RESUMO
The Ca2+ and ADP ribose (ADPR)-activated cation channel TRPM2 is the closest homolog of the cold sensor TRPM8 but serves as a deep-brain warmth sensor. To unravel the molecular mechanism of heat sensing by the TRPM2 protein, we study here temperature dependence of TRPM2 currents in cell-free membrane patches across ranges of agonist concentrations. We find that channel gating remains strictly agonist-dependent even at 40°C: heating alone or in combination with just Ca2+, just ADPR, Ca2+ + cyclic ADPR, or H2O2 pretreatment only marginally activates TRPM2. For fully liganded TRPM2, pore opening is intrinsically endothermic, due to ~10-fold larger activation enthalpy for opening (~200 kJ/mol) than for closure (~20 kJ/mol). However, the temperature threshold is too high (>40°C) for unliganded but too low (<15°C) for fully liganded channels. Thus, warmth sensitivity around 37°C is restricted to narrow ranges of agonist concentrations. For ADPR, that range matches, but for Ca2+, it exceeds bulk cytosolic values. The supraphysiological [Ca2+] needed for TRPM2 warmth sensitivity is provided by Ca2+ entering through the channel's pore. That positive feedback provides further strong amplification to the TRPM2 temperature response (Q10 ~ 1,000), enabling the TRPM2 protein to autonomously respond to tiny temperature fluctuations around 37°C. These functional data together with published structures suggest a molecular mechanism for opposite temperature dependences of two closely related channel proteins.
Assuntos
Canais de Cátion TRPM , Canais de Cátion TRPM/metabolismo , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Cálcio/metabolismo , Adenosina Difosfato Ribose/metabolismoRESUMO
The use of fixed fibroblasts from familial and sporadic Alzheimer's disease patients has previously indicated an upregulation of mitochondria-ER contacts (MERCs) as a hallmark of Alzheimer's disease. Despite its potential significance, the relevance of these results is limited because they were not extended to live neurons. Here we performed a dynamic in vivo analysis of MERCs in hippocampal neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed 'lipid-MERCs' (gap width <10â nm), while 'Ca2+-MERCs' (10-20â nm gap width) were unchanged. In situ TEM showed no significant differences in the lipid-MERCs:total MERCs or lipid-MERCs:mitochondria ratios; however, the average length of lipid-MERCs was significantly decreased in neurons from transgenic rats as compared to controls. In accordance with FRET results, untargeted lipidomics showed significant decreases in levels of 12 lipids and bioenergetic analysis revealed respiratory dysfunction of mitochondria from transgenic rats. Thus, our results reveal changes in MERC structures coupled with impaired mitochondrial functions in Alzheimer's disease-related neurons.This article has an associated First Person interview with the first author of the paper.
Assuntos
Doença de Alzheimer , Retículo Endoplasmático , Mitocôndrias , Neurônios , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Modelos Animais de Doenças , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Ratos , Ratos TransgênicosRESUMO
AIMS: MICU1 encodes the gatekeeper of the mitochondrial Ca2+ uniporter, MICU1 and biallelic loss-of-function mutations cause a complex, neuromuscular disorder in children. Although the role of the protein is well understood, the precise molecular pathophysiology leading to this neuropaediatric phenotype has not been fully elucidated. Here we aimed to obtain novel insights into MICU1 pathophysiology. METHODS: Molecular genetic studies along with proteomic profiling, electron-, light- and Coherent anti-Stokes Raman scattering microscopy and immuno-based studies of protein abundances and Ca2+ transport studies were employed to examine the pathophysiology of MICU1 deficiency in humans. RESULTS: We describe two patients carrying MICU1 mutations, two nonsense (c.52C>T; p.(Arg18*) and c.553C>T; p.(Arg185*)) and an intragenic exon 2-deletion presenting with ataxia, developmental delay and early onset myopathy, clinodactyly, attention deficits, insomnia and impaired cognitive pain perception. Muscle biopsies revealed signs of dystrophy and neurogenic atrophy, severe mitochondrial perturbations, altered Golgi structure, vacuoles and altered lipid homeostasis. Comparative mitochondrial Ca2+ transport and proteomic studies on lymphoblastoid cells revealed that the [Ca2+ ] threshold and the cooperative activation of mitochondrial Ca2+ uptake were lost in MICU1-deficient cells and that 39 proteins were altered in abundance. Several of those proteins are linked to mitochondrial dysfunction and/or perturbed Ca2+ homeostasis, also impacting on regular cytoskeleton (affecting Spectrin) and Golgi architecture, as well as cellular survival mechanisms. CONCLUSIONS: Our findings (i) link dysregulation of mitochondrial Ca2+ uptake with muscle pathology (including perturbed lipid homeostasis and ER-Golgi morphology), (ii) support the concept of a functional interplay of ER-Golgi and mitochondria in lipid homeostasis and (iii) reveal the vulnerability of the cellular proteome as part of the MICU1-related pathophysiology.
