Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences

Voltage-dependent potassium (Kv) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of Kv channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K+ currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric Kv1.3/Kv1.5 channels, which generate the main Kv in macrophages. An increase in K+ current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in Kv1.3 subunits in the Kv1.3/Kv1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in Kv1.3 expression. Neither of these treatments apparently altered the expression of Kv1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in Kv1.3/Kv1.5 hybrid channels by altering the subunit stoichiometry

A novel optical sensor system for the automatic classification of mosquitoes by genus and sex with high levels of accuracy

Background: Every year, more than 700,000 people die from vector-borne diseases, mainly transmitted by mosqui‑ toes. Vector surveillance plays a major role in the control of these diseases and requires accurate and rapid taxo‑ nomical identifcation. New approaches to mosquito surveillance include the use of acoustic and optical sensors in combination with machine learning techniques to provide an automatic classifcation of mosquitoes based on their fight characteristics, including wingbeat frequency. The development and application of these methods could enable the remote monitoring of mosquito populations in the feld, which could lead to signifcant improvements in vector surveillance. Methods: A novel optical sensor prototype coupled to a commercial mosquito trap was tested in laboratory conditions for the automatic classifcation of mosquitoes by genus and sex. Recordings of > 4300 laboratory-reared mosquitoes of Aedes and Culex genera were made using the sensor. The chosen genera include mosquito species that have a major impact on public health in many parts of the world. Five features were extracted from each recording to form balanced datasets and used for the training and evaluation of fve diferent machine learning algorithms to achieve the best model for mosquito classifcation. Results: The best accuracy results achieved using machine learning were: 94.2% for genus classifcation, 99.4% for sex classifcation of Aedes, and 100% for sex classifcation of Culex. The best algorithms and features were deep neural network with spectrogram for genus classifcation and gradient boosting with Mel Frequency Cepstrum Coefcients among others for sex classifcation of either genus. Conclusions: To our knowledge, this is the frst time that a sensor coupled to a standard mosquito suction trap has provided automatic classifcation of mosquito genus and sex with high accuracy using a large number of unique samples with class balance. This system represents an improvement of the state of the art in mosquito surveillance and encourages future use of the sensor for remote, real-time characterization of mosquito populations.info:eu-repo/semantics/publishedVersio

Voltage-dependent Na+ channel phenotype changes in myoblasts. Consequences for cardiac repair

Objective: Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na + channels (Na v ) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Na v during proliferation and differentiation in skeletal myocytes. Methods and results: We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Na v 1.4 and Na v 1.5, although neither protein has an apparent role in cell growth. During myogenesis, Na v1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Na v1.5 accounts for almost 90% of the Na + current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Nav 1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Na v 1.5 in skeletal myocytes. Conclusion: Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Na v1.5 acts as an underlying molecular mechanism

Comparison of Plasma Lipoprotein Composition and Function in Cerebral Amyloid Angiopathy and Alzheimer’s Disease

Malaltia d'Alzheimer; Apolipoproteïnes; Angiopatia amiloide cerebralEnfermedad de Alzheimer; Apolipoproteínas; Angiopatía amiloide cerebralAlzheimer’s disease; Apolipoproteins; Cerebral amyloid angiopathyCerebral amyloid angiopathy (CAA) refers to beta-amyloid (Aβ) deposition in brain vessels and is clinically the main cause of lobar intracerebral hemorrhage (ICH). Aβ can also accumulate in brain parenchyma forming neuritic plaques in Alzheimer’s disease (AD). Our study aimed to determine whether the peripheral lipid profile and lipoprotein composition are associated with cerebral beta-amyloidosis pathology and may reflect biological differences in AD and CAA. For this purpose, lipid and apolipoproteins levels were analyzed in plasma from 51 ICH-CAA patients (collected during the chronic phase of the disease), 60 AD patients, and 60 control subjects. Lipoproteins (VLDL, LDL, and HDL) were isolated and their composition and pro/antioxidant ability were determined. We observed that alterations in the lipid profile and lipoprotein composition were remarkable in the ICH-CAA group compared to control subjects, whereas the AD group presented no specific alterations compared with controls. ICH-CAA patients presented an atheroprotective profile, which consisted of lower total and LDL cholesterol levels. Plasma from chronic ICH-CAA patients also showed a redistribution of ApoC-III from HDL to VLDL and a higher ApoE/ApoC-III ratio in HDL. Whether these alterations reflect a protective response or have a causative effect on the pathology requires further investigation.This research was funded by Instituto de Salud Carlos III (co-financed by the European Regional Development Fund FEDER “Una manera de hacer Europa”), grant numbers PI13/00364, PI16/00471, PI14/01134 and PI17/00275. A.R.-U. was funded by Instituto de Salud Carlos III predoctoral contract FI17/00031. The Neurovascular Research Laboratory is part of the INVICTUS+ network, ISCIII, Spain [RD16/0019/0021]. J.L.S.-Q. is a member of the CIBER of Diabetes and Metabolism (CIBERDEM), ISCIII, Spain. M.H.-G. is supported by the Miguel Servet programme, ISCIII, Spain [CPII17/00010]. A.R.-U., N.P., S.B. and J.L.S.-Q. are members of the Quality Research Group 2017-SGR-1149 from Generalitat de Catalunya. A.R.-U., N.P., S.B. and J.L.S.-Q. are members of the Group of Vascular Biology from the Spanish Atherosclerosis Society

