Xuexin Zhang

TWIK-1 and TREK-1 Are Potassium Channels Contributing Significantly to Astrocyte Passive Conductance in Rat Hippocampal Slices

Expression of a linear current–voltage (I–V) relationship (passive) K+ membrane conductance is a hallmark of mature hippocampal astrocytes. However, the molecular identifications of the K+ channels underlying this passive conductance remain unknown. We provide the following evidence supporting significant contribution of the two-pore domain K+ channel (K2P) isoforms, TWIK-1 and TREK-1, to this conductance. First, both passive astrocytes and the cloned rat TWIK-1 and TREK-1 channels expressed in CHO cells conduct significant amounts of Cs+ currents, but vary in their relative PCs/PK permeability, 0.43, 0.10, and 0.05, respectively. Second, quinine, which potently inhibited TWIK-1 (IC50 = 85 μm) and TREK-1 (IC50 = 41 μm) currents, also inhibited astrocytic passive conductance by 58% at a concentration of 200 μm. Third, a moderate sensitivity of passive conductance to low extracellular pH (6.0) supports a combined expression of acid-insensitive TREK-1, and to a lesser extent, acid-sensitive TWIK-1. Fourth, the astrocyte passive conductance showed low sensitivity to extracellular Ba2+, and extracellular Ba2+ blocked TWIK-1 channels at an IC50 of 960 μm and had no effect on TREK-1 channels. Finally, an immunocytochemical study showed colocalization of TWIK-1 and TREK-1 proteins with the astrocytic markers GLAST and GFAP in rat hippocampal stratum radiatum. In contrast, another K2P isoform TASK-1 was mainly colocalized with the neuronal marker NeuN in hippocampal pyramidal neurons and was expressed at a much lower level in astrocytes. These results support TWIK-1 and TREK-1 as being the major components of the long-sought K+ channels underlying the passive conductance of mature hippocampal astrocytes.

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca2+ sensor stromal interacting molecule 1 (STIM1) and the Ca2+ channel Orai1 mediate the ubiquitous store-operated Ca2+ entry (SOCE) pathway activated by depletion of internal Ca2+ stores and mediated through the highly Ca2+-selective, Ca2+ release-activated Ca2+ (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca2+ currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca2+ imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

Ryanodine Receptor Blockade Reduces Amyloid-β Load and Memory Impairments in Tg2576 Mouse Model of Alzheimer Disease

In Alzheimer disease (AD), the perturbation of the endoplasmic reticulum (ER) calcium (Ca2+) homeostasis has been linked to presenilins, the catalytic core in γ-secretase complexes cleaving the amyloid precursor protein (APP), thereby generating amyloid-β (Aβ) peptides. Here we investigate whether APP contributes to ER Ca2+ homeostasis and whether ER Ca2+ could in turn influence Aβ production. We show that overexpression of wild-type human APP (APP695), or APP harboring the Swedish double mutation (APPswe) triggers increased ryanodine receptor (RyR) expression and enhances RyR-mediated ER Ca2+ release in SH-SY5Y neuroblastoma cells and in APPswe-expressing (Tg2576) mice. Interestingly, dantrolene-induced lowering of RyR-mediated Ca2+ release leads to the reduction of both intracellular and extracellular Aβ load in neuroblastoma cells as well as in primary cultured neurons derived from Tg2576 mice. This Aβ reduction can be accounted for by decreased Thr-668-dependent APP phosphorylation and β- and γ-secretases activities. Importantly, dantrolene diminishes Aβ load, reduces Aβ-related histological lesions, and slows down learning and memory deficits in Tg2576 mice. Overall, our data document a key role of RyR in Aβ production and learning and memory performances, and delineate RyR-mediated control of Ca2+ homeostasis as a physiological paradigm that could be targeted for innovative therapeutic approaches.

Orai3 is an estrogen receptor α-regulated Ca2+ channel that promotes tumorigenesis

Store‐operated Ca2+ entry (SOCE) encoded by Orai1 proteins is a ubiquitous Ca2+‐selective conductance involved in cellular proliferation and migration. We recently described up‐regulation of Orai3 channels that selectively mediate SOCE in estrogen receptor α‐expressing (ERα+) breast cancer cells. However, the connection between ERα and Orai3 and the role of Orai3 in tumorigenesis remain unknown. Here, we show that ERα knockdown decreases Orai3 mRNA (by ~63%) and protein (by ~44%) with no effect on Orai1. ERα knockdown decreases Orai3‐mediated SOCE (by ~43%) and the corresponding Ca2+ release‐activated Ca2+ (CRAC) current (by ~42%) in ERα+ MCF7 cells. The abrogation of SOCE in MCF7 cells on ERα knockdown can be rescued by ectopic expression of Orai3. ERα activation increased Orai3 expression and SOCE in MCF7 cells. Epidermal growth factor (EGF) and thrombin stimulate Ca2+ influx into MCF7 cells through Orai3. Orai3 knockdown inhibited SOCE‐dependent phosphorylation of extracellular signal‐regulated kinase (ERK1/2; by ~44%) and focal adhesion kinase (FAK; by ~46%) as well as transcriptional activity of nuclear factor for activated T cells (NFAT; by ~49%). Significantly, Orai3 knockdown selectively decreased anchorage‐independent growth (by ~58%) and Matrigel invasion (by ~44%) of ERα+ MCF7 cells with no effect on ERα– MDA‐MB231 cells. Moreover, Orai3 knockdown inhibited ERα+ cell tumorigenesis in immunodeficient mice (~66% reduction in tumor volume). These data establish Orai3 as an ERα‐regulated channel and a potential selective therapeutic target for ERα+ breast cancers.—Motiani, R. K., Zhang, X., Harmon, K. E., Keller, R. S., Matrougui, K., Bennett, J. A., Trebak, M. Orai3 is an estrogen receptor α‐regulated Ca2+ channel that promotes tumorigenesis. FASEB J. 27, 63–75 (2013). www.fasebj.org

