Non-excitable Cells Research Articles

Alkaloid ligands enable function of homomeric human α10 nicotinic acetylcholine receptors.

In the nervous system, nicotinic acetylcholine receptors (nAChRs) rapidly transduce a chemical signal into one that is electrical via ligand-gated ion flux through the central channel of the receptor. However, some nAChR subunits are expressed by non-excitable cells where signal transduction apparently occurs through non-ionic mechanisms. One such nAChR subunit, α10, is present in a discreet subset of immune cells and has been implicated in pathologies including cancer, neuropathic pain, and chronic inflammation. Longstanding convention holds that human α10 subunits require co-assembly with α9 subunits for function. Here we assessed whether cholinergic ligands can enable or uncover ionic functions from homomeric α10 nAChRs. Xenopus laevis oocytes expressing human α10 subunits were exposed to a panel of ligands and examined for receptor activation using voltage-clamp electrophysiology. Functional expression of human α10 nAChRs was achieved by exposing the oocytes to the alkaloids strychnine, brucine, or methyllycaconitine. Furthermore, acute exposure to the alkaloid ligands significantly enhanced ionic responses. Acetylcholine-gated currents mediated by α10 nAChRs were potently inhibited by the snake toxins α-bungarotoxin and α-cobratoxin but not by α-conotoxins that target α9 and α9α10 nAChRs. Our findings indicate that human α10 homomers are expressed in oocytes and exposure to certain ligands can enable ionic functions. To our knowledge, this is the first demonstration that human α10 subunits can assemble as functional homomeric nAChRs. These findings have potential implications for receptor regulatory-mechanisms and will enable structural, functional, and further pharmacological characterization of human α10 nAChRs.

Blockage of Orai1-Nucleolin interaction meditated calcium influx attenuates breast cancer cells growth

As an important second messenger, calcium (Ca2+) regulates a wide variety of physiological processes. Disturbance of intracellular calcium homeostasis implicated in the occurrence of multiple types of diseases. Orai1 is the major player in mediating store-operated calcium entry (SOCE) and regulates calcium homeostasis in non-excitable cells. Over-expression and activation of Orai1 have been reported in breast cancer. However, its molecular mechanisms are still not very clear. Here, we demonstrated that Nucleolin (NCL) was a novel interacting partner of Orai1. NCL is a multifunctional nucleocytoplasmic protein and is upregulated in human breast tumors. The binding of C-termini of NCL (NCL-CT) to N-termini of Orai1 (Orai1-NT) is critical for mediating calcium influx and proliferation of breast cancer cells. Blocking the NCL-Orai1 interaction by synthesized Orai1 peptide can effectively reduce the intracellular calcium influx and suppress the proliferation of breast cancer cells in vitro and in vivo. Our findings reveal a novel activation mechanism of Orai1 via direct interaction with NCL, which may lead to calcium homeostasis imbalance and promote the proliferation of breast cancer cells. Blocking NCL-Orai1 interaction might be an effective treatment of breast cancer.

T-Type Calcium Channels: A Mixed Blessing.

The role of T-type calcium channels is well established in excitable cells, where they preside over action potential generation, automaticity, and firing. They also contribute to intracellular calcium signaling, cell cycle progression, and cell fate; and, in this sense, they emerge as key regulators also in non-excitable cells. In particular, their expression may be considered a prognostic factor in cancer. Almost all cancer cells express T-type calcium channels to the point that it has been considered a pharmacological target; but, as the drugs used to reduce their expression are not completely selective, several complications develop, especially within the heart. T-type calcium channels are also involved in a specific side effect of several anticancer agents, that act on microtubule transport, increase the expression of the channel, and, thus, the excitability of sensory neurons, and make the patient more sensitive to pain. This review puts into context the relevance of T-type calcium channels in cancer and in chemotherapy side effects, considering also the cardiotoxicity induced by new classes of antineoplastic molecules.

