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16 pages, 2610 KiB

Open AccessArticle

Ca_v1.2 Activity and Downstream Signaling Pathways in the Hippocampus of An Animal Model of Depression

by Cristian Moreno, Tamara Hermosilla, Paulina Hardy, Víctor Aballai, Patricio Rojas and Diego Varela

Cells 2020, 9(12), 2609; https://doi.org/10.3390/cells9122609 - 4 Dec 2020

Cited by 9 | Viewed by 2218

Abstract

Functional and morphological modifications in the brain caused by major mood disorders involve many brain areas, including the hippocampus, leading to cognitive and mood alterations. Ca_v1.2 channel expression has been found to increase in animals with depressive-like behaviors. Calcium influx through [...] Read more.

Functional and morphological modifications in the brain caused by major mood disorders involve many brain areas, including the hippocampus, leading to cognitive and mood alterations. Ca_v1.2 channel expression has been found to increase in animals with depressive-like behaviors. Calcium influx through these channels is associated with changes in excitation-transcriptional coupling by several intracellular signal pathways that are regulated by its C-terminus region. However, which of these signaling pathways is activated during the development of depressive-like behaviors is not known. Here, we evaluate the phosphorylation and expression levels of crucial kinases and transcription factors at the hippocampus of rats after 21 days of chronic restraint stress. Our results show that rats subjected to CRS protocol achieve less body weight, have heavier adrenal glands, and exhibit depression-like behaviors such as anhedonia, behavioral despair and decreased social interaction. Ca_v1.2 mRNA and protein expression levels, plus l-type calcium current amplitude, are also increased in treated rats when compared with control animals. Out of the three main signaling pathways activated by l-type currents, we only observed an increment of CaM-NFAT axis activity with the concomitant increment in Fas ligand expression. Thus, our results suggest that CRS activates specific pathways, and the increased expression of Ca_v1.2 could lead to neuronal death in the hippocampus. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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16 pages, 1164 KiB

Open AccessArticle

Comprehensive Exonic Sequencing of Hemiplegic Migraine-Related Genes in a Cohort of Suspected Probands Identifies Known and Potential Pathogenic Variants

by Heidi G. Sutherland, Neven Maksemous, Cassie L. Albury, Omar Ibrahim, Robert A. Smith, Rod A. Lea, Larisa M. Haupt, Bronwyn Jenkins, Benjamin Tsang and Lyn R. Griffiths

Cells 2020, 9(11), 2368; https://doi.org/10.3390/cells9112368 - 28 Oct 2020

Cited by 16 | Viewed by 3736

Abstract

Hemiplegic migraine (HM) is a rare migraine disorder with aura subtype including temporary weakness and visual, sensory, and/or speech symptoms. To date, three main genes—CACNA1A, ATP1A2, and SCN1A—have been found to cause HM. These encode ion channels or transporters, [...] Read more.

Hemiplegic migraine (HM) is a rare migraine disorder with aura subtype including temporary weakness and visual, sensory, and/or speech symptoms. To date, three main genes—CACNA1A, ATP1A2, and SCN1A—have been found to cause HM. These encode ion channels or transporters, important for regulating neuronal ion balance and synaptic transmission, leading to HM being described as a channelopathy. However, <20% of HM cases referred for genetic testing have mutations in these genes and other genes with roles in ion and solute transport, and neurotransmission has also been implicated in some HM cases. In this study, we performed whole exome sequencing for 187 suspected HM probands referred for genetic testing, but found to be negative for CACNA1A, ATP1A2, and SCN1A mutations, and applied targeted analysis of whole exome sequencing data for rare missense or potential protein-altering variants in the PRRT2, PNKD, SLC1A3, SLC2A1, SLC4A4, ATP1A3, and ATP1A4 genes. We identified known mutations and some potentially pathogenic variants in each of these genes in specific cases, suggesting that their screening improves molecular diagnosis for the disorder. However, the majority of HM patients were found not to have candidate mutations in any of the previously reported HM genes, suggesting that additional genetic factors contributing to the disorder are yet to be identified. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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24 pages, 4682 KiB

Open AccessArticle

K⁺ Channel Tetramerization Domain 5 (KCTD5) Protein Regulates Cell Migration, Focal Adhesion Dynamics and Spreading through Modulation of Ca²⁺ Signaling and Rac1 Activity

by Jimena Canales, Pablo Cruz, Nicolás Díaz, Denise Riquelme, Elías Leiva-Salcedo and Oscar Cerda

Cells 2020, 9(10), 2273; https://doi.org/10.3390/cells9102273 - 12 Oct 2020

Cited by 9 | Viewed by 3675

Abstract

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca²⁺ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination [...] Read more.

