RESUMEN
Novel KCNMA1 variants, encoding the BK K+ channel, are associated with a debilitating dyskinesia and epilepsy syndrome. Neurodevelopmental delay, cognitive disability, and brain and structural malformations are also diagnosed at lower incidence. More than half of affected individuals present with a rare negative episodic motor disorder, paroxysmal nonkinesigenic dyskinesia (PNKD3). The mechanistic relationship of PNKD3 to epilepsy and the broader spectrum of KCNMA1-associated symptomology is unknown. This review summarizes patient-associated KCNMA1 variants within the BK channel structure, functional classifications, genotype-phenotype associations, disease models, and treatment. Patient and transgenic animal data suggest delineation of gain-of-function (GOF) and loss-of-function KCNMA1 neurogenetic disease, validating two heterozygous alleles encoding GOF BK channels (D434G and N999S) as causing seizure and PNKD3. This discovery led to a variant-defined therapeutic approach for PNKD3, providing initial insight into the neurological basis. A comprehensive clinical definition of monogenic KCNMA1-linked disease and the neuronal mechanisms currently remain priorities for continued investigation.
Asunto(s)
Canalopatías , Corea , Epilepsia , Animales , Humanos , Canales de Potasio de Gran Conductancia Activados por el Calcio , Canalopatías/genética , Epilepsia/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genéticaRESUMEN
Large conductance potassium (BK) channels are among the most sensitive molecular targets of ethanol and genetic variations in the channel-forming α subunit have been nominally associated with alcohol use disorders. However, whether the action of ethanol at BK α influences the motivation to drink alcohol remains to be determined. To address this question, we first tested the effect of systemically administered BK channel modulators on voluntary alcohol consumption in C57BL/6J males. Penitrem A (blocker) exerted dose-dependent effects on moderate alcohol intake, while paxilline (blocker) and BMS-204352 (opener) were ineffective. Because pharmacological manipulations are inherently limited by non-specific effects, we then sought to investigate the behavioral relevance of ethanol's direct interaction with BK α by introducing in the mouse genome a point mutation known to render BK channels insensitive to ethanol while preserving their physiological function. The BK α K361N substitution prevented ethanol from reducing spike threshold in medial habenula neurons. However, it did not alter acute responses to ethanol in vivo, including ataxia, sedation, hypothermia, analgesia, and conditioned place preference. Furthermore, the mutation did not have reproducible effects on alcohol consumption in limited, continuous, or intermittent access home cage two-bottle choice paradigms conducted in both males and females. Notably, in contrast to previous observations made in mice missing BK channel auxiliary ß subunits, the BK α K361N substitution had no significant impact on ethanol intake escalation induced by chronic intermittent alcohol vapor inhalation. It also did not affect the metabolic and locomotor consequences of chronic alcohol exposure. Altogether, these data suggest that the direct interaction of ethanol with BK α does not mediate the alcohol-related phenotypes examined here in mice.
Asunto(s)
Consumo de Bebidas Alcohólicas , Etanol , Ratones Endogámicos C57BL , Animales , Etanol/farmacología , Masculino , Ratones , Consumo de Bebidas Alcohólicas/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Conducta Animal/efectos de los fármacos , FemeninoRESUMEN
Voltage-gated potassium channels are critical in modulating cellular excitability, with Slo (slowpoke) channels forming a unique family characterized by their large conductance and dual regulation by electrical signals and intracellular messengers. Despite their structural and evolutionary similarities, Slo1 and Slo3 channels exhibit significant differences in their voltage-gating properties. This study investigates the molecular determinants that differentiate the voltage-gating properties of human Slo1 and mouse Slo3 channels. Utilizing Slo1/Slo3 chimeras, we pinpointed the selectivity filter region as a key factor in the Slo3 channel's reduced conductance at negative voltages. The S6 transmembrane (TM) segment was identified as pivotal for the Slo3 channel's biphasic deactivation kinetics at these voltages. Additionally, the S4 and S6 TM segments were found to be responsible for the gradual slope in the Slo3 channel's conductance-voltage relationship, while multiple TM regions appear to be involved in the Slo3 channel's requirement of strong depolarization for activation. Mutations in the Slo1's S5 and S6 TM segments revealed three residues (I233, L302, and M304) that likely play a crucial role in the allosteric coupling between the voltage sensors and the pore gate.
