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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 Ca(2+)-activated K(+) channels (K(Ca)3.1). Because Kv1.3 is a very good target for autoimmune therapies, the effects of diclofenac on Kv1.3 are of high pharmacological relevance.

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.

Evidence for a role of angiopoietin-like 7 (ANGPTL7) in extracellular matrix formation of the human trabecular meshwork: implications for glaucoma

The trabecular meshwork tissue controls the drainage of the aqueous humor of the eye. A dysfunctional trabecular meshwork leads to an altered fluid resistance, which results in increased intraocular pressure (IOP). IOP is the major risk factor of glaucoma, the second‐leading cause of blindness in the developed world. In the search for genes altered by glaucomatous insults, we identified angiopoietin‐like7 (ANGPTL7), a member of the ANGPTL family. Although structurally related to the angiopoietins, ANGPTL7′s function is poorly understood. Because ANGPTL7 is secreted and because extracellular matrix (ECM) deposition and organization is critical for aqueous humor resistance, we investigated the effect of ANGPTL7 on relevant trabecular meshwork ECM genes and proteins. We find that overexpression of ANGPTL7 in primary human trabecular meshwork cells altered the expression of fibronectin, collagens type I, IV & V, myocilin, versican, and MMP1. ANGPTL7 also interfered with the fibrillar assembly of fibronectin. Finally, we find that silencing ANGPTL7 during the glucocorticoid insult significantly affected the expression of other steroid‐responsive proteins. These results indicate that ANGPTL7 modulates the trabecular meshwork’s ECM as well as the response of this tissue to steroids. Together with previous findings, these properties strengthen ANGPTL7′s candidacy for the regulation of IOP and glaucoma.

The voltage-dependent K+ channels Kv1.3 and Kv1.5 in human cancer

Voltage-dependent K+ channels (Kv) are involved in a number of physiological processes, including immunomodulation, cell volume regulation, apoptosis as well as differentiation. Some Kv channels participate in the proliferation and migration of normal and tumor cells, contributing to metastasis. Altered expression of Kv1.3 and Kv1.5 channels has been found in several types of tumors and cancer cells. In general, while the expression of Kv1.3 apparently exhibits no clear pattern, Kv1.5 is induced in many of the analyzed metastatic tissues. Interestingly, evidence indicates that Kv1.5 channel shows inversed correlation with malignancy in some gliomas and non-Hodgkins lymphomas. However, Kv1.3 and Kv1.5 are similarly remodeled in some cancers. For instance, expression of Kv1.3 and Kv1.5 correlates with a certain grade of tumorigenicity in muscle sarcomas. Differential remodeling of Kv1.3 and Kv1.5 expression in human cancers may indicate their role in tumor growth and their importance as potential tumor markers. However, despite of this increasing body of information, which considers Kv1.3 and Kv1.5 as emerging tumoral markers, further research must be performed to reach any conclusion. In this review, we summarize what it has been lately documented about Kv1.3 and Kv1.5 channels in human cancer.

Targeting the Voltage-Dependent K+ Channels Kv1.3 and Kv1.5 as Tumor Biomarkers for Cancer Detection and Prevention

Potassium channels (KCh) are a diverse group of membrane proteins that participate in the control of the membrane potential. More than eighty different KCh genes have been identified, which are expressed in virtually all living cells. In addition to nerve and cardiac action potentials, these proteins are involved in a number of physiological processes, including cell volume regulation, apoptosis, immunomodulation and differentiation. Furthermore, many KCh have been reported to play a role in proliferation and cell cycle progression in mammalian cells, and an important number of studies report the involvement of KCh in cancer progression. The voltage dependent potassium (Kv) channels, in turn, form the largest family of human KCh, which comprises about 40 genes. Because Kv1.3 and Kv1.5 channels modulate proliferation of different mammalian cells, these proteins have been analyzed in a number of tumors and cancer cells. In most cancers, the expression patterns of Kv1.3 and Kv1.5 are remodeled, and in some cases, a correlation has been established between protein abundance and grade of tumor malignancy. The list of cancers evaluated is constantly growing, indicating that these proteins may be future targets for treatment. The aim of this review is to provide an updated overview of Kv1.3 and Kv1.5 channels during cancer development. Unlike Kv1.5, Kv1.3 is characterized by a very selective and potent pharmacology, which could lead to specific pharmacological targeting. Because potassium channels may play a pivotal role in tumor cell proliferation, these proteins should be taken into account when designing new cancer treatment strategies.