Assuntos
Proteínas de Ligação ao Cálcio/deficiência , Cálcio/metabolismo , Proteínas de Transporte de Cátions/deficiência , Proteínas de Transporte da Membrana Mitocondrial/genética , Doenças Musculares/genética , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/deficiência , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Doenças Musculares/patologia , ProteômicaRESUMO
BACKGROUND: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.
Assuntos
Condrogênese/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/análise , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Condrogênese/efeitos dos fármacos , Ácido Glutâmico/análise , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
This communication reports the structural and functional characterization of urotoxin, the first K(+) channel toxin isolated from the venom of the Australian scorpion Urodacus yaschenkoi. It is a basic peptide consisting of 37 amino acids with an amidated C-terminal residue. Urotoxin contains eight cysteines forming four disulfide bridges with sequence similarities resembling the α-potassium channel toxin 6 (α-KTx-6) subfamily of peptides; it was assigned the systematic number of α-KTx-6.21. Urotoxin is a potent blocker of human voltage-gated potassium channel (Kv)1.2 channels, with an IC50 of 160 pM, whereas its affinity for other channels tested was in the nanomolar range (hKv1.1, IC50 = 253 nM; hKv1.3, IC50 = 91 nM; and hKCa3.1, IC50 = 70 nM). The toxin had no effect on hKv1.4, hKv1.5, human ether-à-go-go-related gene type 1 (hERG1), or human ether-à-go-go-like (hELK2) channels. Multiple sequence alignments from the venom gland transcriptome showed the existence of four other new peptides similar to urotoxin. Computer modeling of urotoxin's three-dimensional structure suggests the presence of the α/ß-scaffold characteristic of other scorpion toxins, although very likely forming an uncommon disulfide pairing pattern. Using molecular dynamics, a model for the binding of this peptide to human Kv1.2 and hKv1.1 channels is presented, along with the binding of an in silico mutant urotoxin (Lys25Ala) to both channels. Urotoxin enriches our knowledge of K(+) channel toxins and, due to its high affinity for hKv1.2 channels, it may be a good candidate for the development of pharmacologic tools to study the physiologic functions of K(+) channels or related channelopathies and for restoring axonal conduction in demyelinated axons.
Assuntos
Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Escorpiões/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Linhagem Celular , Chlorocebus aethiops , Cricetulus , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Peso Molecular , Alinhamento de SequênciaRESUMO
We would like to present the case of a young woman (age 34) who was admitted to our department due to unbearable colicky pain, which started one week prior to her hospitalization. Examinations revealed mechanical obstruction, which is very unusual in her age without surgical history. During emergency surgery, we found descending colon tumour which was invading the abdominal wall with pelvic carcinomatosis at the border of the sigmoid colon. Due to extreme colonic dilation and impending rupture of the colonic serosa, we performed a subtotal colectomy with ileosigmoid anastomosis. In addition, pelvic peritonectomy was carried out, too. The histopathological examination of the resected part demonstrated adenocarcinoma of the descending-sigmoid colon, as well as another - histologically different - tumour (a well-differentiated neuroendocrine carcinoma [NEC]), which would not have been discovered, because it was invisible and impalpable. However, this latter tumour was responsible for the peritoneal metastases.