Cell migration is critical for several physiological and pathophysiological processes. It depends on the coordinated action of kinases, phosphatases, Rho-GTPases proteins, and Ca²⁺ signaling. Interestingly, ubiquitination events have emerged as regulatory elements of migration. Thus, the role of proteins involved in ubiquitination processes could be relevant to a complete understanding of pro-migratory mechanisms. KCTD5 is a member of Potassium Channel Tetramerization Domain (KCTD) proteins that have been proposed as a putative adaptor for Cullin3-E3 ubiquitin ligase and a novel regulatory protein of TRPM4 channels. Here, we study whether KCTD5 participates in cell migration-associated mechanisms, such as focal adhesion dynamics and cellular spreading. Our results show that KCTD5 CRISPR/Cas9- and shRNA-based depletion in B16-F10 cells promoted an increase in cell migration and cell spreading, and a decrease in the focal adhesion area, consistent with an increased focal adhesion disassembly rate. The expression of a dominant-negative mutant of Rho-GTPases Rac1 precluded the KCTD5 depletion-induced increase in cell spreading. Additionally, KCTD5 silencing decreased the serum-induced Ca²⁺ response, and the reversion of this with ionomycin abolished the KCTD5 knockdown-induced decrease in focal adhesion size. Together, these data suggest that KCTD5 acts as a regulator of cell migration by modulating cell spreading and focal adhesion dynamics through Rac1 activity and Ca²⁺ signaling, respectively. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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15 pages, 1460 KiB

Open AccessArticle

Short Chain Fatty Acids Effect on Chloride Channel ClC-2 as a Possible Mechanism for Lubiprostone Intestinal Action

by Marcelo A. Catalán, Francisca Julio-Kalajzić, María Isabel Niemeyer, Luis Pablo Cid and Francisco V. Sepúlveda

Cells 2020, 9(8), 1781; https://doi.org/10.3390/cells9081781 - 26 Jul 2020

Cited by 5 | Viewed by 2673

Abstract

Lubiprostone, a 20-carbon synthetic fatty acid used for the treatment of constipation, is thought to act through an action on Cl⁻ channel ClC-2. Short chain fatty acids (SCFAs) are produced and absorbed in the distal intestine. We explore whether SCFAs affect ClC-2, [...] Read more.

Lubiprostone, a 20-carbon synthetic fatty acid used for the treatment of constipation, is thought to act through an action on Cl⁻ channel ClC-2. Short chain fatty acids (SCFAs) are produced and absorbed in the distal intestine. We explore whether SCFAs affect ClC-2, re-examine a possible direct effect of lubiprostone on ClC-2, and use mice deficient in ClC-2 to stringently address the hypothesis that the epithelial effect of lubiprostone targets this anion channel. Patch-clamp whole cell recordings of ClC-2 expressed in mammalian cells are used to assay SCFA and lubiprostone effects. Using chamber measurements of ion current in mice deficient in ClC-2 or CFTR channels served to analyze the target of lubiprostone in the distal intestinal epithelium. Intracellular SCFAs had a dual action on ClC-2, partially inhibiting conduction but, importantly, facilitating the voltage activation of ClC-2. Intra- or extracellular lubiprostone had no effect on ClC-2 currents. Lubiprostone elicited a secretory current across colonic epithelia that was increased in mice deficient in ClC-2, consistent with the channel’s proposed proabsorptive function, but absent from those deficient in CFTR. Whilst SCFAs might exert a physiological effect on ClC-2 as part of their known proabsorptive effect, ClC-2 plays no part in the lubiprostone intestinal effect that appears mediated by CFTR activation. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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16 pages, 2734 KiB

Open AccessArticle

TRPV4 Inhibition and CRISPR-Cas9 Knockout Reduce Inflammation Induced by Hyperphysiological Stretching in Human Annulus Fibrosus Cells

by Elena Cambria, Matthias J. E. Arlt, Sandra Wandel, Olga Krupkova, Wolfgang Hitzl, Fabian S. Passini, Oliver N. Hausmann, Jess G. Snedeker, Stephen J. Ferguson and Karin Wuertz-Kozak

Cells 2020, 9(7), 1736; https://doi.org/10.3390/cells9071736 - 21 Jul 2020

Cited by 18 | Viewed by 5476

Abstract

Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We [...] Read more.

