Ion Channels in Cell Proliferation and Apoptotic Cell Death

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl⁻ and K⁺ channels. Besides regulating cytosolic Cl⁻ activity, osmolyte flux and, thus, cell volume, most Cl⁻ channels allow HCO₃⁻ exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K⁺ exit through K⁺ channels may decrease intracellular K⁺ concentration, which in turn favors apoptotic cell death. K⁺ channel activity further maintains the cell membrane potential, a critical determinant of Ca²⁺ entry through Ca²⁺ channels. Cytosolic Ca²⁺ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca²⁺ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.     

昆虫离子通道及其抗药性研究进展

昆虫细胞膜离子通道是多种杀虫剂的作用靶标,通道功能特性的变异等与害虫抗药性密切相关.电压钳及膜片钳等电生理技术在离子通道功能研究中具有独特优势,在杀虫剂作用机理及害虫抗性机理研究中越来越受到重视.昆虫细胞膜离子通道主要包括配体门控通道和电压门控通道两大类.配体门控通道主要包括乙酰胆碱受体、GABA和谷氨酸受体通道等.电压门控通道主要有钠、钾和钙通道等,其中钠通道研究成果较多,与害虫抗性关系密切.由于钙离子的重要生理功能,随着研究深入,钙通道将成为研究重点.     

Role of Potassium Channels in Amyloid-Induced Cell Death

Abstract: Basal forebrain cholinergic neurons are severely depleted early in Alzheimer's disease and appear particularly susceptible to amyloid β-peptide (Aβ) toxicity in vivo. To model this effect in vitro, a cholinergic septal cell line (SN56) was exposed to Aβ. SN56 cells exhibited a tetraethylammonium (TEA)-sensitive outward K⁺ current with delayed rectifier characteristics. Increases of 64% (±19; p < 0.02) and 44% (±12; p < 0.02) in K⁺ current density were noted 6–12 and 12–18 h following the addition of Aβ to SN56 cell cultures, respectively. Morphological observation and staining for cell viability showed that 25 ± 4 and 39 ± 4% of SN56 cells were dead after 48- and 96-h exposures to Aβ, respectively. Perfusion of SN56 cells with 10–20 mM TEA blocked 71 ± 6 to 92 ± 2% of the outward currents, widened action potentials, elevated [Ca²⁺]_i, and inhibited 89 ± 14 and 68 ± 14% of the Aβ toxicity. High [K⁺]_o, which depolarizes cell membranes and increases [Ca²⁺]_i, also protected SN56 cells from Aβ toxicity. This effect appeared specific since glucose deprivation of SN56 cells did not alter K⁺ current density and TEA did not protect these cells from hypoglycemic cell death. Furthermore, Aβ was toxic to a dopaminergic cell line (MES23.5) that expressed a K⁺ current with delayed rectifier characteristics; K⁺ current density was not altered by Aβ and MES23.5 cells were not protected by TEA from Aβ toxicity. In contrast, a noncholinergic septal cell line (SN48) that shows minimal outward K⁺ currents was resistant to the toxicity of Aβ. These data suggest that a K⁺ channel with delayed rectifier characteristics may play an important role in Aβ-mediated toxicity for septal cholinergic cells.     

Unstructural Biology of TRP Ion Channels: The Role of Intrinsically Disordered Regions in Channel Function and Regulation

The first genuine high-resolution single particle cryo-electron microscopy structure of a membrane protein determined was a transient receptor potential (TRP) ion channel, TRPV1, in 2013. This methodical breakthrough opened up a whole new world for structural biology and ion channel aficionados alike. TRP channels capture the imagination due to the sheer endless number of tasks they carry out in all aspects of animal physiology. To date, structures of at least one representative member of each of the six mammalian TRP channel subfamilies as well as of a few non-mammalian families have been determined. These structures were instrumental for a better understanding of TRP channel function and regulation. However, all of the TRP channel structures solved so far are incomplete since they miss important information about highly flexible regions found mostly in the channel N- and C-termini. These intrinsically disordered regions (IDRs) can represent between a quarter to almost half of the entire protein sequence and act as important recruitment hubs for lipids and regulatory proteins. Here, we analyze the currently available TRP channel structures with regard to the extent of these “missing” regions and compare these findings to disorder predictions. We discuss select examples of intra- and intermolecular crosstalk of TRP channel IDRs with proteins and lipids as well as the effect of splicing and post-translational modifications, to illuminate their importance for channel function and to complement the prevalently discussed structural biology of these versatile and fascinating proteins with their equally relevant ’unstructural’ biology.     