Individual molecular response to elevated intraocular pressure in perfused postmortem human eyes

Elevated intraocular pressure (IOP) is the major risk factor for glaucoma. In the clinic, the response to elevated pressure and thus the risk for development of glaucoma differs among individuals. We took advantage of our ability to subject postmortem human eyes from the same individual to physiological and elevated pressure in a perfused outflow model and compared individual patterns of gene expression under pressure. The architecture of the trabecular meshwork, tissue responsible for the maintenance of IOP, was conserved. We performed two sets of experiments. The first set (n = 5, 10 eyes) used Affymetrix GeneChips, identified the 20 most pressure-altered genes in each individual, and compared their pressure response in the other four. The second set (n = 5, 10 eyes) selected 21 relevant trabecular meshwork genes and examined, by real-time TaqMan-PCR, the rank of their abundance and of their pressure differential expression in each individual. The majority of the up- and downregulated top-changers of each individual showed an individual response trend. Few genes were general responders. Individual responders included STATH, FBN2, TF, OGN, IL6, IGF1, CRYAB, and ELAM1 (marker for glaucoma). General responders included MMP1, MMP10, CXCL2, and PDPN. In addition, we found that although the relative abundance of selected genes was very similar among nonstressed individuals, the response to pressure of those same genes had a marked individual component. Our results offer the first molecular insight on the variation of the individual response to IOP observed in the clinical setting.

Impact of KCNE subunits on KCNQ1 (Kv7.1) channel membrane surface targeting

The KCNQ1 (Kv7.1) channel plays an important role in cardiovascular physiology. Cardiomyocytes co‐express KCNQ1 with KCNE1‐5 proteins. KCNQ1 may co‐associate with multiple KCNE regulatory subunits to generate different biophysically and pharmacologically distinct channels. Increasing evidence indicates that the location and targeting of channels are important determinants of their function. In this context, the presence of K+ channels in sphingolipid–cholesterol‐enriched membrane microdomains (lipid rafts) is under investigation. Lipid rafts are important for cardiovascular functioning. We aimed to determine whether KCNE subunits modify the localization and targeting of KCNQ1 channels in lipid rafts microdomains. HEK‐293 cells were transiently transfected with KCNQ1 and KCNE1–5, and their traffic and presence in lipid rafts were analyzed. Only KCNQ1 and KCNE3, when expressed alone, co‐localized in raft fractions. In addition, while KCNE2 and KCNE5 notably stained the cell surface, KCNQ1 and the rest of the KCNEs showed strong intracellular retention. KCNQ1 targets multiple membrane surface microdomains upon association with KCNE peptides. Thus, while KCNQ1/KCNE1 and KCNQ1/KCNE2 channels target lipid rafts, KCNQ1 associated with KCNE3–5 did not. Channel membrane dynamics, analyzed by fluorescence recovery after photobleaching (FRAP) experiments, further supported these results. In conclusion, the trafficking and targeting pattern of KCNQ1 can be influenced by its association with KCNEs. Since KCNQ1 is crucial for cardiovascular physiology, the temporal and spatial regulations that different KCNE subunits may confer to the channels could have a dramatic impact on membrane electrical activity and putative endocrine regulation. J. Cell. Physiol. 225: 692–700, 2010.

Functional implications of KCNE subunit expression for the Kv7.5 (KCNQ5) channel.