Mechanical loading and inflammation interact to cause degenerative disc disease and low back pain (LBP). However, the underlying mechanosensing and mechanotransductive pathways are poorly understood. This results in untargeted pharmacological treatments that do not take the mechanical aspect of LBP into account. We investigated the role of the mechanosensitive ion channel TRPV4 in stretch-induced inflammation in human annulus fibrosus (AF) cells. The cells were cyclically stretched to 20% hyperphysiological strain. TRPV4 was either inhibited with the selective TRPV4 antagonist GSK2193874 or knocked out (KO) via CRISPR-Cas9 gene editing. The gene expression, inflammatory mediator release and MAPK pathway activation were analyzed. Hyperphysiological cyclic stretching significantly increased the IL6, IL8, and COX2 mRNA, PGE2 release, and activated p38 MAPK. The TRPV4 pharmacological inhibition significantly attenuated these effects. TRPV4 KO further prevented the stretch-induced upregulation of IL8 mRNA and reduced IL6 and IL8 release, thus supporting the inhibition data. We provide novel evidence that TRPV4 transduces hyperphysiological mechanical signals into inflammatory responses in human AF cells, possibly via p38. Additionally, we show for the first time the successful gene editing of human AF cells via CRISPR-Cas9. The pharmacological inhibition or CRISPR-based targeting of TRPV4 may constitute a potential therapeutic strategy to tackle discogenic LBP in patients with AF injury. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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15 pages, 3355 KiB

Open AccessArticle

Regulation of TMEM16A by CK2 and Its Role in Cellular Proliferation

by Madalena C. Pinto, Rainer Schreiber, Joana Lerias, Jiraporn Ousingsawat, Aires Duarte, Margarida Amaral and Karl Kunzelmann

Cells 2020, 9(5), 1138; https://doi.org/10.3390/cells9051138 - 5 May 2020

Cited by 14 | Viewed by 3479

Abstract

Casein kinase 2 (CK2) is a highly ubiquitous and conserved serine/threonine kinase that forms a tetramer consisting of a catalytic subunit (CK2α) and a regulatory subunit (CK2β). Despite being ubiquitous, CK2 is commonly found at higher expression levels in cancer cells, where it [...] Read more.

Casein kinase 2 (CK2) is a highly ubiquitous and conserved serine/threonine kinase that forms a tetramer consisting of a catalytic subunit (CK2α) and a regulatory subunit (CK2β). Despite being ubiquitous, CK2 is commonly found at higher expression levels in cancer cells, where it inhibits apoptosis, and supports cell migration and proliferation. The Ca²⁺-activated chloride channel TMEM16A shows similar effects in cancer cells: TMEM16A increases cell proliferation and migration and is highly expressed in squamous cell carcinoma of the head and neck (HNSCC) as well as other malignant tumors. A microscopy-based high-throughput screening was performed to identify proteins that regulate TMEM16A. Within this screen, CK2 was found to be required for proper membrane expression of TMEM16A. small interfering (si) RNA-knockdown of CK2 reduced plasma membrane expression of TMEM16A and inhibited TMEM16A whole cell currents in (cystic fibrosis bronchial epithelial) CFBE airway epithelial cells and in the head and neck cancer cell lines Cal33 and BHY. Inhibitors of CK2, such as TBB and the preclinical compound CX4549 (silmitasertib), also blocked membrane expression of TMEM16A and Ca²⁺-activated whole cell currents. siRNA-knockout of CK2 and its pharmacological inhibition, as well as knockdown or inhibition of TMEM16A by either niclosamide or Ani9, attenuated cell proliferation. Simultaneous inhibition of CK2 and TMEM16A strongly potentiated inhibition of cell proliferation. Although membrane expression of TMEM16A is reduced by inhibition of CK2, our data suggest that the antiproliferative effects by inhibition of CK2 are mostly independent of TMEM16A. Simultaneous inhibition of TMEM16A by niclosamide and inhibition of CK2 by silmitasertib was additive with respect to blocking cell proliferation, while cytotoxicity was reduced when compared to solely blockade of CK2. Therefore, parallel blockade TMEM16A by niclosamide may assist with anticancer therapy by silmitasertib. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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17 pages, 4905 KiB