精子膜表面配体依赖性离子通道与精子顶体反应

配体依赖性离子通道是一类由神经递质调控的跨膜离子通道。研究发现它们在精子的顶体反应中起了重要作用,顶体反应是精子完成受精的一个关键步骤。至今已发现3种配体依赖性离子通道受体存在于精子头部的质膜上,它们是乙酰胆碱受体、甘氨酸受体和GABAa受体。尽管乙酰胆碱受体和甘氨酸受体已被清楚的证明参与了ZP3诱导的顶体反应,GABAa受体的功能则相对复杂,需进一步研究。这类受体在精子膜电压变化中起的作用和由此导致的膜电位改变对于精子顶体反应的重要性,为精子顶体反应提供了一个可能的信号传递途径。     

Role of TRPM7 Channels in Hyperglycemia-Mediated Injury of Vascular Endothelial Cells

This study investigated the change of transient receptor potential melastatin 7 (TRPM7) expression by high glucose and its role in hyperglycemia induced injury of vascular endothelial cells. Human umbilical vein endothelial cells (HUVECs) were incubated in the presence or absence of high concentrations of D-glucose (HG) for 72h. RT-PCR, Real-time PCR, Western blotting, Immunofluorescence staining and whole-cell patch-clamp recordings showed that TRPM7 mRNA, TRPM7 protein expression and TRPM7-like currents were increased in HUVECs following exposure to HG. In contrast to D-glucose, exposure of HUVECs to high concentrations of L-glucose had no effect. HG increased reactive oxygen species (ROS) generation, cytotoxicity and decreased endothelial nitric oxide synthase protein expression, which could be attenuated by knockdown of TRPM7 with TRPM7 siRNA. The protective effect of silencing TRPM7 against HG induced endothelial injury was abolished by U0126, an inhibitor of the extracellular signal-regulated kinase signaling pathway. These observations suggest that TRPM7 channels play an important role in hyperglycemia-induced injury of vascular endothelial cells.     

Amphotericin B Membrane Action: Role for Two Types of Ion Channels in Eliciting Cell Survival and Lethal Effects

The formation of aqueous pores by the polyene antibiotic amphotericin B (AmB) is at the basis of its fungicidal and leishmanicidal action. However, other types of nonlethal and dose-dependent biphasic effects that have been associated with the AmB action in different cells, including a variety of survival responses, are difficult to reconcile with the formation of a unique type of ion channel by the antibiotic. In this respect, there is increasing evidence indicating that AmB forms nonaqueous (cation-selective) channels at concentrations below the threshold at which aqueous pores are formed. The main foci of this review will be (1) to provide a summary of the evidence supporting the formation of cation-selective ion channels and aqueous pores by AmB in lipid membrane models and in the membranes of eukaryotic cells; (2) to discuss the influence of membrane parameters such as thickness fluctuations, the type of sterol present and the existence of sterol-rich specialized lipid raft microdomains in the formation process of such channels; and (3) to develop a cell model that serves as a framework for understanding how the intracellular K(+) and Na(+) concentration changes induced by the cation-selective AmB channels enhance multiple survival response pathways before they are overcome by the more sustained ion fluxes, Ca(2+)-dependent apoptotic events and cell lysis effects that are associated with the formation of AmB aqueous pores.     

Blockade by Neurotransmitter Antagonists of Veratridine-Activated Ion Channels in Neuronal Cell Lines