Association of Kv1.5 and Kv1.3 contributes to the major voltage-dependent K+ channel in macrophages

Voltage-dependent K(+) (Kv) currents in macrophages are mainly mediated by Kv1.3, but biophysical properties indicate that the channel composition could be different from that of T-lymphocytes. K(+) currents in mouse bone marrow-derived and Raw-264.7 macrophages are sensitive to Kv1.3 blockers, but unlike T-cells, macrophages express Kv1.5. Because Shaker subunits (Kv1) may form heterotetrameric complexes, we investigated whether Kv1.5 has a function in Kv currents in macrophages. Kv1.3 and Kv1.5 co-localize at the membrane, and half-activation voltages and pharmacology indicate that K(+) currents may be accounted for by various Kv complexes in macrophages. Co-expression of Kv1.3 and Kv1.5 in human embryonic kidney 293 cells showed that the presence of Kv1.5 leads to a positive shift in K(+) current half-activation voltages and that, like Kv1.3, Kv1.3/Kv1.5 heteromers are sensitive to r-margatoxin. In addition, both proteins co-immunoprecipitate and co-localize. Fluorescence resonance energy transfer studies further demonstrated that Kv1.5 and Kv1.3 form heterotetramers. Electrophysiological and pharmacological studies of different ratios of Kv1.3 and Kv1.5 co-expressed in Xenopus oocytes suggest that various hybrids might be responsible for K(+) currents in macrophages. Tumor necrosis factor-alpha-induced activation of macrophages increased Kv1.3 with no changes in Kv.1.5, which is consistent with a hyperpolarized shift in half-activation voltage and a lower IC(50) for margatoxin. Taken together, our results demonstrate that Kv1.5 co-associates with Kv1.3, generating functional heterotetramers in macrophages. Changes in the oligomeric composition of functional Kv channels would give rise to different biophysical and pharmacological properties, which could determine specific cellular responses

Immunomodulatory effects of diclofenac in leukocytes through the targeting of Kv1.3 voltage-dependent potassium channels

Kv1.3 plays a crucial role in the activation and proliferation of T-lymphocytes and macrophages. While Kv1.3 is responsible for the voltage-dependent potassium current in T-cells, in macrophages this K+ current is generated by the association of Kv1.3 and Kv1.5. Patients with autoimmune diseases show a high number of effector memory T cells that are characterized by a high expression of Kv1.3 and Kv1.3 antagonists ameliorate autoimmune disorders in vivo. Diclofenac is a non-steroidal anti-inflammatory drug (NSAID) used in patients who suffer from painful autoimmune diseases such as rheumatoid arthritis. In this study, we show that diclofenac impairs immune response via a mechanism that involves Kv1.3. While diclofenac inhibited Kv1.3 expression in activated macrophages and T-lymphocytes, Kv1.5 remained unaffected. Diclofenac also decreased iNOS levels in Raw 264.7 cells, impairing their activation in response to lipopolysaccharide (LPS). LPS-induced macrophage migration and IL-2 production in stimulated Jurkat T-cells were also blocked by pharmacological doses of diclofenac. These effects were mimicked by Margatoxin, a specific Kv1.3 inhibitor, and Charybdotoxin, which blocks both Kv1.3 and Ca2+-activated K+ channels (KCa3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance. © 2010 Elsevier Inc.This work was supported by the Ministerio Ciencia e Innovación (MICINN), Spain (BFU2008-00431 and CSD2008-00005 to AF, SAF2007-65868 and FIS D06/0014/0006 to CV, and BFU2009-07501 to CS). NV and JB held a fellowship from the Ministerio Ciencia e Innovación (MICINN). MD held a FIS RD06/0014/0006 contract. NC and TG were supported by the Juan de la Cierva program (MICINN).Peer Reviewe