Ivabradine treatment lowers blood pressure and promotes cardiac and renal protection in spontaneously hypertensive rats

Hypertension is linked to hyperpolarization-activated cyclic nucleotide-gated (HCN) function, expressed in excitable and non-excitable cells. Considering that the reduction in heart rate (HR) improves coronary perfusion and cardiac performance, ivabradine (IVA) emerged as an important drug for the treatment of cardiovascular diseases. AimEvaluate if IVA chronic treatment effect can mitigate hypertension and reverse the cardiac and renal damage in SHR. Main methodsRats were divided into 4 groups treated for 14 days with PBS (1 ml/kg; i.p) or IVA (1 mg/kg; i.p): 1) WKY PBS; 2) SHR PBS; 3) WKY IVA; and 4) SHR IVA. The systolic blood pressure (SBP) was measured, indirectly, before and during the treatment period with IVA (day 0, 1, 7 and 11). Rats were subjected to artery cannulation for direct blood pressure (BP) measurement. Morphofunctional and gene expression were evaluated in the heart and kidneys. Key findingsIVA reduced SBP only in SHR on the 7th day. Direct blood pressure measurement showed that IVA chronic treatment reduced HR in the SHR. Interestingly, mean arterial pressure (MAP) was reduced in SHR IVA when compared to SHR PBS. Serum and urinary biochemical data were not altered by IVA. Moreover, IVA reduced the renal inflammatory infiltrates and increased glomerular density, besides preventing the cardiac inflammatory and hypertrophic responses. SignificanceIVA treatment lowered blood pressure, improved cardiac remodeling and inflammation, as well as decreasing renal damage in SHR. Further, IVA increased renal HCN2 mRNA and reduced cardiac HCN4 mRNA.

Glutamate Signaling and Filopodiagenesis of Astrocytoma Cells in Brain Cancers: Survey and Questions.

Astrocytes are non-excitable cells in the CNS that can cause life-threatening astrocytoma tumors when they transform to cancerous cells. Perturbed homeostasis of the neurotransmitter glutamate is associated with astrocytoma tumor onset and progression, but the factors that govern this phenomenon are less known. Herein, we review possible mechanisms by which glutamate may act in facilitating the growth of projections in astrocytic cells. This review discusses the similarities and differences between the morphology of astrocytes and astrocytoma cells, and the role that dysregulation in glutamate and calcium signaling plays in the aberrant morphology of astrocytoma cells. Converging reports suggest that ionotropic glutamate receptors and voltage-gated calcium channels expressed in astrocytes may be responsible for the abnormal filopodiagenesis or process extension leading to astrocytoma cells’ infiltration throughout the brain.

Exosomal MiR-4261 mediates calcium overload in RBCs by downregulating the expression of ATP2B4 in multiple myeloma.

BackgroundHypercalcemia induced by multiple myeloma (MM) affects the biological functions of excitable and non-excitable cells. However, red blood cells (RBCs) regulatory effect on calcium in hypercalcemia is still not fully understood.MethodsA total of 113 patients with MM osteolytic lesions were studied retrospectively. Flow cytometry and atomic absorption spectroscopy were used to detect calcium content. Immunofluorescence and Western blotting were used to investigate protein expression. GEO and miRNA databases were used to screen miRNAs. Exosomal miR-4261 migration was investigated by Transwell assay. Dual-luciferase assays confirmed the targeting relationship between miR-4261 and ATP2B4. An RBC oxidative stress model was constructed, and Omega-Agatoxin IVA was used to study the role of plasma membrane Ca2+-ATPase 4 (PMCA4) in RBCs.ResultsThe results showed that MM RBCs had calcium overload, and serum calcium levels increased as the number of RBCs decreased. The expression of PMCA4 in MM RBCs was significantly lower than in normal RBCs. The exosomal miR-4261 produced by MM cells could be transferred to RBCs to downregulate the expression of ATP2B4.ConclusionsStudies have confirmed that RBCs experience calcium overload in MM with osteolytic lesions, which is related to the downregulation of ATP2B4 by MM exosomal miR-4261.

The Kv10.1 Channel: A Promising Target in Cancer

Carcinogenesis is a multistage process involving the dysregulation of multiple genes, proteins, and pathways that make any normal cell acquire a cancer cell phenotype. Therefore, it is no surprise that numerous ion channels could be involved in this process. Since their discovery and subsequent cloning, ion channels have been established as therapeutic targets in excitable cell pathologies (e.g., cardiac arrhythmias or epilepsy); however, their involvement in non-excitable cell pathologies is relatively recent. Among all ion channels, the voltage-gated potassium channels Kv10.1 have been established as a promising target in cancer treatment due to their high expression in tumoral tissues compared to low levels in healthy tissues.

Ion Channels in Multiple Myeloma: Pathogenic Role and Therapeutic Perspectives.