Abstract: The voltage-dependent Na⁺ ionophore of various neuronal cells is permeable not only to Na⁺ ions but also to guanidinium ions. Therefore, the veratridine-(or aconitine-) stimulated influx of [¹⁴C]guanidinium in neuroblastoma × glioma hybrid cells was measured to characterize the Na⁺ ionophore of these cells. Half-maximal stimulation of guanidinium uptake was seen at 30 μ M veratridine. At 1 m M guanidinium, the veratridine-stimulated uptake of guanidinium was lowered to 50% by approximately 60 m M Li⁺, Na⁺, or K⁺ and by a few millimolar Mn²⁺, Co²⁺, or Ni²⁺. The basal, as well as the veratridine-stimulated, uptake of guanidinium was inhibited by the cholinergic antagonists (+)-tubocurarine ( K_i = 50 to 500 n M ) and atropine ( K_i = 5 to 30 μ M ) and the adrenergic antagonists phentolamine ( K_i = 5 μ M ) and propranolol ( K_i = 60 μ M ). The specificity of the inhibitory effects of these agents is stressed by the ineffectiveness of various other neurotransmitter antagonists. However, the corresponding ionophore in neuroblastoma cells (clone N1E-115) seems to be regulated differently. While phentolamine and propranolol inhibit the veratridine-activated uptake as in the hybrid cells, (+)-tubocurarine and atropine exert only a slight effect.     

Acidosis,Acid-Sensing Ion Channels,and Neuronal Cell Death

Acidosis is a common feature of many neuronal diseases and often accompanied with adverse consequences such as pain and neuronal injury. Before the discovery of acid-sensing ion channels (ASICs), protons were usually considered as a modulator of other ion channels, such as voltage-gated calcium channels, N-methyl-d-aspartate, and γ-amino butyric acid(A) receptor channels. Accordingly, the functional effects of acidosis were considered as consequences of modulations of these channels. Since the first cloning of ASICs in 1997, the conventional view on acidosis-mediated pain and cell injury has been dramatically changed. To date, ASICs, which are directly activated by extracellular protons, are shown to mediate most of the acidosis-associated physiological and pathological functions. For example, ASIC1a channels are reported to mediate acidosis-induced ischemic neuronal death. In this article, we will review the possible mechanisms that underlie ASIC1a channel-mediated neuronal death and discuss ASIC1a channel modulators involved in this process.     

Double-Stranded RNA Attenuates the Barrier Function of Human Pulmonary Artery Endothelial Cells

Circulating RNA may result from excessive cell damage or acute viral infection and can interact with vascular endothelial cells. Despite the obvious clinical implications associated with the presence of circulating RNA, its pathological effects on endothelial cells and the governing molecular mechanisms are still not fully elucidated. We analyzed the effects of double stranded RNA on primary human pulmonary artery endothelial cells (hPAECs). The effect of natural and synthetic double-stranded RNA (dsRNA) on hPAECs was investigated using trans-endothelial electric resistance, molecule trafficking, calcium (Ca²⁺) homeostasis, gene expression and proliferation studies. Furthermore, the morphology and mechanical changes of the cells caused by synthetic dsRNA was followed by in-situ atomic force microscopy, by vascular-endothelial cadherin and F-actin staining. Our results indicated that exposure of hPAECs to synthetic dsRNA led to functional deficits. This was reflected by morphological and mechanical changes and an increase in the permeability of the endothelial monolayer. hPAECs treated with synthetic dsRNA accumulated in the G1 phase of the cell cycle. Additionally, the proliferation rate of the cells in the presence of synthetic dsRNA was significantly decreased. Furthermore, we found that natural and synthetic dsRNA modulated Ca²⁺ signaling in hPAECs by inhibiting the sarco-endoplasmic Ca²⁺-ATPase (SERCA) which is involved in the regulation of the intracellular Ca²⁺ homeostasis and thus cell growth. Even upon synthetic dsRNA stimulation silencing of SERCA3 preserved the endothelial monolayer integrity. Our data identify novel mechanisms by which dsRNA can disrupt endothelial barrier function and these may be relevant in inflammatory processes.     

A Role for Exocyst in Maturation and Bactericidal Function of Staphylococci‐Containing Endothelial Cell Phagosomes

Bacteria that invade human endothelial cells can be efficiently eliminated in phagolysosomes. We investigated the role of vesicle tethering exocyst complex in maturation and function of endothelial cell phagosomes harbouring staphylococci or latex beads. Exocyst complex proteins (Sec5, ‐8, ‐10, Exo70) together with recycling endosome marker Rab11 were detected in vesicles that dynamically interacted and seemingly fused with endothelial cell phagosomes. Knockdown of exocyst proteins Sec8 and Exo70 inhibited the accumulation of Rab11‐positive vesicles at the phagosomes. Furthermore, knockdown of exocyst proteins and Rab11 greatly reduced acidification of phagosomes and significantly diminished the elimination of invaded staphylococci in endothelial cells. The inhibitory effect of Exo70 knockdown on bacterial elimination could be rescued by constitutively active Rab11‐Q70L. Our data suggest that exocyst complex controls the interaction of recycling endocytic vesicles with phagosomes and this process is involved in maturation and functioning of the phagosomes in endothelial cells.      