Store-Independent Orai1/3 Channels Activated by Intracrine LeukotrieneC4: Role in Neointimal Hyperplasia

Rationale: Through largely unknown mechanisms, Ca2+ signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca2+ entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca2+ entry pathways, its upregulation during VSMC remodeling, and its contribution to neointima formation remain unknown. Objective: The goal of this study was to determine the agonist-evoked Ca2+ entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries. Methods and Results: Ca2+ imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca2+ release-activated Ca2+ channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca2+-selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca2+ store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling, and in vivo Orai3 knockdown inhibits neointima formation. Conclusions: These results demonstrate that distinct native Ca2+-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca2+ signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

STIM1 controls endothelial barrier function independently of Orai1 and Ca2+ entry.

The calcium sensor STIM1 disrupts the endothelial barrier by coupling the thrombin receptor to the actin cytoskeleton. Breaking the Endothelial Barrier Thrombin is an endogenous ligand that induces vasoconstriction and can also disrupt the barrier formed by blood vessel endothelial cells, which leads to increased vascular permeability and leakage of plasma into the tissue. Using the thrombin-induced decrease in transendothelial resistance in two types of cultured endothelial cells as a model of barrier disruption, Shinde et al. found that the calcium-responsive protein STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA and rearrangement of the actin cytoskeleton, which contribute to loss of cell-cell contact. Surprisingly, this role did not involve various cation channels that are targets of STIM1. How STIM1 couples the thrombin receptor to RhoA remains an open question. Endothelial barrier function is critical for tissue fluid homeostasis, and its disruption contributes to various pathologies, including inflammation and sepsis. Thrombin is an endogenous agonist that impairs endothelial barrier function. We showed that the thrombin-induced decrease in transendothelial electric resistance of cultured human endothelial cells required the endoplasmic reticulum–localized, calcium-sensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca2+ entry across the plasma membrane and the Ca2+ release–activated Ca2+ channel protein Orai1, which is the target of STIM1 in the store-operated calcium entry pathway. We found that STIM1 coupled the thrombin receptor to activation of the guanosine triphosphatase RhoA, stimulation of myosin light chain phosphorylation, formation of actin stress fibers, and loss of cell-cell adhesion. Thus, STIM1 functions in pathways that are dependent on and independent of Ca2+ entry.

TWIK-1 two-pore domain potassium channels change ion selectivity and conduct inward leak sodium currents in hypokalemia.

Very low extracellular K+ concentrations, which can trigger cardiac arrhythmias, cause TWIK-1 potassium channels to become permeable to Na+. Losing Selectivity When the serum potassium drops to very low concentrations (pathological hypokalemia), the resting membrane potential of human cardiomyocytes can depolarize, rather than hyperpolarizing as would be predicted by the Nernst equation. This paradoxical depolarization, which is thought to be mediated by an inward sodium current, may contribute to the abnormalities of cardiac rhythm that can occur under conditions of pathological hypokalemia—or even cardiac arrest. Here, Ma et al. show that the ion selectivity of TWIK-1 channels, members of a class of potassium-selective ion channels that help maintain the resting membrane potential, changes at subphysiological extracellular potassium concentrations, so that they become permeable to sodium as well. Ectopic expression of TWIK-1 channels in a mouse cardiomyocyte cell line led to paradoxical depolarization in the presence of low extracellular potassium, whereas loss of TWIK-1 in cultured human cardiomyocytes abolished their paradoxical depolarization. Thus, by losing their selectivity for potassium and becoming permeable to sodium, TWIK-1 channels may contribute to cardiac paradoxical depolarization in pathological hypokalemia. Background potassium (K+) channels, which are normally selectively permeable to K+, maintain the cardiac resting membrane potential at around −80 mV. In subphysiological extracellular K+ concentrations ([K+]o), which occur in pathological hypokalemia, the resting membrane potential of human cardiomyocytes can depolarize to around −50 mV, whereas rat and mouse cardiomyocytes become hyperpolarized, consistent with the Nernst equation for K+. This paradoxical depolarization of cardiomyocytes in subphysiological [K+]o, which may contribute to cardiac arrhythmias, is thought to involve an inward leak sodium (Na+) current. Here, we show that human cardiac TWIK-1 (also known as K2P1) two-pore domain K+ channels change ion selectivity, becoming permeable to external Na+, and conduct inward leak Na+ currents in subphysiological [K+]o. A specific threonine residue (Thr118) within the pore selectivity sequence TxGYG was required for this altered ion selectivity. Mouse cardiomyocyte–derived HL-1 cells exhibited paradoxical depolarization with ectopic expression of TWIK-1 channels, whereas TWIK-1 knockdown in human spherical primary cardiac myocytes eliminated paradoxical depolarization. These findings indicate that ion selectivity of TWIK-1 K+ channels changes during pathological hypokalemia, elucidate a molecular basis for inward leak Na+ currents that could trigger or contribute to cardiac paradoxical depolarization in lowered [K+]o, and identify a mechanism for regulating cardiac excitability.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation.