Asunto(s)
Activación del Canal Iónico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Animales , Humanos , Ratones , Secuencia de Aminoácidos , Membrana Celular/metabolismo , Cinética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Potenciales de la Membrana , Mutación , Dominios ProteicosRESUMEN
Malignant hyperthermia susceptibility (MHS) is an autosomal dominant pharmacogenetic disorder that manifests as a hypermetabolic state when carriers are exposed to halogenated volatile anesthetics or depolarizing muscle relaxants. In animals, heat stress intolerance is also observed. MHS is linked to over 40 variants in RYR1 that are classified as pathogenic for diagnostic purposes. More recently, a few rare variants linked to the MHS phenotype have been reported in CACNA1S, which encodes the voltage-activated Ca2+ channel CaV1.1 that conformationally couples to RyR1 in skeletal muscle. Here, we describe a knock-in mouse line that expresses one of these putative variants, CaV1.1-R174W. Heterozygous (HET) and homozygous (HOM) CaV1.1-R174W mice survive to adulthood without overt phenotype but fail to trigger with fulminant malignant hyperthermia when exposed to halothane or moderate heat stress. All three genotypes (WT, HET, and HOM) express similar levels of CaV1.1 by quantitative PCR, Western blot, [3H]PN200-110 receptor binding and immobilization-resistant charge movement densities in flexor digitorum brevis fibers. Although HOM fibers have negligible CaV1.1 current amplitudes, HET fibers have similar amplitudes to WT, suggesting a preferential accumulation of the CaV1.1-WT protein at triad junctions in HET animals. Never-the-less both HET and HOM have slightly elevated resting free Ca2+ and Na+ measured with double barreled microelectrode in vastus lateralis that is disproportional to upregulation of transient receptor potential canonical (TRPC) 3 and TRPC6 in skeletal muscle. CaV1.1-R174W and upregulation of TRPC3/6 alone are insufficient to trigger fulminant malignant hyperthermia response to halothane and/or heat stress in HET and HOM mice.
Asunto(s)
Halotano , Respuesta al Choque Térmico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Hipertermia Maligna , Animales , Ratones , Calcio/metabolismo , Halotano/farmacología , Respuesta al Choque Térmico/genética , Hipertermia Maligna/genética , Hipertermia Maligna/metabolismo , Hipertermia Maligna/patología , Músculo Esquelético/metabolismo , Mutación , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genéticaRESUMEN
Fast growing solid tumors are frequently surrounded by an acidic microenvironment. Tumor cells employ a variety of mechanisms to survive and proliferate under these harsh conditions. In that regard, acid-sensitive membrane receptors constitute a particularly interesting target, since they can affect cellular functions through ion flow and second messenger cascades. Our knowledge of these processes remains sparse, however, especially regarding medulloblastoma, the most common pediatric CNS malignancy. In this study, using RT-qPCR, whole-cell patch clamp, and Ca2+-imaging, we uncovered several ion channels and a G protein-coupled receptor, which were regulated directly or indirectly by low extracellular pH in DAOY and UW228 medulloblastoma cells. Acidification directly activated acid-sensing ion channel 1a (ASIC1a), the proton-activated Cl- channel (PAC, ASOR, or TMEM206), and the proton-activated G protein-coupled receptor OGR1. The resulting Ca2+ signal secondarily activated the large conductance calcium-activated potassium channel (BKCa). Our analyses uncover a complex relationship of these transmembrane proteins in DAOY cells that resulted in cell volume changes and induced cell death under strongly acidic conditions. Collectively, our results suggest that these ion channels in concert with OGR1 may shape the growth and evolution of medulloblastoma cells in their acidic microenvironment.