Open AccessArticle

Functional Consequences of the Variable Stoichiometry of the Kv1.3-KCNE4 Complex

by Laura Solé, Daniel Sastre, Magalí Colomer-Molera, Albert Vallejo-Gracia, Sara R. Roig, Mireia Pérez-Verdaguer, Pilar Lillo, Michael M. Tamkun and Antonio Felipe

Cells 2020, 9(5), 1128; https://doi.org/10.3390/cells9051128 - 2 May 2020

Cited by 4 | Viewed by 3138

Abstract

The voltage-gated potassium channel Kv1.3 plays a crucial role during the immune response. The channel forms oligomeric complexes by associating with several modulatory subunits. KCNE4, one of the five members of the KCNE family, binds to Kv1.3, altering channel activity and membrane expression. [...] Read more.

The voltage-gated potassium channel Kv1.3 plays a crucial role during the immune response. The channel forms oligomeric complexes by associating with several modulatory subunits. KCNE4, one of the five members of the KCNE family, binds to Kv1.3, altering channel activity and membrane expression. The association of KCNEs with Kv channels is the subject of numerous studies, and the stoichiometry of such associations has led to an ongoing debate. The number of KCNE4 subunits that can interact and modulate Kv1.3 is unknown. KCNE4 transfers important elements to the Kv1.3 channelosome that negatively regulate channel function, thereby fine-tuning leukocyte physiology. The aim of this study was to determine the stoichiometry of the functional Kv1.3-KCNE4 complex. We demonstrate that as many as four KCNE4 subunits can bind to the same Kv1.3 channel, indicating a variable Kv1.3-KCNE4 stoichiometry. While increasing the number of KCNE4 subunits steadily slowed the activation of the channel and decreased the abundance of Kv1.3 at the cell surface, the presence of a single KCNE4 peptide was sufficient for the cooperative enhancement of the inactivating function of the channel. This variable architecture, which depends on KCNE4 availability, differentially affects Kv1.3 function. Therefore, our data indicate that the physiological remodeling of KCNE4 triggers functional consequences for Kv1.3, thus affecting cell physiology. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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52 pages, 2530 KiB

Open AccessReview

TRPM Channels in Human Diseases

by Ivanka Jimenez, Yolanda Prado, Felipe Marchant, Carolina Otero, Felipe Eltit, Claudio Cabello-Verrugio, Oscar Cerda and Felipe Simon

Cells 2020, 9(12), 2604; https://doi.org/10.3390/cells9122604 - 4 Dec 2020

Cited by 40 | Viewed by 5709

Abstract

The transient receptor potential melastatin (TRPM) subfamily belongs to the TRP cation channels family. Since the first cloning of TRPM1 in 1989, tremendous progress has been made in identifying novel members of the TRPM subfamily and their functions. The TRPM subfamily is composed [...] Read more.

The transient receptor potential melastatin (TRPM) subfamily belongs to the TRP cation channels family. Since the first cloning of TRPM1 in 1989, tremendous progress has been made in identifying novel members of the TRPM subfamily and their functions. The TRPM subfamily is composed of eight members consisting of four six-transmembrane domain subunits, resulting in homomeric or heteromeric channels. From a structural point of view, based on the homology sequence of the coiled-coil in the C-terminus, the eight TRPM members are clustered into four groups: TRPM1/M3, M2/M8, M4/M5 and M6/M7. TRPM subfamily members have been involved in several physiological functions. However, they are also linked to diverse pathophysiological human processes. Alterations in the expression and function of TRPM subfamily ion channels might generate several human diseases including cardiovascular and neurodegenerative alterations, organ dysfunction, cancer and many other channelopathies. These effects position them as remarkable putative targets for novel diagnostic strategies, drug design and therapeutic approaches. Here, we review the current knowledge about the main characteristics of all members of the TRPM family, focusing on their actions in human diseases. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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28 pages, 2480 KiB

