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Recent breakthroughs in the store-operated calcium (Ca 2+ ) entry (SOCE) pathway have identified Stim1 as the endoplasmic reticulum Ca 2+ sensor and Orai1 as the pore forming subunit of the highly Ca 2+ -selective CRAC channel expressed in hematopoietic cells. Previous studies, however, have suggested that endothelial cell (EC) SOCE is mediated by the nonselective canonical transient receptor potential channel (TRPC) family, TRPC1 or TRPC4. Here, we show that passive store depletion by thapsigargin or receptor activation by either thrombin or the vascular endothelial growth factor activates the same pathway in primary ECs with classical SOCE pharmacological features. ECs possess the archetypical Ca 2+ release-activated Ca 2+ current (I CRAC ), albeit of a very small amplitude. Using a maneuver that amplifies currents in divalent-free bath solutions, we show that EC CRAC has similar characteristics to that recorded from rat basophilic leukemia cells, namely a similar time course of activation, sensitivity to 2-aminoethoxydiphenyl borate, and low concentrations of lanthanides, and large Na + currents displaying the typical depotentiation. RNA silencing of either Stim1 or Orai1 essentially abolished SOCE and I CRAC in ECs, which were rescued by ectopic expression of either Stim1 or Orai1, respectively. Surprisingly, knockdown of either TRPC1 or TRPC4 proteins had no effect on SOCE and I CRAC . Ectopic expression of Stim1 in ECs increased their I CRAC to a size comparable to that in rat basophilic leukemia cells. Knockdown of Stim1, Stim2, or Orai1 inhibited EC proliferation and caused cell cycle arrest at S and G2/M phase, although Orai1 knockdown was more efficient than that of Stim proteins. These results are first to our knowledge to establish the requirement of Stim1/Orai1 in the endothelial SOCE pathway.

Microglia are the resident immune cells of the CNS, which are important for preserving neural tissue functions, but may also contribute to neurodegeneration. Activation of these cells in infection, inflammation, or trauma leads to the release of various toxic molecules, including reactive oxygen species (ROS) and the excitatory amino acid glutamate. In this study, we used an electrophysiologic approach and a D-[(3)H]aspartate (glutamate) release assay to explore the ROS-dependent regulation of glutamate-permeable volume-regulated anion channels (VRACs). Exposure of rat microglia to hypo-osmotic media stimulated Cl(-) currents and D-[(3)H]aspartate release, both of which were inhibited by the selective VRAC blocker, DCPIB. Exogenously applied H(2)O(2) potently increased swelling-activated glutamate release. Stimulation of microglia with zymosan triggered production of endogenous ROS and strongly enhanced glutamate release via VRAC in swollen cells. The effects of zymosan were attenuated by the ROS scavenger, MnTMPyP, and by two inhibitors of NADPH oxidase (NOX), diphenyliodonium and thioridazine. However, zymosan-stimulated glutamate release was insensitive to other NOX blockers, apocynin and HEBSF. This pharmacologic profile pointed to the potential involvement of apocynin-insensitive NOX4. Using RT-PCR we confirmed that NOX4 is expressed in rat microglial cells along with NOX1 and NOX2. To check for potential involvement of phagocytic NOX2, we stimulated this isoform using protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate or inhibited it with the broad spectrum PKC blocker, Gö6983. Both agents potently modulated endogenous ROS production by NOX2 but not VRAC activity. Taken together, these data suggest that the anion channel VRAC may contribute to microglial glutamate release and that its activity is regulated by endogenous ROS originating from NOX4.

Ubiquitously expressed volume-regulated anion channels (VRACs) are chloride channels which are permeable to a variety of small organic anions, including the excitatory amino acids (EAAs) glutamate and aspartate. Broad spectrum anion channel blockers strongly reduce EAA release in cerebral ischaemia and other pathological states associated with prominent astrocytic swelling. However, it is uncertain whether VRAC serves as a major pathway for EAA release from swollen cells. In the present study, we measured swelling-activated release of EAAs as d-[3H]aspartate efflux, and VRAC-mediated Cl− currents by whole-cell patch clamp in cultured rat astrocytes. We compared the pharmacological profiles of the swelling-activated EAA release pathway and Cl− currents. The expression of candidate Cl− channels was confirmed by RT-PCR. The maxi Cl− channel (p-VDAC) blocker Gd3+, the ClC-2 inhibitor Cd2+, and the MDR-1 blocker verapamil did not affect EAA release or VRAC currents. An antagonist of calcium-sensitive Cl− channels (CaCC), niflumic acid, had little effect on EAA release and only partially inhibited swelling-activated Cl− currents. The phorbol ester PDBu, which blocks ClC-3-mediated Cl− currents, had no effect on VRAC currents and up-regulated EAA release. In contrast, DCPIB, which selectively inhibits VRACs, potently suppressed both EAA release and VRAC currents. Two other relatively selective VRAC inhibitors, tamoxifen and phloretin, also blocked the VRAC currents and strongly reduced EAA release. Taken together, our data suggest that (i) astrocytic volume-dependent EAA release is largely mediated by the VRAC, and (ii) the ClC-2, ClC-3, ClC-4, ClC-5, VDAC, CaCC, MDR-1 and CFTR gene products do not contribute to EAA permeability.