Asunto(s)
Canales Iónicos Sensibles al Ácido , Meduloblastoma , Receptores Acoplados a Proteínas G , Humanos , Canales Iónicos Sensibles al Ácido/metabolismo , Canales Iónicos Sensibles al Ácido/genética , Meduloblastoma/metabolismo , Meduloblastoma/patología , Línea Celular Tumoral , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Concentración de Iones de Hidrógeno , Tamaño de la Célula , Muerte Celular , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Calcio/metabolismo , Neoplasias Cerebelosas/metabolismo , Neoplasias Cerebelosas/patologíaRESUMEN
The pore-forming α-subunit of the large-conductance K+ (BK) channel is encoded by a single gene, KCNMA1. BK channel-mediated K+ secretion in the kidney is crucial for overall renal K+ homeostasis in both physiological and pathological conditions. BK channels achieve phenotypic diversity by various mechanisms, including substantial exon rearrangements at seven major alternative splicing sites. However, KCNMA1 alternative splicing in the kidney has not been characterized. The present study aims to identify the major splice variants of mouse Kcnma1 in whole kidney and distal nephron segments. We designed primers that specifically cross exons within each alternative splice site of mouse Kcnma1 and performed real-time quantitative RT-PCR (RT-qPCR) to quantify relative abundance of each splice variant. Our data suggest that Kcnma1 splice variants within mouse kidney are less diverse than in the brain. During postnatal kidney development, most Kcnma1 splice variants at site 5 and the COOH terminus increase in abundance over time. Within the kidney, the regulation of Kcnma1 alternative exon splicing within these two sites by dietary K+ loading is both site and sex specific. In microdissected distal tubules, the Kcnma1 alternative splicing profile, as well as its regulation by dietary K+, are distinctly different than in the whole kidney, suggesting segment and/or cell type specificity in Kcnma1 splicing events. Overall, our data provide evidence that Kcnma1 alternative splicing is regulated during postnatal development and may serve as an important adaptive mechanism to dietary K+ loading in mouse kidney.NEW & NOTEWORTHY We identified the major Kcnma1 splice variants that are specifically expressed in the whole mouse kidney or aldosterone-sensitive distal nephron segments. Our data suggest that Kcnma1 alternative splicing is developmentally regulated and subject to changes in dietary K+.
Asunto(s)
Empalme Alternativo , Riñón , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Potasio en la Dieta , Animales , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Potasio en la Dieta/metabolismo , Riñón/metabolismo , Ratones Endogámicos C57BL , Ratones , Masculino , Regulación del Desarrollo de la Expresión Génica , Exones , FemeninoRESUMEN
The large-conductance, voltage-gated, calcium (Ca2+)-activated potassium channel (BKCa) is one of the most abundant potassium channels in the myometrium. Previous work conducted by our group has identified a link between inflammation, BKCa channels and excitability of myometrial smooth muscle cells. Here, we investigate the role of BKCa channels in spontaneous and lipopolysaccharide (LPS)-stimulated uterine contraction to gain a better understanding of the relationship between the BKCa channel and uterine contraction in basal and inflammatory states. Uteri of C57BL/6 J mice on gestational day 18.5 (GD18.5) were obtained and either fixed in formalin or used immediately for tension recording or isolation of primary myocytes for patch-clamp. Paraffin sections were used for immunofluorescenctdetection of BKCa and Toll-like receptor (TLR4). For tension recordings, LPS was administered to determine its effect on uterine contractions. Paxilline, a BKCa inhibitor, was used to dissect the role of BKCa in uterine contraction in basal and inflammatory states. Finally, patch-clamp recordings were performed to investigate the relationship between LPS, the BKCa channel and membrane currents in mouse myometrial smooth muscle cells (mMSMCs). We confirmed the expression of BKCa and TLR4 in the myometrium of GD18.5 mice and found that inhibiting BKCa channels with paxilline suppressed both spontaneous and LPS-stimulated uterine contractions. Furthermore, application of BKCa inhibitors (paxilline or iberiotoxin) after LPS inhibited BKCa channel activity in mMSMCs. Moreover, pretreatment with BKCa inhibitor or the TLR4 inhibitor suppressed LPS-activated BKCa currents. Our study demonstrates that BKCa channels are involved in both basal and LPS-stimulated uterine contraction in pregnant mice.