Cell cycle-dependent expression of Kv1.5 is involved in myoblast proliferation

Voltage-dependent K+ channels (Kv) are involved in the proliferation of many types of cells, but the mechanisms by which their activity is related to cell growth remain unclear. Kv antagonists inhibit the proliferation of mammalian cells, which is of physiological relevance in skeletal muscle. Although myofibres are terminally differentiated, some resident myoblasts may re-enter the cell cycle and proliferate. Here we report that the expression of Kv1.5 is cell-cycle dependent during myoblast proliferation. In addition to Kv1.5 other Kv, such as Kv1.3, are also up-regulated. However, pharmacological evidence mainly implicates Kv1.5 in myoblast growth. Thus, the presence of S0100176, a Kv antagonist, but not margatoxin and dendrotoxin, led to cell cycle arrest during the G1-phase. The use of selective cell cycle blockers showed that Kv1.5 was transiently accumulated during the early G1-phase. Furthermore, while myoblasts treated with S0100176 expressed low levels of cyclin A and D1, the expression of p21cip-1 and p27kip1, two cyclin-dependent kinase inhibitors, increased. Our results indicate that the cell cycle-dependent expression of Kv1.5 is involved in skeletal muscle cell proliferation. © 2008 Elsevier B.V. All rights reserved.This work was supported by the Ministerio de Educación y Ciencia (MEC), Spain (BFI2002-00764 and BFU2005-00695 to AF, SAF2007-65868 to CV, and BFU2006-06076 to CS), Generalitat de Catalunya 2005SGR00308 to AF and FIS D06/0014/0006 to CV. NV and RMM hold fellowships from the MEC and MRF from the Generalitat de Catalunya.Peer Reviewe

Voltage-dependent Na+ channel phenotype changes in myoblasts. Consequences for cardiac repair

[Objective]: Cellular cardiomyoplasty using skeletal myoblasts is a promising therapy for myocardial infarct repair. Once transplanted, myoblasts grow, differentiate and adapt their electrophysiological properties towards more cardiac-like phenotypes. Voltage-dependent Na+ channels (Nav) are the main proteins involved in the propagation of the cardiac action potential, and their phenotype affects cardiac performance. Therefore, we examined the expression of Nav during proliferation and differentiation in skeletal myocytes. [Methods and results]: We used the rat neonatal skeletal myocyte cell line L6E9. Proliferation of L6E9 cells induced Nav1.4 and Nav1.5, although neither protein has an apparent role in cell growth. During myogenesis, Nav1.5 was largely induced. Electrophysiological and pharmacological properties, as well as mRNA expression, indicate that cardiac-type Nav1.5 accounts for almost 90% of the Na+ current in myotubes. Unlike in proliferation, this protein plays a pivotal role in myogenesis. The adoption of a cardiac-like phenotype is further supported by the increase in Nav1.5 colocalization in caveolae. Finally, we demonstrate that the treatment of myoblasts with neuregulin further increased Nav1.5 in skeletal myocytes. [Conclusion]: Our results indicate that skeletal myotubes adopt a cardiac-like phenotype in cell culture conditions and that the expression of Nav1.5 acts as an underlying molecular mechanism. © 2007 Elsevier B.V. All rights reserved.Supported by the Ministerio de Educación y Ciencia (MEC), Spain (BFI2002-00764 and BFU2005-00695 to AF; SAF2005-00489 to AG; SAF2004-06856, CAM GR/SAL/0854/2004 and FIS RD06/0014/0006 to CV). RMM, RS and NV hold fellowships from the MEC and MRF from the Generalitat de Catalunya.Peer Reviewe

Immunomodulation of voltage-dependent K+ channels in macrophages: molecular and biophysical consequences

Voltage-dependent potassium (K(v)) channels play a pivotal role in the modulation of macrophage physiology. Macrophages are professional antigen-presenting cells and produce inflammatory and immunoactive substances that modulate the immune response. Blockage of K(v) channels by specific antagonists decreases macrophage cytokine production and inhibits proliferation. Numerous pharmacological agents exert their effects on specific target cells by modifying the activity of their plasma membrane ion channels. Investigation of the mechanisms involved in the regulation of potassium ion conduction is, therefore, essential to the understanding of potassium channel functions in the immune response to infection and inflammation. Here, we demonstrate that the biophysical properties of voltage-dependent K(+) currents are modified upon activation or immunosuppression in macrophages. This regulation is in accordance with changes in the molecular characteristics of the heterotetrameric K(v)1.3/K(v)1.5 channels, which generate the main K(v) in macrophages. An increase in K(+) current amplitude in lipopolysaccharide-activated macrophages is characterized by a faster C-type inactivation, a greater percentage of cumulative inactivation, and a more effective margatoxin (MgTx) inhibition than control cells. These biophysical parameters are related to an increase in K(v)1.3 subunits in the K(v)1.3/K(v)1.5 hybrid channel. In contrast, dexamethasone decreased the C-type inactivation, the cumulative inactivation, and the sensitivity to MgTx concomitantly with a decrease in K(v)1.3 expression. Neither of these treatments apparently altered the expression of K(v)1.5. Our results demonstrate that the immunomodulation of macrophages triggers molecular and biophysical consequences in K(v)1.3/K(v)1.5 hybrid channels by altering the subunit stoichiometry.This work was supported by the Ministerio de Ciencia e Innovación (MICINN), Spain (grants BFU2005-00695, BFU2008-00431, and CSD2008-00005 to A. Felipe, and grants SAF2007-65868 and FIS D06/0014/0006 to C. Valenzuela). N. Villalonga and J. Bielanska held fellowships from the MICINN. M. David held an FIS RD06/0014/0006 contract. N. Comes is supported by the Juan de la Cierva program (MICINN