Ion channels are pore-forming proteins that allow ions to flow across plasma membranes and intracellular organelles in both excitable and non-excitable cells. They are involved in the regulation of several biological processes (i.e., proliferation, cell volume and shape, differentiation, migration, and apoptosis). Recently, the aberrant expression of ion channels has emerged as an important step of malignant transformation, tumor progression, and drug resistance, leading to the idea of “onco-channelopathy”. Here, we review the contribution of ion channels and transporters in multiple myeloma (MM), a hematological neoplasia characterized by the expansion of tumor plasma cells (MM cells) in the bone marrow (BM). Deregulation of ion channels sustains MM progression by modulating intracellular pathways that promote MM cells’ survival, proliferation, and drug resistance. Finally, we focus on the promising role of ion channels as therapeutic targets for the treatment of MM patients in a combination strategy with currently used anti-MM drugs to improve their cytotoxic activity and reduce adverse effects.

Mechanisms Underlying C-type Inactivation in Kv Channels: Lessons From Structures of Human Kv1.3 and Fly Shaker-IR Channels.

Voltage-gated potassium (Kv) channels modulate the function of electrically-excitable and non-excitable cells by using several types of “gates” to regulate ion flow through the channels. An important gating mechanism, C-type inactivation, limits ion flow by transitioning Kv channels into a non-conducting inactivated state. Here, we highlight two recent papers, one on the human Kv1.3 channel and the second on the Drosophila Shaker Kv channel, that combined cryogenic electron microscopy and molecular dynamics simulation to define mechanisms underlying C-type inactivation. In both channels, the transition to the non-conducting inactivated conformation begins with the rupture of an intra-subunit hydrogen bond that fastens the selectivity filter to the pore helix. The freed filter swings outwards and gets tethered to an external residue. As a result, the extracellular end of the selectivity filter dilates and K+ permeation through the pore is impaired. Recovery from inactivation may entail a reversal of this process. Such a reversal, at least partially, is induced by the peptide dalazatide. Binding of dalazatide to external residues in Kv1.3 frees the filter to swing inwards. The extracellular end of the selectivity filter narrows allowing K+ to move in single file through the pore typical of conventional knock-on conduction. Inter-subunit hydrogen bonds that stabilize the outer pore in the dalazatide-bound structure are equivalent to those in open-conducting conformations of Kv channels. However, the intra-subunit bond that fastens the filter to the pore-helix is absent, suggesting an incomplete reversal of the process. These mechanisms define how Kv channels self-regulate the flow of K+ by changing the conformation of the selectivity filter.

A computational model of organism development and carcinogenesis resulting from cells’ bioelectric properties and communication

A sound theory of biological organization is clearly missing for a better interpretation of observational results and faster progress in understanding life complexity. The availability of such a theory represents a fundamental progress in explaining both normal and pathological organism development. The present work introduces a computational implementation of some principles of a theory of organism development, namely that the default state of cells is proliferation and motility, and includes the principle of variation and organization by closure of constraints. In the present model, the bioelectric context of cells and tissue is the field responsible for organization, as it regulates cell proliferation and the level of communication driving the system’s evolution. Starting from a depolarized (proliferative) cell, the organism grows to a certain size, limited by the increasingly polarized state after successive proliferation events. The system reaches homeostasis, with a depolarized core (proliferative cells) surrounded by a rim of polarized cells (non-proliferative in this condition). This state is resilient to cell death (random or due to injure) and to limited depolarization (potentially carcinogenic) events. Carcinogenesis is introduced through a localized event (a spot of depolarized cells) or by random depolarization of cells in the tissue, which returns cells to their initial proliferative state. The normalization of the bioelectric condition can reverse this out-of-equilibrium state to a new homeostatic one. This simplified model of embryogenesis, tissue organization and carcinogenesis, based on non-excitable cells’ bioelectric properties, can be made more realistic with the introduction of other components, like biochemical fields and mechanical interactions, which are fundamental for a more faithful representation of reality. However, even a simple model can give insight for new approaches in complex systems and suggest new experimental tests, focused in its predictions and interpreted under a new paradigm.

GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice.

Long bones are formed and repaired through the process of endochondral ossification. Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth. G protein-gated inwardly-rectifying K+ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2-/-, Girk3-/- and Girk2/3-/- mice. Bones from 12-week-old Girk2-/- mice were normal in length, but femurs and tibiae from Girk3-/- and Girk2/3-/- mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3-/- mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3-/- chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3-/- mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.