Asunto(s)
Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Contracción Uterina , Animales , Femenino , Ratones , Embarazo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Lipopolisacáridos/farmacología , Ratones Endogámicos C57BL , Receptor Toll-Like 4/metabolismo , Contracción Uterina/efectos de los fármacos , Contracción Uterina/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismoRESUMEN
BACKGROUND: Many long noncoding RNAs (lncRNAs) with altered expression significantly influence colorectal cancer (CRC) progression and behavior. The functions of many lncRNAs in CRC are not clear yet. This study aimed to discover novel lncRNA entities and comprehensively examine and validate their roles and underlying molecular mechanisms in CRC. METHODS: Tissue samples, both tumourous and non-tumourous, from three CRC patients were submitted for sequencing. Following expression validation in samples from ten patients and four CRC cell lines. The lncRNA KCNMA1-AS2 was synthesized by In-vitro transcription RNA synthesis and the lncRNA was directly transfected into CRC cell lines to overexpress. Functional assays including MTT proliferation assay, Annexin-V/propidium iodide apoptosis assay, wound healing migration assay and cell cycle assays were performed to evaluate the effect of overexpression of KCNMA1-AS2. Furthermore, the binding of KCNMA1-AS2 to miR-1227-5p was confirmed using dual luciferase reporter assays and qPCR analyses. Subsequent bioinformatics analyses identified 58 potential downstream targets of miR-1227-5p across three databases. RESULTS: In this study, we identified the lncRNA KCNMA1-AS2, the expression of which was down-regulated consistently in cancer tissues and CRC cell lines compared to non-cancerous tissues. The overexpression of lncRNA KCNMA1-AS2 led to significant reduction in CRC cell proliferation and migration, increase in cell apoptosis, and more cells arrested in S phase. Additionally, the interaction between KCNMA1-AS2 and miR-1227-5p was confirmed through dual luciferase reporter assay and qPCR analysis. It is also putatively predicted that MTHFR and ST8SIA2 may be linked to CRC based on bioinformatics analyses. CONCLUSIONS: LncRNA KCNMA1-AS2 exhibited distinct gene expression patterns in both CRC tissue and cell lines, impacting various cellular functions while also acting as a sponge for miR-1227-5p.The findings spotlight lncRNA KCNMA1-AS2 as a potential marker for diagnosis and treatment of CRC.
Asunto(s)
Apoptosis , Movimiento Celular , Proliferación Celular , Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , MicroARNs , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/patología , Neoplasias Colorrectales/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Proliferación Celular/genética , Apoptosis/genética , Movimiento Celular/genética , Línea Celular Tumoral , Femenino , Masculino , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Persona de Mediana EdadRESUMEN
Uncontrolled diabetes causes a catabolic state with multi-organic complications, of which impairment on skeletal muscle contributes to the damaged mobility. Kcnma1 gene encodes the pore-forming α-subunit of Ca2+ - and voltage-gated K+ channels of large conductance (BK channels), and loss-of-function mutations in Kcnma1 are in regards to impaired myogenesis. Herein, we observed a time-course reduction of Kcnma1 expression in the tibialis anterior muscles of leptin receptor-deficient (db/db) diabetic mice. To investigate the role of Kcnma1 in diabetic muscle atrophy, muscle-specific knockdown of Kcnma1 was achieved by mice receiving intravenous injection of adeno-associated virus-9 (AAV9)-encoding shRNA against Kcnma1 under the muscle creatine kinase (MCK) promoter. Impairment on muscle mass and myogenesis were observed in m/m mice with AAV9-shKcnma1 intervention, while this impairment was more obvious in diabetic db/db mice. Simultaneously, damaged mitochondrial dynamics and biogenesis showed much severer in db/db mice with AAV9-shKcnma1 intervention. RNA sequencing revealed the large transcriptomic changes resulted by Kcnma1 knockdown, and changes in mitochondrial homeostasis-related genes were validated. Besides, the artificial alteration of Kcnma1 in mouse C2C12 myoblasts was achieved with an adenovirus vector. Consistent results were demonstrated by Kcnma1 knockdown in palmitate-treated cells, whereas opposite results were exhibited by Kcnma1 overexpression. Collectively, we document Kcnma1 as a potential keeper of mitochondrial homeostasis, and the loss of Kcnma1 is a critical event in priming skeletal muscle loss in diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Canales de Potasio de Gran Conductancia Activados por el Calcio , Ratones , Animales , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Músculo Esquelético/metabolismo , HomeostasisRESUMEN
The mechanisms behind renal vasodilatation elicited by stimulation of ß-adrenergic receptors are not clarified. As several classes of K channels are potentially activated, we tested the hypothesis that KV7 and BKCa channels contribute to the decreased renal vascular tone in vivo and in vitro. Changes in renal blood flow (RBF) during ß-adrenergic stimulation were measured in anaesthetized rats using an ultrasonic flow probe. The isometric tension of segmental arteries from normo- and hypertensive rats and segmental arteries from wild-type mice and mice lacking functional KV7.1 channels was examined in a wire-myograph. The ß-adrenergic agonist isoprenaline increased RBF significantly in vivo. Neither activation nor inhibition of KV7 and BKCa channels affected the ß-adrenergic RBF response. In segmental arteries from normo- and hypertensive rats, inhibition of KV7 channels significantly decreased the ß-adrenergic vasorelaxation. However, inhibiting BKCa channels was equally effective in reducing the ß-adrenergic vasorelaxation. The ß-adrenergic vasorelaxation was not different between segmental arteries from wild-type mice and mice lacking KV7.1 channels. As opposed to rats, inhibition of KV7 channels did not affect the murine ß-adrenergic vasorelaxation. Although inhibition and activation of KV7 channels or BKCa channels significantly changed baseline RBF in vivo, none of the treatments affected ß-adrenergic vasodilatation. In isolated segmental arteries, however, inhibition of KV7 and BKCa channels significantly reduced the ß-adrenergic vasorelaxation, indicating that the regulation of RBF in vivo is driven by several actors in order to maintain an adequate RBF. Our data illustrates the challenge in extrapolating results from in vitro to in vivo conditions.