The Role of TRP Channels in Oxidative Stress-induced Cell Death

The transient receptor potential (TRP) protein superfamily is a diverse group of voltage-independent calcium-permeable cation channels expressed in mammalian cells. These channels have been divided into six subfamilies, and two of them, TRPC and TRPM, have members that are widely expressed and activated by oxidative stress. TRPC3 and TRPC4 are activated by oxidants, which induce Na⁺ and Ca²⁺ entry into cells through mechanisms that are dependent on phospholipase C. TRPM2 is activated by oxidative stress or TNFα, and the mechanism involves production of ADP-ribose, which binds to an ADP-ribose binding cleft in the TRPM2 C-terminus. Treatment of HEK 293T cells expressing TRPM2 with H₂O₂ resulted in Ca²⁺ influx and increased susceptibility to cell death, whereas coexpression of the dominant negative isoform TRPM2-S suppressed H₂O₂-induced Ca²⁺ influx, the increase in [Ca²⁺]_i, and onset of apoptosis. U937-ecoR monocytic cells expressing increased levels of TRPM2 also exhibited significantly increased [Ca²⁺]_i and increased apoptosis after treatment with H₂O₂ or TNFα. A dramatic increase in caspase 8, 9, 3, 7, and PARP cleavage was observed in TRPM2-expressing cells, demonstrating a downstream mechanism through which cell death is mediated. Inhibition of endogenous TRPM2 function through three approaches, depletion of TRPM2 by RNA interference, blockade of the increase in [Ca²⁺]_i through TRPM2 by calcium chelation, or expression of the dominant negative splice variant TRPM2-S protected cell viability. H₂O₂ and amyloid β-peptide also induced cell death in primary cultures of rat striatal cells, which endogenously express TRPM2. TRPM7 is activated by reactive oxygen species/nitrogen species, resulting in cation conductance and anoxic neuronal cell death, which is rescued by suppression of TRPM7 expression. TRPM2 and TRPM7 channels are physiologically important in oxidative stress-induced cell death.     

Structure,Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels

Voltage-gated ion channels are crucial for both neuronal and cardiac excitability. Decades of research have begun to unravel the intriguing machinery behind voltage sensitivity. Although the details regarding the arrangement and movement in the voltage-sensor domain are still debated, consensus is slowly emerging. There are three competing conceptual models: the helical-screw, the transporter, and the paddle model. In this review we explore the structure of the activated voltage-sensor domain based on the recent X-ray structure of a chimera between Kv1.2 and Kv2.1. We also present a model for the closed state. From this we conclude that upon depolarization the voltage sensor S4 moves approximately 13 A outwards and rotates approximately 180 degrees, thus consistent with the helical-screw model. S4 also moves relative to S3b which is not consistent with the paddle model. One interesting feature of the voltage sensor is that it partially faces the lipid bilayer and therefore can interact both with the membrane itself and with physiological and pharmacological molecules reaching the channel from the membrane. This type of channel modulation is discussed together with other mechanisms for how voltage-sensitivity is modified. Small effects on voltage-sensitivity can have profound effects on excitability. Therefore, medical drugs designed to alter the voltage dependence offer an interesting way to regulate excitability.     