Potassium Channel KCNH1 Activating Variants Cause Altered Functional and Morphological Ciliogenesis

The primary cilium is a non-motile sensory organelle that extends from the surface of most vertebrate cells and transduces signals regulating proliferation, differentiation, and migration. Primary cilia dysfunctions have been observed in cancer and in a group of heterogeneous disorders called ciliopathies, characterized by renal and liver cysts, skeleton and limb abnormalities, retinal degeneration, intellectual disability, ataxia, and heart disease and, recently, in autism spectrum disorder, schizophrenia, and epilepsy. The potassium voltage-gated channel subfamily H member 1 (KCNH1) gene encodes a member of the EAG (ether-à-go-go) family, which controls potassium flux regulating resting membrane potential in both excitable and non-excitable cells and is involved in intracellular signaling, cell proliferation, and tumorigenesis. KCNH1 missense variants have been associated with syndromic neurodevelopmental disorders, including Zimmermann-Laband syndrome 1 (ZLS1, MIM #135500), Temple-Baraitser syndrome (TMBTS, MIM #611816), and, recently, with milder phenotypes as epilepsy. In this work, we provide evidence that KCNH1 localizes at the base of the cilium in pre-ciliary vesicles and ciliary pocket of human dermal fibroblasts and retinal pigment epithelial (hTERT RPE1) cells and that the pathogenic missense variants (L352V and R330Q; NP_002229.1) perturb cilia morphology, assembly/disassembly, and Sonic Hedgehog signaling, disclosing a multifaceted role of the protein. The study of KCNH1 localization, its functions related to primary cilia, and the alterations introduced by mutations in ciliogenesis, cell cycle coordination, cilium morphology, and cilia signaling pathways could help elucidate the molecular mechanisms underlying neurological phenotypes and neurodevelopmental disorders not considered as classical ciliopathies but for which a significant role of primary cilia is emerging.

Differential Expression of Genes Regulating Store-operated Calcium Entry in Conjunction With Mitochondrial Dynamics as Potential Biomarkers for Cancer: A Single-Cell RNA Analysis.

Regulation of intracellular concentration of calcium levels is crucial for cell signaling, homeostasis, and in the pathology of diseases including cancer. Agonist-induced entry of calcium ions into the non-excitable cells is mediated by store-operated calcium channels (SOCs). This pathway is activated by the release of calcium ions from the endoplasmic reticulum and further regulated by the calcium uptake through mitochondria leading to calcium-dependent inactivation of calcium-release activated calcium channels (CARC). SOCs including stromal interaction molecules (STIM) and ORAI proteins have been implicated in tumor growth, progression, and metastasis. In the present study, we analyzed the mRNA and protein expression of genes mediating SOCs—STIM1, STIM2, ORAI1, ORAI2, ORAI3, TRPC1, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7, TRPV1, TRPV2, TRPM1, and TRPM7 in head and neck squamous cell cancer (HNSC) patients using TCGA and CPTAC analysis. Further, our in silico analysis showed a significant correlation between the expression of SOCs and genes involved in the mitochondrial dynamics (MDGs) both at mRNA and protein levels. Protein-protein docking results showed lower binding energy for SOCs with MDGs. Subsequently, we validated these results using gene expression and single-cell RNA sequencing datasets retrieved from Gene Expression Omnibus (GEO). Single-cell gene expression analysis of HNSC tumor tissues revealed that SOCs expression is remarkably associated with the MDGs expression in both cancer and fibroblast cells.

Involvement of ORAI1/SOCE in Human AML Cell Lines and Primary Cells According to ABCB1 Activity, LSC Compartment and Potential Resistance to Ara-C Exposure.

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

A neuropsin-based optogenetic tool for precise control of Gq signaling.

Gq-coupled receptors regulate numerous physiological processes by activating enzymes and inducing intracellular Ca2+ signals. There is a strong need for an optogenetic tool that enables powerful experimental control over Gq signaling. Here, we present chicken opsin 5 (cOpn5) as the long sought-after, single-component optogenetic tool that mediates ultra-sensitive optical control of intracellular Gq signaling with high temporal and spatial resolution. Expressing cOpn5 in HEK 293T cells and primary mouse astrocytes enables blue light-triggered, Gq-dependent Ca2+ release from intracellular stores and protein kinase C activation. Strong Ca2+ transients were evoked by brief light pulses of merely 10 ms duration and at 3 orders lower light intensity of that for common optogenetic tools. Photostimulation of cOpn5-expressing cells at the subcellular and single-cell levels generated fast intracellular Ca2+ transition, thus demonstrating the high spatial precision of cOpn5 optogenetics. The cOpn5-mediated optogenetics could also be applied to activate neurons and control animal behavior in a circuit-dependent manner. cOpn5 optogenetics may find broad applications in studying the mechanisms and functional relevance of Gq signaling in both non-excitable cells and excitable cells in all major organ systems.