Asunto(s)
Riñón , Vasodilatación , Animales , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiología , Masculino , Ratas , Ratones , Riñón/metabolismo , Riñón/irrigación sanguínea , Canal de Potasio KCNQ1/metabolismo , Isoproterenol/farmacología , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Agonistas Adrenérgicos beta/farmacología , Ratones Noqueados , Receptores Adrenérgicos beta/metabolismo , Circulación Renal/efectos de los fármacos , Circulación Renal/fisiología , Ratones Endogámicos C57BL , Ratas Wistar , Hipertensión/fisiopatología , Hipertensión/metabolismoRESUMEN
BRAF mutations have been found in gliomas which exhibit abnormal electrophysiological activities, implying their potential links with the ion channel functions. In this study, we identified the Drosophila potassium channel, Slowpoke (Slo), the ortholog of human KCNMA1, as a critical factor involved in dRafGOF glioma progression. Slo was upregulated in dRafGOF glioma. Knockdown of slo led to decreases in dRafGOF levels, glioma cell proliferation, and tumor-related phenotypes. Overexpression of slo in glial cells elevated dRaf expression and promoted cell proliferation. Similar mutual regulations of p-BRAF and KCNMA1 levels were then recapitulated in human glioma cells with the BRAF mutation. Elevated p-BRAF and KCNMA1 were also observed in HEK293T cells upon the treatment of 20 mM KCl, which causes membrane depolarization. Knockdown KCNMA1 in these cells led to a further decrease in cell viability. Based on these results, we conclude that the levels of p-BRAF and KCNMA1 are co-dependent and mutually regulated. We propose that, in depolarized glioma cells with BRAF mutations, high KCNMA1 levels act to repolarize membrane potential and facilitate cell growth. Our study provides a new strategy to antagonize the progression of gliomas as induced by BRAF mutations.
Asunto(s)
Glioma , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Proteínas Proto-Oncogénicas B-raf , Animales , Humanos , Drosophila/metabolismo , Glioma/genética , Células HEK293 , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Canales de Potasio/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismoRESUMEN
The tumor suppressor gene F-box and WD repeat domain-containing (FBXW) 7 reduces cancer stemness properties by promoting the protein degradation of pluripotent stem cell markers. We recently demonstrated the transcriptional repression of FBXW7 by the three-dimensional (3D) spheroid formation of several cancer cells. In the present study, we found that the transcriptional activity of FBXW7 was promoted by the inhibition of the Ca2+-activated K+ channel, KCa1.1, in a 3D spheroid model of human prostate cancer LNCaP cells through the Akt-Nrf2 signaling pathway. The transcriptional activity of FBXW7 was reduced by the siRNA-mediated inhibition of the CCAAT-enhancer-binding protein C/EBP δ (CEBPD) after the transfection of miR223 mimics in the LNCaP spheroid model, suggesting the transcriptional regulation of FBXW7 through the Akt-Nrf2-CEBPD-miR223 transcriptional axis in the LNCaP spheroid model. Furthermore, the KCa1.1 inhibition-induced activation of FBXW7 reduced (1) KCa1.1 activity and protein levels in the plasma membrane and (2) the protein level of the cancer stem cell (CSC) markers, c-Myc, which is a molecule degraded by FBXW7, in the LNCaP spheroid model, indicating that KCa1.1 inhibition-induced FBXW7 activation suppressed CSC conversion in KCa1.1-positive cancer cells.