Cell Migration in BeWo Cells and the Role of Epithelial Sodium Channels

Cell migration/proliferation processes associated with wound healing were measured in BeWo cells at 6 h, when mitosis is still scarce. Cells were cultured in medium with 1% fetal bovine serum to minimize proliferation. BeWo cell migration covered 20.6 ± 7.0%, 38.0 ± 5.4%, 16.6 ± 4.8% and 13.7 ± 3.6% of the wound when cultivated under control, aldosterone (100 nM, 12 h), aldosterone plus amiloride (10 μM) and amiloride treatments, respectively. When BeWo cells were treated with aldosterone, there was an increase in wound healing (P < 0.05), which was prevented by adding the ENaC blocker amiloride (P < 0.05, n = 16). Immunocytochemistry studies showed that the three ENaC subunits showed greater expression at the leading edge of the wound 3 h after injury, supporting the notion that these proteins participate in a postinjury signal. Antisense oligonucleotides directed against the α-ENaC subunit decreased the migratory response of the cells compared to the sense treated cells or the cells without oligonucleotides (P < 0.001, n = 16): 30.2 ± 3.7%, 17.6 ± 1.3%, 27.5 ± 1.5% and 20.2 ± 1.5% reinvasion of the wound with aldosterone, aldosterone plus antisense, aldosterone plus sense treatments and control conditions, respectively. Aldosterone and amiloride influence wound healing in BeWo cells, probably by their effects upon ENaCs, transmitting a signal to the cell cytoplasm for the release of several agents that promote cell migration.     

多不饱和脂肪酸对钾通道的调控作用及机理

多不饱和脂肪酸具有包括离子通道在内的众多作用靶点,通过作用于这些靶点,可以有效保护免疫系统、神经系统和心血管系统的功能,在一定程度上保护人体健康。电压门控钾离子通道家族K_V7通道和大电导钙离子激活的钾离子通道(BK_Ca)广泛表达于机体的各类组织中,具有重要的生理或病理功能。本综述围绕K_V7和BK_Ca通道,根据对已有报道的汇总,多不饱和脂肪酸可以增大K_V7和BK_Ca通道的电流幅值,其中对K_V7通道电流的影响主要是改变其电压依赖特性和最大电导值,而对BK_Ca通道电流的影响主要是改变其孔道区域关闭态的构象。此外,多不饱和脂肪酸对K_V7和BK_Ca通道功能的调节也会受到共表达的辅助亚基影响,但相关机制有待进一步阐明。深入理解多不饱和脂肪酸对K_V7和BK_Ca通道调节作用效果和分子机制,有助于全面理解K_V7和BK     

细胞因子对内皮祖细胞功能影响的研究进展

内皮祖细胞(EPCs)是一种具有较强增殖能力的前体细胞,血管损伤或者缺血会刺激骨髓EPCs动员,迁移、归巢于相应的靶位,然后分化为内皮细胞(ECs),从而参与血管修复和血管新生。因此,EPCs的成功发现为缺血性和血管损伤性疾病的治疗提供了新策略。但是EPCs存在动员率低、靶向性较差和功能不全等问题。大量研究显示细胞因子对EPCs的动员、归巢、增殖和分化等均起着重要的调节作用,同时,通过调控细胞因子能改善EPCs的功能活性,因此选择合适的细胞因子来提高EPCs功能变得非常重要。现总结了近年来细胞因子对EPCs功能影响的研究进展,并提出有待解决的问题和作一定的展望。     

Compartmentalizing Proximal FGFR1 Signaling in Ovine Placental Artery Endothelial Cell Caveolae

Caveolae orchestrate the dominant placental angiogenic growth factor fibroblast growth factor 2 (FGF2) signaling primarily via FGF receptor 1 (FGFR1) in placental artery endothelial cells; however, how the proximal FGF2/FGFR1 signaling is organized in the caveolae is obscure. We have shown in the present study that the FGFR substrate 2alpha (FRS2alpha) is physically associated with FGFR1, and both are targeted to the caveolae via interaction with caveolin-1 in ovine fetoplacental artery endothelial cells. Treatment with FGF2 rapidly stimulated time- and concentration-dependent FRS2alpha tyrosine phosphorylation and recruited the cytosolic growth factor receptor-bound protein 2 (GRB2)-GRB2-associated binding protein 1 (GAB1) complex to the caveolae, where they formed a ternary complex with FRS2alpha. Disruption of caveolae by cholesterol depletion with methyl-beta-cyclodextrin inhibited FGF2-induced FRS2alpha tyrosine phosphorylation, and it blocked the FGF2-induced recruitment of GRB2 and GAB1 to the caveolae and formation of the FRS2alpha-GRB2-GAB1 complex in the caveolae, as well as activation of the PI3K/AKT1 and MAPK1/2 pathways. Thus, these findings have demonstrated that the proximal fibroblast growth factor (FGF2/FGFR1) signaling is compartmentalized in the placental endothelial caveolae via the FGFR substrate 2α that mediates formation of a FRS2α-GRB2-GAB1 complex.