Exaggerated Store‐Operated Calcium Entry: A Mechanism for High Glucose‐induced Podocyte Injury and Mitochondria Damage

Purpose and hypothesisPodocyte injury induced by hyperglycemia is the key factor contributing to proteinuria and kidney dysfunction in diabetic nephropathy (DN). Accumulating evidence suggests that mitochondria dysfunction leads to DN‐associated podocyte injury. However, the mechanism of podocyte mitochondria injury by high glucose (HG) is poorly understood. Store‐operated calcium entry (SOCE) is essential for multiple cell functions in both excitable and non‐excitable cells. However, the role of this Ca2+ signaling in podocytopathy remains unknown. The present study was carried out to test the hypothesis that enhanced SOCE mediated HG‐induced podocyte injury and mitochondria damage.MethodsAll experiments were carried out using cultured immortalized human podocytes. The conventional whole‐cell patch‐clamp was performed to measure Orai1 channel currents. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and STIM1. Calcium imaging was used to evaluate SOCE. Confocal microscopy was used to visualize podocyte actin arrangement. Mitochondrial function was evaluated by measuring: 1) the mitochondria membrane potential (MMP) using TMRE fluorescence; 2) generation of mitochondria reactive oxygen species (ROS) using MitoSox Red Mitochondrial Superoxide Indicator; 3) ATP production using ATP assay kit. In addition, CRISPR‐CAS 9 approach was used to delete Orai1 in podocytes.ResultsThapsigargin (TG, 1 µM), the activator of Orai1 channel induced robust inward currents which were blocked by La3+ (2 µM), an inhibitor of Orai1 channel. Ang II (1 µM) also evoked inward currents, which were significantly blunted by BTP2 (10 µM), an Orai1 channel blocker. Orai1 protein abundance significantly increased by HG (25 mM) treatment for the time periods ranging from 2 to 12 h. This HG induced Orai1 increase was dose‐dependent. Consistently, Ca2+ imaging experiment showed that HG treatment for 12 h significantly increased podocyte SOCE. Thus, the biochemical and functional data suggested an enhancement of SOCE by HG. Furthermore, HG treatment resulted in podocyte cytoskeleton rearrangement by formation of cortical F‐actin, an indication of podocyte injury. This HG response was significantly blunted by BTP2 and Orai1 CRISPR‐CAS 9 lentivirus. Moreover, HG treatment significantly decreased podocyte MMP, ATP production and increased mitochondrial ROS generation, all of which were significantly inhibited by BTP2. Furthermore, activation of SOCE by TG significantly increased mitochondrial ROS generation, which was prevented by pre‐treatment of BTP2.ConclusionEnhanced SOCE contributed to HG induced podocyte injury and mitochondrial dysfunction.

STIM and Orai Mediated Regulation of Calcium Signaling in Age-Related Diseases.

Tight spatiotemporal regulation of intracellular Ca2+ plays a critical role in regulating diverse cellular functions including cell survival, metabolism, and transcription. As a result, eukaryotic cells have developed a wide variety of mechanisms for controlling Ca2+ influx and efflux across the plasma membrane as well as Ca2+ release and uptake from intracellular stores. The STIM and Orai protein families comprising of STIM1, STIM2, Orai1, Orai2, and Orai3, are evolutionarily highly conserved proteins that are core components of all mammalian Ca2+ signaling systems. STIM1 and Orai1 are considered key players in the regulation of Store Operated Calcium Entry (SOCE), where release of Ca2+ from intracellular stores such as the Endoplasmic/Sarcoplasmic reticulum (ER/SR) triggers Ca2+ influx across the plasma membrane. SOCE, which has been widely characterized in non-excitable cells, plays a central role in Ca2+-dependent transcriptional regulation. In addition to their role in Ca2+ signaling, STIM1 and Orai1 have been shown to contribute to the regulation of metabolism and mitochondrial function. STIM and Orai proteins are also subject to redox modifications, which influence their activities. Considering their ubiquitous expression, there has been increasing interest in the roles of STIM and Orai proteins in excitable cells such as neurons and myocytes. While controversy remains as to the importance of SOCE in excitable cells, STIM1 and Orai1 are essential for cellular homeostasis and their disruption is linked to various diseases associated with aging such as cardiovascular disease and neurodegeneration. The recent identification of splice variants for most STIM and Orai isoforms while complicating our understanding of their function, may also provide insight into some of the current contradictions on their roles. Therefore, the goal of this review is to describe our current understanding of the molecular regulation of STIM and Orai proteins and their roles in normal physiology and diseases of aging, with a particular focus on heart disease and neurodegeneration.