Assuntos
Adenocarcinoma/cirurgia , Colectomia , Obstrução Intestinal/cirurgia , Neoplasias Primárias Múltiplas/cirurgia , Tumores Neuroendócrinos/cirurgia , Neoplasias Peritoneais/cirurgia , Neoplasias do Colo Sigmoide/cirurgia , Adenocarcinoma/complicações , Adenocarcinoma/patologia , Adulto , Anastomose Cirúrgica , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimioterapia Adjuvante , Colectomia/métodos , Colo Sigmoide/cirurgia , Feminino , Humanos , Íleo/cirurgia , Obstrução Intestinal/etiologia , Neoplasias Primárias Múltiplas/complicações , Neoplasias Primárias Múltiplas/patologia , Tumores Neuroendócrinos/complicações , Tumores Neuroendócrinos/secundário , Neoplasias Peritoneais/secundário , Tomografia por Emissão de Pósitrons , Neoplasias do Colo Sigmoide/complicações , Neoplasias do Colo Sigmoide/patologia , Tomografia Computadorizada por Raios X , Resultado do TratamentoRESUMO
The Transient Receptor Potential Melastatin 2 (TRPM2) channel is a homotetrameric ligand-gated cation channel opened by the binding of cytosolic ADP ribose (ADPR) and Ca2+. In addition, strong temperature dependence of its activity has lately become a center of attention for both physiological and biophysical studies. TRPM2 temperature sensitivity has been affirmed to play a role in central and peripheral thermosensation, pancreatic insulin secretion, and immune cell function. On the other hand, a number of different underlying mechanisms have been proposed from studies in intact cells. This review summarizes available information on TRPM2 temperature sensitivity, with a focus on recent mechanistic insight obtained in a cell-free system. Those biophysical results outline TRPM2 as a channel with an intrinsically endothermic opening transition, a temperature threshold strongly modulated by cytosolic agonist concentrations, and a response steepness greatly enhanced through a positive feedback loop generated by Ca2+ influx through the channel's pore. Complex observations in intact cells and apparent discrepancies between studies using in vivo and in vitro models are discussed and interpreted in light of the intrinsic biophysical properties of the channel protein.
Assuntos
Cálcio , Canais de Cátion TRPM , Cálcio/metabolismo , Canais de Cátion TRPM/metabolismo , Adenosina Difosfato Ribose/metabolismo , Insulina/metabolismo , Secreção de InsulinaRESUMO
The transient receptor potential melastatin 2 (TRPM2) channel plays a central role in connecting redox state with calcium signaling in living cells. This coupling makes TRPM2 essential for physiological functions such as pancreatic insulin secretion or cytokine production, but also allows it to contribute to pathological processes, including neuronal cell death or ischemia-reperfusion injury. Genetic deletion of the channel, albeit not lethal, alters physiological functions in mice. In humans, population genetic studies and whole-exome sequencing have identified several common and rare genetic variants associated with mental disorders and neurodegenerative diseases, including single nucleotide variants (SNVs) in exonic regions. In this review, we summarize available information on the four best-documented SNVs: one common (rs1556314) and three rare genetic variants (rs139554968, rs35288229, and rs145947009), manifested in amino acid substitutions D543E, R707C, R755C, and P1018L respectively. We discuss existing evidence supporting or refuting the associations between SNVs and disease. Furthermore, we aim to interpret the molecular impacts of these amino acid substitutions based on recently published structures of human TRPM2. Finally, we formulate testable hypotheses and suggest means to investigate them. Studying the function of proteins with rare mutations might provide insight into disease etiology and delineate new drug targets.
Assuntos
Doenças Neurodegenerativas , Canais de Cátion TRPM , Humanos , Camundongos , Animais , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , Insulina/metabolismo , Secreção de Insulina , Oxirredução , Cálcio/metabolismoRESUMO
INTRODUCTION: Colorectal cancer is the second most common cause of cancer related death in Hungary. AIM: The authors examined retrospectively the ratio and the clinical course of patients under the age of 50 years operated by themselves between January, 2006 and May, 2012. METHOD: During this period of time 667 patients underwent surgery for colorectal carcinoma and 44 (6.6%) were less than 50 years old. 22.2% of the operations were performed for emergency reasons. The surgical technique, TNM stage, differentiation state and localization of the tumours were analyzed as well as the histological types with unfavourable prognosis. In order to analyse risk factors a questionnaire was sent to patients of young age group. RESULTS: The majority of patients, including those under 50 years of age were diagnosed as having TNM stages III and IV. Only half of the patients returned the questionnaire. CONCLUSIONS: Due to a relatively low response rate, the importance of risk factors for colorectal carcinoma in patients under 50 years of age remains to be further investigated.
