1.3 was localized in the Golgi. Denaturing and reducing immunoblot analysis identified an expected Kv1.3 monomer and an unexpected Kv1.3 dimer/aggregate. These two forms had different protein half-lives: that of the monomer form T1/2 was 5.1 h, whereas the dimer/aggregate form was stable over the 8-h measurement period. The Kv1.3 dimer/aggregate form on immunoblots appeared to be correlated with its Golgi retention, based on examination with several cell types that expressed Kv1.3. Glycosidase treatment showed that Kv1.3 contained complex-type N-glycans terminated with sialic acids, suggesting that Kv1.3 had traveled to the trans-Golgi network for sialylation before it was recycled to the cis-Golgi for retention. Inhibition of N-glycosylation did not affect Kv1.3 localization, indicating that N-glycans did not play a role in its Golgi retention. Thus, Kv1.3 appears to be distributed to the cis-Golgi membrane of rat astrocytes in a similar way as a Golgi resident protein, and this unusual distribution appears to be correlated with its SDS/2-mercaptoethanol-resistant dimer/aggregate forms on immunoblots.

Asunto(s)

Astrocitos/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Canal de Potasio Kv1.3/metabolismo , Canal de Potasio Kv1.6/metabolismo , Animales , Línea Celular Tumoral , Glicosilación , Humanos , Membranas Intracelulares/metabolismo , Células MCF-7 , Isoformas de Proteínas/metabolismo , Multimerización de Proteína , Procesamiento Proteico-Postraduccional , Estabilidad Proteica , Transporte de Proteínas , Ratas

RESUMEN

BACKGROUND: LEF1/TCF transcription factors and their activator ß-catenin are effectors of the canonical Wnt pathway. Although Wnt/ß-catenin signaling has been implicated in neurodegenerative and psychiatric disorders, its possible role in the adult brain remains enigmatic. To address this issue, we sought to identify the genetic program activated by ß-catenin in neurons. We recently showed that ß-catenin accumulates specifically in thalamic neurons where it activates Cacna1g gene expression. In the present study, we combined bioinformatics and experimental approaches to find new ß-catenin targets in the adult thalamus. RESULTS: We first selected the genes with at least two conserved LEF/TCF motifs within the regulatory elements. The resulting list of 428 putative LEF1/TCF targets was significantly enriched in known Wnt targets, validating our approach. Functional annotation of the presumed targets also revealed a group of 41 genes, heretofore not associated with Wnt pathway activity, that encode proteins involved in neuronal signal transmission. Using custom polymerase chain reaction arrays, we profiled the expression of these genes in the rat forebrain. We found that nine of the analyzed genes were highly expressed in the thalamus compared with the cortex and hippocampus. Removal of nuclear ß-catenin from thalamic neurons in vitro by introducing its negative regulator Axin2 reduced the expression of six of the nine genes. Immunoprecipitation of chromatin from the brain tissues confirmed the interaction between ß-catenin and some of the predicted LEF1/TCF motifs. The results of these experiments validated four genes as authentic and direct targets of ß-catenin: Gabra3 for the receptor of GABA neurotransmitter, Calb2 for the Ca(2+)-binding protein calretinin, and the Cacna1g and Kcna6 genes for voltage-gated ion channels. Two other genes from the latter cluster, Cacna2d2 and Kcnh8, appeared to be regulated by ß-catenin, although the binding of ß-catenin to the regulatory sequences of these genes could not be confirmed. CONCLUSIONS: In the thalamus, ß-catenin regulates the expression of a novel group of genes that encode proteins involved in neuronal excitation. This implies that the transcriptional activity of ß-catenin is necessary for the proper excitability of thalamic neurons, may influence activity in the thalamocortical circuit, and may contribute to thalamic pathologies.

Asunto(s)

Canales de Calcio Tipo T/genética , Canal de Potasio Kv1.6/genética , Neuronas/metabolismo , Receptores de GABA-A/genética , Proteína G de Unión al Calcio S100/genética , Tálamo/metabolismo , beta Catenina/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Sitios de Unión , Calbindina 2 , Canales de Calcio Tipo T/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Hipocampo/citología , Hipocampo/metabolismo , Canal de Potasio Kv1.6/metabolismo , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Masculino , Neuronas/citología , Neurotransmisores , Cultivo Primario de Células , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Receptores de GABA-A/metabolismo , Proteína G de Unión al Calcio S100/metabolismo , Transducción de Señal , Tálamo/citología , Activación Transcripcional , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , beta Catenina/metabolismo

RESUMEN

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 µM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.

Asunto(s)

Péptidos/genética , Péptidos/aislamiento & purificación , Péptidos/farmacología , Venenos de Araña/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Canal de Potasio Kv.1.1/metabolismo , Canal de Potasio Kv1.6/metabolismo , Datos de Secuencia Molecular , Mutación , Oocitos/efectos de los fármacos , Oocitos/fisiología , Técnicas de Placa-Clamp , Péptidos/metabolismo , Filogenia , Canales de Potasio con Entrada de Voltaje/metabolismo , Estructura Secundaria de Proteína , Escorpiones/química , Homología de Secuencia de Aminoácido , Proteínas de Xenopus/metabolismo , Xenopus laevis

RESUMEN

KCNA1, KCNA2, KCNA6 and KCNQ2 are associated with peripheral nerve hyperexcitability in humans. In order to determine if these genes are also involved in Jack Russell Terriers with a similar syndrome characterized by myokymia and neuromyotonia, their predicted canine orthologs were first validated experimentally. They were found either incompletely or even incorrectly annotated, mainly due to gaps in the canine genomic sequence and insufficient transcript data. Canine KCNQ2 was found to contain 20 coding exons, of which three are not described in humans. It encodes for at least 14 different transcript variants in the frontal cortex of a single dog, of which only four are also described in humans. Mutation detection in Jack Russell Terriers diagnosed with peripheral nerve hyperexcitability revealed no pathogenetic relevant structural mutations. However, the four missense sequence variations and the 14 transcript variants of KCNQ2 will contribute to the study of the functional diversity of voltage-gated potassium channels.

Asunto(s)

Enfermedades de los Perros/genética , Canal de Potasio KCNQ2/genética , Canal de Potasio Kv.1.1/genética , Canal de Potasio Kv.1.2/genética , Canal de Potasio Kv1.6/genética , Enfermedades del Sistema Nervioso Periférico/veterinaria , Animales , Perros , Estudios de Asociación Genética , Síndrome de Isaacs/genética , Síndrome de Isaacs/veterinaria , Mutación , Miocimia/genética , Miocimia/veterinaria , Enfermedades del Sistema Nervioso Periférico/genética