Asunto(s)
Proteína 7 que Contiene Repeticiones F-Box-WD , Regulación Neoplásica de la Expresión Génica , Factor 2 Relacionado con NF-E2 , Neoplasias de la Próstata , Transducción de Señal , Esferoides Celulares , Humanos , Proteína 7 que Contiene Repeticiones F-Box-WD/metabolismo , Proteína 7 que Contiene Repeticiones F-Box-WD/genética , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Esferoides Celulares/metabolismo , Línea Celular Tumoral , Regulación hacia Arriba , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
BACKGROUND: Large conductance calcium-activated potassium (BKCa) channels have been implicated in the neurobiological underpinnings of migraine. Considering the clinical similarities between migraine and persistent post-traumatic headache (PPTH), we aimed to examine whether MaxiPost (a BKCa channel opener) could induce migraine-like headache in persons with PPTH. METHODS: This is a randomized double-blind, placebo-controlled, two-way crossover study from September 2023 to December 2023. Eligible participants were adults with PPTH after mild traumatic brain injury who reported having no personal history of migraine. The randomized participants received a single dose of either MaxiPost (0.05 mg/min) or placebo (isotonic saline) that was infused intravenously over 20 minutes. The two experiment sessions were scheduled at least one week apart to avoid potential carryover effects. The primary endpoint was the induction of migraine-like headache after MaxiPost as compared to placebo within 12 hours of drug administration. The secondary endpoint was the area under the curve (AUC) values for headache intensity scores between MaxiPost and placebo over the same 12-hour observation period. RESULTS: Twenty-one adult participants (comprising 14 females and 7 males) with PPTH were enrolled and completed both experiment sessions. The proportion of participants who developed migraine-like headache was 11 (52%) of 21 participants after MaxiPost infusion, in contrast to four (19%) participants following placebo (P = .02). Furthermore, the median headache intensity scores, represented by AUC values, were higher following MaxiPost than after placebo (P < .001). CONCLUSIONS: Our results indicate that BKCa channel opening can elicit migraine-like headache in persons with PPTH. Thus, pharmacologic blockade of BKCa channels might present a novel avenue for drug discovery. Additional investigations are nonetheless needed to confirm these insights and explore the therapeutic prospects of BKCa channel blockers in managing PPTH. GOV IDENTIFIER: NCT05378074.
Asunto(s)
Estudios Cruzados , Cefalea Postraumática , Humanos , Femenino , Masculino , Adulto , Método Doble Ciego , Cefalea Postraumática/tratamiento farmacológico , Cefalea Postraumática/etiología , Trastornos Migrañosos/tratamiento farmacológico , Persona de Mediana Edad , Conmoción Encefálica/complicaciones , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Adulto Joven , Canales de Potasio de Gran Conductancia Activados por el CalcioRESUMEN
Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.
Asunto(s)
Antiarrítmicos , Péptidos y Proteínas de Señalización Intercelular , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Neurotoxinas , Péptidos , Venenos de Araña , Animales , Antiarrítmicos/química , Antiarrítmicos/farmacología , Antiarrítmicos/uso terapéutico , Fibrilación Atrial/tratamiento farmacológico , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/farmacología , Péptidos y Proteínas de Señalización Intercelular/uso terapéutico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Lípidos , Ratones , Neurotoxinas/química , Neurotoxinas/farmacología , Péptidos/química , Péptidos/farmacología , Venenos de Araña/química , Venenos de Araña/farmacología , Venenos de Araña/uso terapéuticoRESUMEN
Human BK channels are large voltage and Ca2+-activated K+ channels, involved in several important functions within the body. The core channel is a tetramer of α subunits, and its function is modulated by the presence of ß and γ accessory subunits. BK channels composed of α subunits, as well as BK channels composed of α and ß1 subunits, were successfully solubilised from HEK cells with styrene maleic acid (SMA) polymer and purified by nickel affinity chromatography. Native SMA-PAGE analysis of the purified proteins showed the α subunits were extracted as a tetramer. In the presence of ß1 subunits, they were co-extracted with the α subunits as a heteromeric complex. Purified SMA lipid particles (SMALPs) containing BK channel could be inserted into planar lipid bilayers (PLB) and single channel currents recorded, showing a high conductance (≈260â pS), as expected. The open probability was increased in the presence of co-purified ß1 subunits. However, voltage-dependent gating of the channel was restricted. In conclusion, we have demonstrated that SMA can be used to effectively extract and purify large, complex, human ion channels, from low expressing sources. That these large channels can be incorporated into PLB from SMALPs and display voltage-dependent channel activity. However, the SMA appears to reduce the voltage dependent gating of the channels.