Assuntos
Carcinoma/diagnóstico , Carcinoma/epidemiologia , Colectomia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/epidemiologia , Adulto , Distribuição por Idade , Fatores Etários , Carcinoma/complicações , Carcinoma/patologia , Carcinoma/cirurgia , Colectomia/métodos , Colectomia/estatística & dados numéricos , Neoplasias Colorretais/complicações , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Procedimentos Cirúrgicos Eletivos/métodos , Procedimentos Cirúrgicos Eletivos/estatística & dados numéricos , Tratamento de Emergência/métodos , Tratamento de Emergência/estatística & dados numéricos , Comportamento Alimentar , Feminino , Humanos , Hungria/epidemiologia , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Cuidados Paliativos/métodos , Cuidados Paliativos/estatística & dados numéricos , Estudos Retrospectivos , Fatores de Risco , Distribuição por Sexo , Inquéritos e Questionários , Fatores de TempoRESUMO
Introduction: The bowel preparation before elective colorectal surgery is a controversial topic in the surgical practice. During the last 15 years numerous publications concerning the necessity and options of the bowel preparation were published. The aim of our team was to perform a survey about the current, domestic practice as well as to give a short overview of the relevant literature and recommendations. Methods: 50 surgical departments performing colorectal surgeries routinely were asked to fill out an online, anonymous survey. The data of the survey filled out by 40 surgical departments were analyzed separately for the surgeries of the right colon, the left colon, and the rectum. Results: Based on the results, there is a high heterogeneity concerning the mode of the bowel preparation: in case of the right colon 7 different combinations, in case of the left colon and rectum 3-3 different combinations of bowel preparation methods are used. Conclusions: In the current domestic practice there is a high heterogeneity regarding the modes of the bowel preparation before the elective colorectal surgery, which are presumably based mainly on individual experience. The methods of the bowel preparation used by the majority of the surgical centers are not in accordance with the current international recommendations. A preparation of a Hungarian consensus document in this topic would be beneficial.
Assuntos
Cirurgia Colorretal , Procedimentos Cirúrgicos do Sistema Digestório , Humanos , Cuidados Pré-Operatórios/métodos , Procedimentos Cirúrgicos Eletivos , Reto/cirurgia , Colo/cirurgia , Infecção da Ferida Cirúrgica/tratamento farmacológico , Infecção da Ferida Cirúrgica/cirurgia , Antibacterianos/uso terapêuticoRESUMO
Dysregulation of mitochondrial Ca2+ homeostasis has been linked to neurodegenerative diseases. Mitochondrial Ca2+ uptake is mediated via the calcium uniporter complex that is primarily regulated by MICU1, a Ca2+-sensing gatekeeper. Recently, human patients with MICU1 loss-of-function mutations were diagnosed with neuromuscular and cognitive impairments. While studies in patient-derived cells revealed altered mitochondrial calcium signaling, the neuronal pathogenesis was difficult to study. To fill this void, we created a neuron-specific MICU1-KO mouse model. These animals show progressive, abnormal motor and cognitive phenotypes likely caused by the degeneration of motor neurons in the spinal cord and the cortex. We found increased susceptibility to mitochondrial Ca2+ overload-induced excitotoxic insults and cell death in MICU1-KO neurons and MICU1-deficient patient-derived cells, which can be blunted by inhibiting the mitochondrial permeability transition pore. Thus, our study identifies altered neuronal mitochondrial Ca2+ homeostasis as causative in the clinical symptoms of MICU1-deficient patients and highlights potential therapeutic targets.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Transporte da Membrana Mitocondrial , Doenças Neurodegenerativas , Animais , Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Camundongos , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologiaRESUMO
Endoplasmic reticulum-mitochondria contacts (ERMCs) are restructured in response to changes in cell state. While this restructuring has been implicated as a cause or consequence of pathology in numerous systems, the underlying molecular dynamics are poorly understood. Here, we show means to visualize the capture of motile IP3 receptors (IP3Rs) at ERMCs and document the immediate consequences for calcium signaling and metabolism. IP3Rs are of particular interest because their presence provides a scaffold for ERMCs that mediate local calcium signaling, and their function outside of ERMCs depends on their motility. Unexpectedly, in a cell model with little ERMC Ca2+ coupling, IP3Rs captured at mitochondria promptly mediate Ca2+ transfer, stimulating mitochondrial oxidative metabolism. The Ca2+ transfer does not require linkage with a pore-forming protein in the outer mitochondrial membrane. Thus, motile IP3Rs can traffic in and out of ERMCs, and, when 'parked', mediate calcium signal propagation to the mitochondria, creating a dynamic arrangement that supports local communication.