The cellular and molecular processes underlying the regulation of ryanodine receptor (RyR) Ca 2+ release in smooth muscle cells (SMCs) are incompletely understood. Here we show that FKBP12.6 proteins are expressed in pulmonary artery (PA) smooth muscle and associated with type-2 RyRs (RyR2), but not RyR1, RyR3, or IP 3 receptors (IP 3 Rs) in PA sarcoplasmic reticulum. Application of FK506, which binds to FKBPs and dissociates these proteins from RyRs, induced an increase in [Ca 2+ ] i and Ca 2+ -activated Cl − and K + currents in freshly isolated PASMCs, whereas cyclosporin, an agent known to inhibit calcineurin but not to interact with FKBPs, failed to induce an increase in [Ca 2+ ] i . FK506-induced [Ca 2+ ] i increase was completely blocked by the RyR antagonist ruthenium red and ryanodine, but not the IP 3 R antagonist heparin. Hypoxic Ca 2+ response and hypoxic vasoconstriction were significantly enhanced in FKBP12.6 knockout mouse PASMCs. FK506 or rapamycin pretreatment also enhanced hypoxic increase [Ca 2+ ] i , but did not alter caffeine-induced Ca 2+ release (SR Ca 2+ content) in PASMCs. Norepinephrine-induced Ca 2+ release and force generation were also markedly enhanced in PASMCs from FKBP12.6 null mice. These findings suggest that FKBP12.6 plays an important role in hypoxia- and neurotransmitter-induced Ca 2+ and contractile responses by regulating the activity of RyRs in PASMCs.

A hypotonic challenge, but not cAMP stimulation, was found to induce release of ATP measured by the luciferin-luciferase assay from both the murine mammary carcinoma cell line C127i and C127 cells stably transfected with the cDNA for human cystic fibrosis transmembrane conductance regulator (CFTR) protein (C127/CFTR). CFTR expression augmented swelling-induced ATP release by 10–20 times under hypotonic conditions (≤ 80 % osmolality). Glibenclamide failed to suppress swelling-induced ATP release from C127/CFTR cells. In contrast, whole-cell patch-clamp recordings showed that both the cAMP-activated ohmic Cl− currents and volume-sensitive outwardly rectifying (VSOR) Cl− currents were prominently suppressed by glibenclamide. Gd3+ markedly blocked swelling-induced ATP release but failed to suppress both cAMP- and swelling-activated Cl− currents in the CFTR-expressing cells. Even after pretreatment and during treatment with Gd3+, VSOR Cl− currents were activated normally. The continuous presence of an ATP-hydrolysing enzyme, apyrase, in the bathing solution did not prevent activation of VSOR Cl− currents in C127/CFTR cells. The rate of regulatory volume decrease (RVD) in C127/CFTR cells was much faster than that in C127i cells. When apyrase was added to the bathing solution, the RVD rate was retarded in C127/CFTR cells. On balance, the following conclusions can be deduced. First, swelling-induced ATP release is augmented by expression of CFTR but is not mediated by the CFTR Cl− channel. Second, swelling-induced ATP release is not mediated by the VSOR Cl− channel. Third, the released ATP facilitated the RVD process but is not involved in the activation of VSOR Cl− channels in C127/CFTR cells. Cystic fibrosis transmembrane conductance regulator (CFTR) belongs to a superfamily of ATP-binding cassette (ABC) transporters (Hyde et al. 1990), most members of which form ATP-dependent pumps for a wide variety of substances (Higgins, 1992). Unlike other members of the ABC transporter family, CFTR functions as a cAMP/protein kinase A-activated Cl− channel regulated by intracellular ATP (Andersen et al. 1991). Evidence has accumulated in support of the concept that CFTR is a regulator of other ion channels (Devidas & Guggino, 1997), such as the outwardly rectifying Cl− channel (ORCC) (Egan et al. 1992; Gabriel et al. 1993; Jovov et al. 1995; Schwiebert et al. 1995), the volume-sensitive Cl− channel (Vennekens et al. 1999), the amiloride-sensitive epithelial Na+ channel (ENaC) (Stutts et al. 1995) and the ATP-dependent inwardly rectifying K+ channel (ROMK) (McNicholas et al. 1996). Some investigators have suggested that CFTR may mediate the plasma membrane permeability (Schwiebert et al. 1995; Prat et al. 1996; Jiang et al. 1998) or conductance of ATP (Reisin et al. 1994; Schwiebert et al. 1995; Cantiello et al. 1998). However, the ‘CFTR = ATP channel hypothesis’ has recently been challenged by the observation that the ATP permeability or conductance is not specifically associated with CFTR using a variety of methods and preparations (Takahashi et al. 1994; Reddy et al. 1996; Li et al. 1996; Grygorczyk et al. 1996; Grygorczyk & Hanrahan, 1997) and that the pore properties of the CFTR Cl− channel and the ATP channel are distinct (Sugita et al. 1998). Thus, a precise relationship between CFTR and ATP release is still a matter for investigation (Devidas & Guggino, 1997). CFTR expression has also been found to enhance ATP release induced by cell shrinkage upon removal of extracellular Cl− (Rotoli et al. 1996) or by osmotic cell swelling (Hazama et al. 1998b; Taylor et al. 1998). Swelling-induced ATP release has been shown to be coupled to activation of a volume-sensitive Cl− conductance which is involved in regulatory shrinkage (regulatory volume decrease, RVD) after osmotic swelling in rat hepatoma cells (Wang et al. 1996; Roman et al. 1997) and in rat and human biliary epithelial cells (Roman et al. 1999). Thus, there is a possibility that swelling-induced ATP release from CFTR-expressing cells is associated with the activity of CFTR Cl− channels and/or volume-sensitive Cl− channels. Therefore, in the present study, this possibility was examined by measuring ATP release and Cl− conductances in a murine mammary carcinoma cell line, C127i, and in C127 cells stably transfected with CFTR cDNA (C127/CFTR). Here we have provided clear evidence that swelling-induced ATP release is mediated neither by the CFTR Cl− channel nor by the volume-sensitive outwardly rectifying (VSOR) Cl− channel, and that the mechanism of activation of the VSOR Cl− channel is independent of the released ATP in the CFTR-expressing cell.