Asunto(s)
Activación del Canal Iónico , Canales de Potasio de Gran Conductancia Activados por el Calcio , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismoRESUMEN
Ca2+/voltage-gated, large conductance K+ channels (BKCa) are formed by homotetrameric association of α (slo1) subunits. Their activity, however, is suited to tissue-specific physiology largely due to their association with regulatory subunits (ß and γ types), chaperone proteins, localized signaling, and the channel's lipid microenvironment. PIP2 and cholesterol can modulate BKCa activity independently of downstream signaling, yet activating Ca2+i levels and regulatory subunits control ligand action. At physiological Ca2+i and voltages, cholesterol and PIP2 reduce and increase slo1 channel activity, respectively. Moreover, slo1 proteins provide sites that seem to recognize cholesterol and PIP2: seven CRAC motifs in the slo1 cytosolic tail and a string of positively charged residues (Arg329, Lys330, Lys331) immediately after S6, respectively. A model that could explain the modulation of BKCa activity by cholesterol and/or PIP2 is hypothesized. The roles of additional sites, whether in slo1 or BKCa regulatory subunits, for PIP2 and/or cholesterol to modulate BKCa function are also discussed.
Asunto(s)
Activación del Canal Iónico , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Citosol/metabolismo , Activación del Canal Iónico/fisiología , Transducción de Señal , Colesterol/metabolismo , Canales de Potasio de Gran Conductancia Activados por el Calcio/químicaRESUMEN
Large-conductance Ca2+ and voltage-activated K+ (BK) channels control membrane excitability in many cell types. BK channels are tetrameric. Each subunit is composed of a voltage sensor domain (VSD), a central pore-gate domain, and a large cytoplasmic domain (CTD) that contains the Ca2+ sensors. While it is known that BK channels are activated by voltage and Ca2+, and that voltage and Ca2+ activations interact, less is known about the mechanisms involved. We explore here these mechanisms by examining the gating contribution of an interface formed between the VSDs and the αB helices located at the top of the CTDs. Proline mutations in the αB helix greatly decreased voltage activation while having negligible effects on gating currents. Analysis with the Horrigan, Cui, and Aldrich model indicated a decreased coupling between voltage sensors and pore gate. Proline mutations decreased Ca2+ activation for both Ca2+ bowl and RCK1 Ca2+ sites, suggesting that both high-affinity Ca2+ sites transduce their effect, at least in part, through the αB helix. Mg2+ activation also decreased. The crystal structure of the CTD with proline mutation L390P showed a flattening of the first helical turn in the αB helix compared to wild type, without other notable differences in the CTD, indicating that structural changes from the mutation were confined to the αB helix. These findings indicate that an intact αB helix/VSD interface is required for effective coupling of Ca2+ binding and voltage depolarization to pore opening and that shared Ca2+ and voltage transduction pathways involving the αB helix may be involved.
Asunto(s)
Calcio/metabolismo , Activación del Canal Iónico/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Dominios Proteicos/genética , Regulación Alostérica , Animales , Cationes Bivalentes/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/ultraestructura , Potenciales de la Membrana , Mutagénesis Sitio-Dirigida , Oocitos , Técnicas de Placa-Clamp , Prolina/genética , Conformación Proteica en Hélice alfa/genética , Relación Estructura-Actividad , Xenopus laevisRESUMEN
The tremorgenic fungal alkaloid paxilline (PAX) is a commonly used specific inhibitor of the large-conductance, voltage- and Ca2+-dependent BK-type K+ channel. PAX inhibits BK channels by selective interaction with closed states. BK inhibition by PAX is best characterized by the idea that PAX gains access to the channel through the central cavity of the BK channel, and that only a single PAX molecule can interact with the BK channel at a time. The notion that PAX reaches its binding site via the central cavity and involves only a single PAX molecule would be consistent with binding on the axis of the permeation pathway, similar to classical open channel block and inconsistent with the observation that PAX selectively inhibits closed channels. To explore the potential sites of interaction of PAX with the BK channel, we undertook a computational analysis of the interaction of PAX with the BK channel pore gate domain guided by recently available liganded (open) and metal-free (closed) Aplysia BK channel structures. The analysis unambiguously identified a preferred position of PAX occupancy that accounts for all previously described features of PAX inhibition, including state dependence, G311 sensitivity, stoichiometry, and central cavity accessibility. This PAX-binding pose in closed BK channels is supported by additional functional results.
