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The Bestrophin family is composed of four human genes that form transmembrane proteins [1-3]. When heteroexpressed in HEK293 cells, all four human Bestrophin genes produce novel and unique Cl− anion currents [4,5]. Mutations in one family member, Bestrophin-1 (BEST1 or VMD2), have been linked to five distinct ocular pathologies in humans: an autosomal dominant form of juvenile blindness known as Best vitelliform macular dystrophy, adult-onset foveomacular vitelliform dystrophy, autosomal dominant vitroretinochoroidopathy, autosomal dominant MRCS (microcornea, rod-cone dystrophy, early-onset cataract, and posterior staphyloma), and autosomal recessive bestrophinopathy (see [1,2,6] and reviews [7-9]). Expression of Best1 is particularly high in the retina and retinal pigment epithelium, consistent with a role in visual system. However, Best1 mRNA expression have also been detected in testes, spinal cord, and the brain [2,3,10]. Emerging studies suggest that in the brain Best1 forms glial Cl−/anion channel and is responsible for release of neurotransmitters GABA and glutamate from astrocytes [10-13]. Best1-mediated GABA release has been shown to contribute to tonic inhibition of neuronal activity in cerebellum and perhaps other brain regions [11]. Homologous Bestrophins genes have been identified throughout the animal kingdom, including in the model organisms Drosophila melanogaster and Caenorhabditis elegans [7]. A high degree of conservation is seen in the N-terminal portion of all Bestrophin proteins, particularly in areas composed of hydrophobic amino acids that are hypothesized to encode transmembrane domains [7,14]. GenBank lists two principal human Best1 variants, Best1V1 and Best1V2, whose protein products differ in both the N- and C-termini due to alternative splicing (first reported by Wistow et al. [15]). While the first exon is common to both variants, it does not contain a translational start site. As translation initiates at a start codon in either exon 2 or exon 3, depending on alternative splicing, inclusion of exon 2 in the Best1V1 variant results in a protein with a longer, unique N-terminus. The amino acid sequence encoded by exon 2 is thought to form a transmembrane domain and lack of this fragment in Best1V2 may have important functional consequences [5,16]. Alternative splicing at the C-terminus of Best1V2 replaces the terminal 5 amino acids of Best1V1 with a unique 84-amino acid sequence. The C-terminus of canonical Best1V1 variant, which is predicted to be located in the cytoplasm, has been shown to play numerous roles. It contains a putative calcium-sensing domain, and both truncations or point mutations in this domain can dramatically reduce Ca2+ activated Cl− currents in heteroexpression systems [17,18]. Furthermore, it has been reported that the N- and C-termini of the Best1 protein allow for inter-domain interactions and binding [19]. In the latter study, the authors proposed that Best1 multimerization via the N-C-termini interactions is critical for channel function. The functional effects of alternative splicing of the C-terminus are not known. In the present study, we cloned several Best1 splice variants from normal and malignant human glial cells. In addition to the previously reported Best1V1 and Best1V2 isoforms, we identified a novel Best1V1 splice variant lacking exon 2 (Best1V1Δex2). This splice variant allowed us to explore the functional significance of the N- and C-terminal portions of the Best1 protein. With this purpose, we heteroexpressed Best1V1, Best1V2, and the novel Best1V1Δex2 in HEK293 cells and compared their ability to form ion channels. Surprisingly, Best1V1 and Best1V1Δex2, but not Best1V2, generated Ca2+- activated Cl− channels. These results indicate that the exon 2-encoded N-terminus is not essential for formation of functional Ca2+-activated Cl− channel. In contrast, the cytoplasmic C-terminus appears to play a critical role, likely via its impact on protein stability.

Multiple Ca(2+) release and entry mechanisms and potential cytoskeletal targets have been implicated in vascular endothelial barrier dysfunction; however, the immediate downstream effectors of Ca(2+) signals in the regulation of endothelial permeability still remain unclear. In the present study, we evaluated the contribution of multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) as a mediator of thrombin-stimulated increases in human umbilical vein endothelial cell (HUVEC) monolayer permeability. For the first time, we identified the CaMKIIdelta(6) isoform as the predominant CaMKII isoform expressed in endothelium. As little as 2.5 nM thrombin maximally increased CaMKIIdelta(6) activation assessed by Thr(287) autophosphorylation. Electroporation of siRNA targeting endogenous CaMKIIdelta (siCaMKIIdelta) suppressed expression of the kinase by >80% and significantly inhibited 2.5 nM thrombin-induced increases in monolayer permeability assessed by electrical cell-substrate impedance sensing (ECIS). siCaMKIIdelta inhibited 2.5 nM thrombin-induced activation of RhoA, but had no effect on thrombin-induced ERK1/2 activation. Although Rho kinase inhibition strongly suppressed thrombin-induced HUVEC hyperpermeability, inhibiting ERK1/2 activation had no effect. In contrast to previous reports, these results indicate that thrombin-induced ERK1/2 activation in endothelial cells is not mediated by CaMKII and is not involved in endothelial barrier hyperpermeability. Instead, CaMKIIdelta(6) mediates thrombin-induced HUVEC barrier dysfunction through RhoA/Rho kinase as downstream intermediates. Moreover, the relative contribution of the CaMKIIdelta(6)/RhoA pathway(s) diminished with increasing thrombin stimulation, indicating recruitment of alternative signaling pathways mediating endothelial barrier dysfunction, dependent upon thrombin concentration.

Store-operated calcium (Ca2+) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca2+ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca2+ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca2+-selective Ca2+ release-activated Ca2+ current (ICRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca2+ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and ICRAC in estrogen receptor-positive (ER+) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER−) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER+ breast cancer cells represent the first example where the native SOCE pathway and ICRAC are mediated by Orai3. Future studies implicating Orai3 in ER+ breast cancer progression might establish Orai3 as a selective target in therapy of ER+ breast tumors.

The identity of store-operated calcium (Ca(2+)) entry (SOCE) channels in vascular smooth muscle cells (VSMCs) remains a highly contentious issue. Whereas previous studies have suggested that SOCE in VSMCs is mediated by the nonselective transient receptor potential canonical (TRPC) 1 protein, the identification of STIM1 and Orai1 as essential components of I(CRAC), a highly Ca(2+)-selective SOCE current in leukocytes, has challenged that view. Here we show that cultured proliferative migratory VSMCs isolated from rat aorta (called "synthetic") display SOCE with classic features, namely inhibition by 2-aminoethoxydiphenyl borate, ML-9, and low concentrations of lanthanides. On store depletion, synthetic VSMCs and A7r5 cells display currents with characteristics of I(CRAC). Protein knockdown of either STIM1 or Orai1 in synthetic VSMCs greatly reduced SOCE, whereas Orai2, Orai3, TRPC1, TRPC4, and TRPC6 knockdown had no effect. Orai1 knockdown reduced I(CRAC) in synthetic VSMCs and A7r5 cells. Synthetic VSMCs showed up-regulated STIM1/Orai1 proteins and SOCE compared with quiescent freshly isolated VSMC. Knockdown of STIM1 and Orai1 inhibited synthetic VSMC proliferation and migration, whereas STIM2, Orai2, and Orai3 knockdown had no effect. To our knowledge, these results are the first to show I(CRAC) in VSMCs and resolve a long-standing controversy by identifying CRAC as the elusive VSMC SOCE channel important for proliferation and migration.

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.

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.

`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, calciumsensing protein stromal interacting molecule 1 (STIM1), but was independent of Ca 2+ entry across the plasma membrane and the Ca 2+ release–activated Ca 2+ 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 Ca 2+ entry.