Open AccessReview

Clinical Importance of the Human Umbilical Artery Potassium Channels

by Margarida Lorigo, Nelson Oliveira and Elisa Cairrao

Cells 2020, 9(9), 1956; https://doi.org/10.3390/cells9091956 - 25 Aug 2020

Cited by 25 | Viewed by 3165

Abstract

Potassium (K⁺) channels are usually predominant in the membranes of vascular smooth muscle cells (SMCs). These channels play an important role in regulating the membrane potential and vessel contractility—a role that depends on the vascular bed. Thus, the activity of K [...] Read more.

Potassium (K⁺) channels are usually predominant in the membranes of vascular smooth muscle cells (SMCs). These channels play an important role in regulating the membrane potential and vessel contractility—a role that depends on the vascular bed. Thus, the activity of K⁺ channels represents one of the main mechanisms regulating the vascular tone in physiological and pathophysiological conditions. Briefly, the activation of K⁺ channels in SMC leads to hyperpolarization and vasorelaxation, while its inhibition induces depolarization and consequent vascular contraction. Currently, there are four different types of K⁺ channels described in SMCs: voltage-dependent K⁺ (K_V) channels, calcium-activated K⁺ (K_Ca) channels, inward rectifier K⁺ (Kir) channels, and 2-pore domain K⁺ (K_2P) channels. Due to the fundamental role of K⁺ channels in excitable cells, these channels are promising therapeutic targets in clinical practice. Therefore, this review discusses the basic properties of the various types of K⁺ channels, including structure, cellular mechanisms that regulate their activity, and new advances in the development of activators and blockers of these channels. The vascular functions of these channels will be discussed with a focus on vascular SMCs of the human umbilical artery. Then, the clinical importance of K⁺ channels in the treatment and prevention of cardiovascular diseases during pregnancy, such as gestational hypertension and preeclampsia, will be explored. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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19 pages, 1023 KiB

Open AccessReview

Endocytosis: A Turnover Mechanism Controlling Ion Channel Function

by Irene Estadella, Oriol Pedrós-Gámez, Magalí Colomer-Molera, Manel Bosch, Alexander Sorkin and Antonio Felipe

Cells 2020, 9(8), 1833; https://doi.org/10.3390/cells9081833 - 4 Aug 2020

Cited by 24 | Viewed by 5523

Abstract

Ion channels (IChs) are transmembrane proteins that selectively drive ions across membranes. The function of IChs partially relies on their abundance and proper location in the cell, fine-tuned by the delicate balance between secretory, endocytic, and degradative pathways. The disruption of this balance [...] Read more.

Ion channels (IChs) are transmembrane proteins that selectively drive ions across membranes. The function of IChs partially relies on their abundance and proper location in the cell, fine-tuned by the delicate balance between secretory, endocytic, and degradative pathways. The disruption of this balance is associated with several diseases, such as Liddle’s and long QT syndromes. Because of the vital role of these proteins in human health and disease, knowledge of ICh turnover is essential. Clathrin-dependent and -independent mechanisms have been the primary mechanisms identified with ICh endocytosis and degradation. Several molecular determinants recognized by the cellular internalization machinery have been discovered. Moreover, specific conditions can trigger the endocytosis of many IChs, such as the activation of certain receptors, hypokalemia, and some drugs. Ligand-dependent receptor activation primarily results in the posttranslational modification of IChs and the recruitment of important mediators, such as β-arrestins and ubiquitin ligases. However, endocytosis is not a final fate. Once internalized into endosomes, IChs are either sorted to lysosomes for degradation or recycled back to the plasma membrane. Rab proteins are crucial participants during these turnover steps. In this review, we describe the major ICh endocytic pathways, the signaling inputs triggering ICh internalization, and the key mediators of this essential cellular process. Full article

(This article belongs to the Special Issue MiNICAD Special Issue: Ion Channels and Human Diseases)

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