Asunto(s)
Indoles/antagonistas & inhibidores , Indoles/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/química , Canales de Potasio de Gran Conductancia Activados por el Calcio/efectos de los fármacos , Animales , Sitios de Unión , Activación del Canal Iónico/efectos de los fármacos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/química , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/efectos de los fármacos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Ratones , Simulación del Acoplamiento Molecular , Conformación Proteica , Dominios ProteicosRESUMEN
Despite a growing number of ion channel genes implicated in hereditary ataxia, it remains unclear how ion channel mutations lead to loss-of-function or death of cerebellar neurons. Mutations in the gene KCNMA1, encoding the α-subunit of the BK channel have emerged as responsible for a variety of neurological phenotypes. We describe a mutation (BKG354S) in KCNMA1, in a child with congenital and progressive cerebellar ataxia with cognitive impairment. The mutation in the BK channel selectivity filter dramatically reduced single-channel conductance and ion selectivity. The BKG354S channel trafficked normally to plasma, nuclear, and mitochondrial membranes, but caused reduced neurite outgrowth, cell viability, and mitochondrial content. Small interfering RNA (siRNA) knockdown of endogenous BK channels had similar effects. The BK activator, NS1619, rescued BKG354S cells but not siRNA-treated cells, by selectively blocking the mutant channels. When expressed in cerebellum via adenoassociated virus (AAV) viral transfection in mice, the mutant BKG354S channel, but not the BKWT channel, caused progressive impairment of several gait parameters consistent with cerebellar dysfunction from 40- to 80-d-old mice. Finally, treatment of the patient with chlorzoxazone, a BK/SK channel activator, partially improved motor function, but ataxia continued to progress. These studies indicate that a loss-of-function BK channel mutation causes ataxia and acts by reducing mitochondrial and subsequently cellular viability.
Asunto(s)
Cerebelo/patología , Clorzoxazona/administración & dosificación , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/genética , Mitocondrias/patología , Degeneraciones Espinocerebelosas/genética , Adolescente , Animales , Animales Recién Nacidos , Línea Celular , Cerebelo/citología , Análisis Mutacional de ADN , Dependovirus/genética , Modelos Animales de Enfermedad , Femenino , Técnicas de Silenciamiento del Gen , Vectores Genéticos/genética , Humanos , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/antagonistas & inhibidores , Subunidades alfa de los Canales de Potasio de Gran Conductancia Activados por Calcio/metabolismo , Mutación con Pérdida de Función , Ratones , Oocitos , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Degeneraciones Espinocerebelosas/diagnóstico , Degeneraciones Espinocerebelosas/tratamiento farmacológico , Degeneraciones Espinocerebelosas/patología , Transfección , Secuenciación del Exoma , XenopusRESUMEN
Melatonin (Mel) promotes sleep through G protein-coupled receptors. However, the downstream molecular target(s) is unknown. We identified the Caenorhabditis elegans BK channel SLO-1 as a molecular target of the Mel receptor PCDR-1-. Knockout of pcdr-1, slo-1, or homt-1 (a gene required for Mel synthesis) causes substantially increased neurotransmitter release and shortened sleep duration, and these effects are nonadditive in double knockouts. Exogenous Mel inhibits neurotransmitter release and promotes sleep in wild-type (WT) but not pcdr-1 and slo-1 mutants. In a heterologous expression system, Mel activates the human BK channel (hSlo1) in a membrane-delimited manner in the presence of the Mel receptor MT1 but not MT2 A peptide acting to release free Gßγ also activates hSlo1 in a MT1-dependent and membrane-delimited manner, whereas a Gßλ inhibitor abolishes the stimulating effect of Mel. Our results suggest that Mel promotes sleep by activating the BK channel through a specific Mel receptor and Gßλ.