Kv7 (KCNQ) proteins form a family of voltage-gated potassium channels that is comprised of five members, Kv7.1-Kv7.5. While Kv7.1 is crucial in the heart, the Kv7.2, Kv7.3, Kv7.4 and Kv7.5 channels contribute to the M-current in the nervous system. In addition to the brain, Kv7.5 is expressed in skeletal and smooth muscle, where its physiological role is currently under evaluation. Kv7 associations with KCNE accessory subunits (KCNE1-5) enhance channel diversity and their interaction provides mechanisms to respond to a variety of stimuli. KCNE peptides control the surface expression, voltage-dependence, kinetics of gating, unitary conductance, ion selectivity and pharmacology of several channels. KCNE subunits have been primarily studied in the heart; however, their activity in the brain and in many other tissues is being increasingly recognized. Here, we found that Kv7.5 and KCNE subunits are present in myoblasts. Therefore, oligomeric associations may underlie some Kv7.5 functional diversity in skeletal muscle. An extensive study in Xenopus oocytes and HEK-293 cells demonstrates that KCNE1 and KCNE3, but none of the other KCNE subunits, affect Kv7.5 currents. While KCNE1 slows activation and suppresses inward rectification, KCNE3 drastically inhibits Kv7.5 currents. In addition, KCNE1 increases Kv7.5 currents in HEK cells. Changes in gating and amplitude indicate functional interactions. Our results have physiological relevance since Kv7.5 is abundant in skeletal and smooth muscle and its association with KCNE peptides may fine-tune cellular responses.

A new KCNQ1 mutation at the S5 segment that impairs its association with KCNE1 is responsible for short QT syndrome

AIMS KCNQ1 and KCNE1 encode Kv7.1 and KCNE1, respectively, the pore-forming and the accessory subunits of the slow delayed rectifier potassium current, IKs. KCNQ1 mutations are associated with long and short QT syndrome. The aim of this study was to characterize the biophysical and cellular phenotype of a KCNQ1 missense mutation, F279I, found in a 23-year-old man with a corrected QT interval (QTc) of 356 ms and a family history of sudden cardiac death. METHODS AND RESULTS Experiments were performed using perforated patch-clamp, western blot, co-immunoprecipitation, biotinylation, and immunocytochemistry techniques in HEK293, COS7 cells and in cardiomyocytes transfected with WT Kv7.1/KCNE1 or F279I Kv7.1/KCNE1 channels. In the absence of KCNE1, F279I Kv7.1 current exhibited a lesser degree of inactivation than WT Kv7.1. Also, functional analysis of F279I Kv7.1 in the presence of KCNE1 revealed a negative shift in the activation curve and an acceleration of the activation kinetics leading to a gain of function in IKs. The co-assembly between F279I Kv7.1 channels and KCNE1 was markedly decreased compared with WT Kv7.1 channels, as revealed by co-immunoprecipitation and Föster Resonance Energy Transfer experiments. All these effects contribute to the increase of IKs when channels incorporate F279I Kv7.1 subunits, as shown by a computer model simulation of these data that predicts a shortening of the action potential (AP) consistent with the patient phenotype. CONCLUSION The F279I mutation induces a gain of function of IKs due to an impaired gating modulation of Kv7.1 induced by KCNE1, leading to a shortening of the cardiac AP.

Involvement of potassium channels in the progression of cancer to a more malignant phenotype.

Potassium channels are a diverse group of pore-forming transmembrane proteins that selectively facilitate potassium flow through an electrochemical gradient. They participate in the control of the membrane potential and cell excitability in addition to different cell functions such as cell volume regulation, proliferation, cell migration, angiogenesis as well as apoptosis. Because these physiological processes are essential for the correct cell function, K+ channels have been associated with a growing number of diseases including cancer. In fact, different K+ channel families such as the voltage-gated K+ channels, the ether à-go-go K+ channels, the two pore domain K+ channels and the Ca2+-activated K+ channels have been associated to tumor biology. Potassium channels have a role in neoplastic cell-cycle progression and their expression has been found abnormal in many types of tumors and cancer cells. In addition, the expression and activity of specific K+ channels have shown a significant correlation with the tumor malignancy grade. The aim of this overview is to summarize published data on K+ channels that exhibit oncogenic properties and have been linked to a more malignant cancer phenotype. This article is part of a Special Issue entitled: Membrane channels and transporters in cancers.