Assuntos
Sinalização do Cálcio , Mitocôndrias , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Sinalização do Cálcio/fisiologia , Mitocôndrias/metabolismo , Respiração Celular , Cálcio/metabolismo , Estresse OxidativoRESUMO
Contact sites of endoplasmic reticulum (ER) and mitochondria locally convey calcium signals between the IP3 receptors (IP3R) and the mitochondrial calcium uniporter, and are central to cell survival. It remains unclear whether IP3Rs also have a structural role in contact formation and whether the different IP3R isoforms have redundant functions. Using an IP3R-deficient cell model rescued with each of the three IP3R isoforms and an array of super-resolution and ultrastructural approaches we demonstrate that IP3Rs are required for maintaining ER-mitochondrial contacts. This role is independent of calcium fluxes. We also show that, while each isoform can support contacts, type 2 IP3R is the most effective in delivering calcium to the mitochondria. Thus, these studies reveal a non-canonical, structural role for the IP3Rs and direct attention towards the type 2 IP3R that was previously neglected in the context of ER-mitochondrial calcium signaling.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Retículo Endoplasmático/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Mitocôndrias/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Galinhas , Células HeLa , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Isoformas de Proteínas/genéticaRESUMO
Mitochondrial Ca2+ uptake through the Ca2+ uniporter supports cell functions, including oxidative metabolism, while meeting tissue-specific calcium signaling patterns and energy needs. The molecular mechanisms underlying tissue-specific control of the uniporter are unknown. Here, we investigated a possible role for tissue-specific stoichiometry between the Ca2+-sensing regulators (MICUs) and pore unit (MCU) of the uniporter. Low MICU1:MCU protein ratio lowered the [Ca2+] threshold for Ca2+ uptake and activation of oxidative metabolism but decreased the cooperativity of uniporter activation in heart and skeletal muscle compared to liver. In MICU1-overexpressing cells, MICU1 was pulled down by MCU proportionally to MICU1 overexpression, suggesting that MICU1:MCU protein ratio directly reflected their association. Overexpressing MICU1 in the heart increased MICU1:MCU ratio, leading to liver-like mitochondrial Ca2+ uptake phenotype and cardiac contractile dysfunction. Thus, the proportion of MICU1-free and MICU1-associated MCU controls these tissue-specific uniporter phenotypes and downstream Ca2+ tuning of oxidative metabolism.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Especificidade de Órgãos , Feminino , Humanos , Fígado/metabolismo , Músculos/metabolismo , Miocárdio/metabolismo , OxirreduçãoRESUMO
Six new peptides were isolated from the venom of the Mexican scorpion Centruroides tecomanus; their primary structures were determined and the effects on ion channels were verified by patch-clamp experiments. Four are K(+)-channel blockers of the α-KTx family, containing 32 to 39 amino acid residues, cross-linked by three disulfide bonds. They all block Kv1.2 in nanomolar concentrations and show various degree of selectivity over Kv1.1, Kv1.3, Shaker and KCa3.1 channels. One peptide has 42 amino acids cross-linked by four disulfides; it blocks ERG-channels and belongs to the γ-KTx family. The sixth peptide has only 32 amino acid residues, three disulfide bonds and has no effect on the ion-channels assayed. It also does not have antimicrobial activity. Systematic numbers were assigned (time of elution on HPLC): α-KTx 10.4 (time 24.1); α-KTx 2.15 (time 26.2); α-KTx 2.16 (time 23.8); α-KTx 2.17 (time 26.7) and γ-KTx 1.9 (elution time 29.6). A partial proteomic analysis of the short chain basic peptides of this venom, which elutes on carboxy-methyl-cellulose column fractionation, is included. The pharmacological properties of the peptides described in this study may provide valuable tools for understanding the structure-function relationship of K(+) channel blocking scorpion toxins.