AIMS Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. METHODS AND RESULTS We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. CONCLUSION Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

Multiple types of calcium channels arising from alternative translation initiation of the Orai1 message

Distinct Orai1 transcripts encode proteins that form Ca2+ channels with distinct properties. One gene for three currents Mammals produce alternative forms of the calcium channel pore-forming protein Orai1 using two different translation initiation start sites in the encoding transcripts. Desai et al. showed that these long and short forms produce calcium channels with distinct properties. Although both forms can participate in channels that respond to depletion of calcium from internal stores, only the long form contributes to a channel that is activated by arachidonic acid and leukotriene C4, lipids that promote inflammation. Their data suggest that characteristics of Orai1 genetic knockout may result from loss of any combination of these different calcium currents, not just loss of store-operated calcium entry. In mammals exclusively, the pore-forming Ca2+ release–activated Ca2+ (CRAC) channel subunit Orai1 occurs in two forms because of alternative translation initiation. The longer, mammal-specific Orai1α contains an additional 63 amino acids upstream of the conserved start site for Orai1β, which occurs at methionine 64 in Orai1α. Orai1 participates in the generation of three distinct Ca2+ currents, including two store-operated currents: Icrac, which involves activation of Orai1 channels by the Ca2+-sensing protein STIM1 (stromal interaction molecule 1), and Isoc, which involves an interaction among Orai1, the transient receptor potential (TRP) family member TRPC1 (TRP canonical 1), and STIM1. Orai1 is also a pore-forming subunit of an arachidonic acid (or leukotriene C4)–regulated current Iarc that involves interactions among Orai1, Orai3, and STIM1. We evaluated the roles of the two Orai1 forms in the Ca2+ currents Icrac, Isoc, and Iarc. We found that Orai1α and Orai1β were largely interchangeable for Icrac and Isoc, although Orai1α exhibited stronger inhibition by Ca2+. Only the mammalian-specific Orai1α functioned in the arachidonic acid–regulated current Iarc. Thus, alternative translation initiation of the Orai1 message produces at least three types of Ca2+ channels with distinct signaling and regulatory properties.

Enhanced Ca2+ entry due to Orai1 plasma membrane insertion increases IL-8 secretion by cystic fibrosis airways

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). The most common mutation, ΔF508, causes retention of CFTR in the endoplasmic reticulum (ER). Some CF abnormalities can be explained by altered Ca2+ homeostasis, although it remains unknown how CFTR influences calcium signaling. This study examined the novel hypothesis that store‐operated calcium entry (SOCE) through Orai1 is abnormal in CF. The significance of Orai1‐mediated SOCE for increased interleukin‐8 (IL‐8) expression in CF was also investigated. CF and non‐CF human airway epithelial cell line and primary cells (obtained at lung transplantation) were used in Ca2+ imaging, electrophysiology, and fluorescence imaging experiments to explore differences in Orai1 function in CF vs. non‐CF cells. Protein expression and localization was assessed by Western blots, cell surface biotinylation, ELISA, and image correlation spectroscopy (ICS). We show here that store‐operated Ca2+ entry (SOCE) is elevated in CF human airway epithelial cells (hAECs; ~1.8‐ and ~2.5‐fold for total Ca2+i increase and Ca2+ influx rate, respectively, and ~2‐fold increase in the ICRAC current) and is caused by increased exocytotic insertion (~2‐fold) of Orai1 channels into the plasma membrane, which is normalized by rescue of ΔF508‐CFTR trafficking to the cell surface. Augmented SOCE in CF cells is a major factor leading to increased IL‐8 secretion (~2‐fold). CFTR normally down‐regulates the Orai1/stromal interaction molecule 1 (STIM1) complex, and loss of this inhibition due to the absence of CFTR at the plasma membrane helps to explain the potentiated inflammatory response in CF cells.—Balghi, H., Robert, R., Rappaz, B., Zhang, X., Wohlhuter‐Haddad, A., Evagelidis, A., Luo, Y., Goepp, J., Ferraro, P., Roméo, P., Trebak, M., Wiseman, P. W., Thomas, D. Y., Hanrahan, J. W. Enhanced Ca2+ entry due to Orai1 plasma membrane insertion increases IL‐8 secretion by cystic fibrosis airways. FASEB J. 25, 4274–4291 (2011). www.fasebj.org