Transient expression of wild-type human cystic fibrosis transmembrane conductance regulator (CFTR) in HEK293T cells resulted in a profound decrease in the amplitude of volume-sensitive outwardly rectifying Cl- channel (VSOR) current without changing the single-channel amplitude. This effect was not mimicked by expression of the DeltaF508 mutant of CFTR, which did not reach the plasma membrane. The VSOR regulation by CFTR was not affected by G551D mutation at first nucleotide-binding domain (NBD1), which is known to impair CFTR interaction with the outwardly rectifying chloride channel, ORCC, epithelial amiloride-sensitive Na-channel, ENaC, and renal potassium channel, ROMK2. The CFTR-VSOR interaction was insensitive to the deletion mutation, DeltaTRL, which is known to impair CFTR-PDZ domain binding. In contrast, the G1349D mutant, which impairs ATP binding at NBD2, effectively abolished the down-regulatory effect of CFTR. Furthermore, the K1250M mutation at the Walker A motif and the D1370N mutation at the Walker B motif, both known to impair ATP hydrolysis at NBD2, completely abolished the VSOR regulation by CFTR. Thus, we conclude that an ATP-hydrolysable conformation of NBD2 is essential for the regulation of the VSOR by the CFTR protein, and that VSOR is a first channel regulated by CFTR through its NBD2.

Gliomas are morbid brain tumors that are extremely resistant to available chemotherapy and radiology treatments. Some studies have suggested that calcium-activated potassium channels contribute to the high proliferative potential of tumor cells, including gliomas. However, other publications demonstrated no role for these channels or even assigned them antitumorogenic properties. In this work we characterized the expression and functional contribution to proliferation of Ca(2+)-activated K(+) channels in human glioblastoma cells. Quantitative RT-PCR detected transcripts for the big conductance (BK), intermediate conductance (IK1), and small conductance (SK2) K(+) channels in two glioblastoma-derived cell lines and a surgical sample of glioblastoma multiforme. Functional expression of BK and IK1 in U251 and U87 glioma cell lines and primary glioma cultures was verified using whole-cell electrophysiological recordings. Inhibitors of BK (paxilline and penitrem A) and IK1 channels (clotrimazole and TRAM-34) reduced U251 and U87 proliferation in an additive fashion, while the selective blocker of SK channels UCL1848 had no effect. However, the antiproliferative properties of BK and IK1 inhibitors were seen at concentrations that were higher than those necessary to inhibit channel activity. To verify specificity of pharmacological agents, we downregulated BK and IK1 channels in U251 cells using gene-specific siRNAs. Although siRNA knockdowns caused strong reductions in the BK and IK1 current densities, neither single nor double gene silencing significantly affected rates of proliferation. Taken together, these results suggest that Ca(2+)-activated K(+) channels do not play a critical role in proliferation of glioma cells and that the effects of pharmacological inhibitors occur through their off-target actions.