Assuntos
Bloqueadores dos Canais de Potássio/química , Venenos de Escorpião/química , Escorpiões , Sequência de Aminoácidos , Animais , Linhagem Celular , Fenômenos Eletrofisiológicos , Humanos , México , Testes de Sensibilidade Microbiana , Peptídeos/química , Peptídeos/isolamento & purificação , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/isolamento & purificação , Bloqueadores dos Canais de Potássio/farmacologia , Proteômica , Venenos de Escorpião/farmacologiaRESUMO
Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin containing four disulfide bridges, is a high affinity blocker of the voltage-gated potassium channel Kv1.3, but also blocks Kv1.2. To improve potential therapeutic use of the toxin, we have designed a double substituted analog, [N17A/F32T]-AnTx, which showed comparable Kv1.3 affinity to the wild-type peptide, but also a 2500-fold increase in the selectivity for Kv1.3 over Kv1.2. In the present study we have achieved the chemical synthesis of a Sec-analog in which all cysteine (Cys) residues have been replaced by selenocysteine (Sec) forming four diselenide bonds. To the best of our knowledge this is the first time to replace, by chemical synthesis, all disulfide bonds with isosteric diselenides in a peptide/protein. Gratifyingly, the key pharmacological properties of the Sec-[N17A/F32T]-AnTx are retained since the peptide is functionally active. We also propose here a combined experimental and theoretical approach including NOE- and 77Se-based NMR supplemented by MD simulations for conformational and dynamic characterization of the Sec-[N17A/F32T]-AnTx. Using this combined approach allowed us to attain unequivocal assignment of all four diselenide bonds and supplemental MD simulations allowed characterization of the conformational dynamics around each disulfide/diselenide bridge.
RESUMO
Ion channels are crucially important for the activation and proliferation of T lymphocytes, and thus, for the function of the immune system. Previous studies on the effects of channel blockers on T cell proliferation reported variable effectiveness due to differing experimental systems. Therefore our aim was to investigate how the strength of the mitogenic stimulation influences the efficiency of cation channel blockers in inhibiting activation, cytokine secretion and proliferation of T cells under standardized conditions. Human peripheral blood lymphocytes were activated via monoclonal antibodies targeting the TCR-CD3 complex and the co-stimulator CD28. We applied the blockers of Kv1.3 (Anuroctoxin), KCa3.1 (TRAM-34) and CRAC (2-Apb) channels of T cells either alone or in combination with rapamycin, the inhibitor of the mammalian target of rapamycin (mTOR). Five days after the stimulation ELISA and flow cytometric measurements were performed to determine IL-10 and IFN-γ secretion, cellular viability and proliferation. Our results showed that ion channel blockers and rapamycin inhibit IL-10 and IFN-γ secretion and cell division in a dose-dependent manner. Simultaneous application of the blockers for each channel along with rapamycin was the most effective, indicating synergy among the various activation pathways. Upon increasing the extent of mitogenic stimulation the anti-proliferative effect of the ion channel blockers diminished. This phenomenon may be important in understanding the fine-tuning of T cell activation.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , Proliferação de Células/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canal de Potássio Kv1.3/antagonistas & inibidores , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Compostos de Boro/farmacologia , Células Cultivadas , Sinergismo Farmacológico , Humanos , Imunossupressores/farmacologia , Interferon gama/metabolismo , Interleucina-4/metabolismo , Mitógenos/imunologia , Pirazóis/farmacologia , Receptores de Antígenos de Linfócitos T/metabolismo , Sirolimo/farmacologia , Linfócitos T/fisiologiaRESUMO
The voltage-gated Kv1.3 K(+) channel plays a key role in the activation of T lymphocytes. Kv1.3 blockers selectively suppress immune responses mediated by effector memory T cells, which indicates the great potential of selective Kv1.3 inhibitors in the therapy of certain autoimmune diseases. Anuroctoxin (AnTx), a 35-amino-acid scorpion toxin is a high affinity blocker of Kv1.3, but also blocks Kv1.2 with similar potency. We designed and produced three AnTx variants: ([F32T]-AnTx, [N17A]-AnTx, [N17A/F32T]-AnTx) using solid-phase synthesis with the goal of improving the selectivity of the toxin for Kv1.3 over Kv1.2 while keeping the high affinity for Kv1.3. We used the patch-clamp technique to determine the blocking potency of the synthetic toxins on hKv1.3, mKv1.1, hKv1.2 and hKCa3.1 channels. Of the three variants [N17A/F32T]-AnTx maintained the high affinity of the natural peptide for Kv1.3 but became more than 16000-fold selective over Kv1.2. NMR data and molecular dynamics simulations suggest that the more rigid structure with restricted conformational space of the double substituted toxin compared to the flexible wild-type one is an important determinant of toxin selectivity. Our results provide the foundation for the possibility of the production and future therapeutic application of additional, even more selective toxins targeting various ion channels.