Store-Operated Calcium Entry and Its Implications in Cancer Stem Cells.

Tumors are composed by a heterogeneous population of cells. Among them, a sub-population of cells, termed cancer stem cells, exhibit stemness features, such as self-renewal capabilities, disposition to differentiate to a more proliferative state, and chemotherapy resistance, processes that are all mediated by Ca2+. Ca2+ homeostasis is vital for several physiological processes, and alterations in the patterns of expressions of the proteins and molecules that modulate it have recently become a cancer hallmark. Store-operated Ca2+ entry is a major mechanism for Ca2+ entry from the extracellular medium in non-excitable cells that leads to increases in the cytosolic Ca2+ concentration required for several processes, including cancer stem cell properties. Here, we focus on the participation of STIM, Orai, and TRPC proteins, the store-operated Ca2+ entry key components, in cancer stem cell biology and tumorigenesis.

Gambierol Blocks a K+ Current Fraction without Affecting Catecholamine Release in Rat Fetal Adrenomedullary Cultured Chromaffin Cells.

Gambierol inhibits voltage-gated K+ (KV) channels in various excitable and non-excitable cells. The purpose of this work was to study the effects of gambierol on single rat fetal (F19–F20) adrenomedullary cultured chromaffin cells. These excitable cells have different types of KV channels and release catecholamines. Perforated whole-cell voltage-clamp recordings revealed that gambierol (100 nM) blocked only a fraction of the total outward K+ current and slowed the kinetics of K+ current activation. The use of selective channel blockers disclosed that gambierol did not affect calcium-activated K+ (KCa) and ATP-sensitive K+ (KATP) channels. The gambierol concentration necessary to inhibit 50% of the K+ current-component sensitive to the polyether (IC50) was 5.8 nM. Simultaneous whole-cell current-clamp and single-cell amperometry recordings revealed that gambierol did not modify the membrane potential following 11s depolarizing current-steps, in both quiescent and active cells displaying repetitive firing of action potentials, and it did not increase the number of exocytotic catecholamine release events, with respect to controls. The subsequent addition of apamin and iberiotoxin, which selectively block the KCa channels, both depolarized the membrane and enhanced by 2.7 and 3.5-fold the exocytotic event frequency in quiescent and active cells, respectively. These results highlight the important modulatory role played by KCa channels in the control of exocytosis from fetal (F19–F20) adrenomedullary chromaffin cells.

Harnessing the Membrane Potential to Combat Cancer Progression.

Rapid fluctuations in the plasma membrane potential (Vm) provide the basis underlying the action potential waveform in electrically excitable cells; however, a growing body of literature shows that the Vm is also functionally instructive in nonexcitable cells, including cancer cells. Various ion channels play a key role in setting and fine tuning the Vm in cancer and stromal cells within the tumor microenvironment (TME), raising the possibility that the Vm could be targeted therapeutically using ion channel-modulating compounds. Emerging evidence points to the Vm as a viable therapeutic target, given its functional significance in regulating cell cycle progression, migration, invasion, immune infiltration, and pH regulation. Several compounds are now undergoing clinical trials and there is increasing interest in therapeutic manipulation of the Vm via application of pulsed electric fields. The purpose of this article is to update the reader on the significant recent and ongoing progress to elucidate the functional significance of Vm regulation in tumors, to highlight key remaining questions and the prospect of future therapeutic targeting. In particular, we focus on key developments in understanding the functional consequences of Vm alteration on tumor development via the activation of small GTPase (K-Ras and Rac1) signaling, as well as the impact of Vm changes within the heterogeneous TME on immune cell function and cancer progression.