Assuntos
Canal de Potássio Kv1.3/metabolismo , Engenharia de Proteínas/métodos , Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Espectroscopia de Ressonância Magnética , Camundongos , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Conformação Proteica , Venenos de Escorpião/síntese química , Venenos de Escorpião/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
Margatoxin (MgTx), an alpha-KTx scorpion toxin, is considered a selective inhibitor of the Kv1.3K + channel. This peptide is widely used in ion channel research; however, a comprehensive study of its selectivity with electrophysiological methods has not been published yet. The lack of selectivity might lead to undesired side effects upon therapeutic application or may lead to incorrect conclusion regarding the role of a particular ion channel in a physiological or pathophysiological response either in vitro or in vivo. Using the patch-clamp technique we characterized the selectivity profile of MgTx using L929 cells expressing mKv1.1 channels, human peripheral lymphocytes expressing Kv1.3 channels and transiently transfected tsA201 cells expressing hKv1.1, hKv1.2, hKv1.3, hKv1.4-IR, hKv1.5, hKv1.6, hKv1.7, rKv2.1, Shaker-IR, hERG, hKCa1.1, hKCa3.1 and hNav1.5 channels. MgTx is indeed a high affinity inhibitor of Kv1.3 (Kd = 11.7 pM) but is not selective, it inhibits the Kv1.2 channel with similar affinity (Kd = 6.4 pM) and Kv1.1 in the nanomolar range (Kd = 4.2 nM). Based on our comprehensive data MgTX has to be considered a non-selective Kv1.3 inhibitor, and thus, experiments aiming at elucidating the significance of Kv1.3 in in vitro or in vivo physiological responses have to be carefully evaluated.
Assuntos
Canal de Potássio Kv1.3/antagonistas & inibidores , Neurotoxinas/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Linhagem Celular , Humanos , Canal de Potássio Kv1.2/antagonistas & inibidores , Canal de Potássio Kv1.2/efeitos dos fármacos , Canal de Potássio Kv1.3/genética , Linfócitos/metabolismo , Técnicas de Patch-Clamp , Venenos de Escorpião/farmacologiaRESUMO
Opisthacanthus cayaporum belongs to the Liochelidae family, and the scorpions from this genus occur in southern Africa, Central America and South America and, therefore, can be considered a true Gondwana heritage. In this communication, the isolation, primary structure characterization, and Kâº-channel blocking activity of new peptide from this scorpion venom are reported. OcyKTx2 is a 34 amino acid long peptide with four disulfide bridges and molecular mass of 3807 Da. Electrophysiological assays conducted with pure OcyKTx2 showed that this toxin reversibly blocks Shaker B Kâº-channels with a Kd of 82 nM, and presents an even better affinity toward hKv1.3, blocking it with a Kd of â¼18 nM. OcyKTx2 shares high sequence identity with peptides belonging to subfamily 6 of α-KTxs that clustered very closely in the phylogenetic tree included here. Sequence comparison, chain length and number of disulfide bridges analysis classify OcyKTx2 into subfamily 6 of the α-KTx scorpion toxins (systematic name, α-KTx6.17).