Drosophila bestrophin-1 chloride current is dually regulated by calcium and cell volume

Mutations in the human bestrophin-1 (hBest1) gene are responsible for Best vitelliform macular dystrophy, however the mechanisms leading to retinal degeneration have not yet been determined because the function of the bestrophin protein is not fully understood. Bestrophins have been proposed to comprise a new family of Cl(-) channels that are activated by Ca(2+). While the regulation of bestrophin currents has focused on intracellular Ca(2+), little is known about other pathways/mechanisms that may also regulate bestrophin currents. Here we show that Cl(-) currents in Drosophila S2 cells, that we have previously shown are mediated by bestrophins, are dually regulated by Ca(2+) and cell volume. The bestrophin Cl(-) currents were activated in a dose-dependent manner by osmotic pressure differences between the internal and external solutions. The increase in the current was accompanied by cell swelling. The volume-regulated Cl(-) current was abolished by treating cells with each of four different RNAi constructs that reduced dBest1 expression. The volume-regulated current was rescued by transfecting with dBest1. Furthermore, cells not expressing dBest1 were severely depressed in their ability to regulate their cell volume. Volume regulation and Ca(2+) regulation can occur independently of one another: the volume-regulated current was activated in the complete absence of Ca(2+) and the Ca(2+)-activated current was activated independently of alterations in cell volume. These two pathways of bestrophin channel activation can interact; intracellular Ca(2+) potentiates the magnitude of the current activated by changes in cell volume. We conclude that in addition to being regulated by intracellular Ca(2+), Drosophila bestrophins are also novel members of the volume-regulated anion channel (VRAC) family that are necessary for cell volume homeostasis.     

Single Cl- channels activated by Ca2+ in Drosophila S2 cells are mediated by bestrophins

Mutations in human bestrophin-1 (VMD2) are genetically linked to several forms of retinal degeneration but the underlying mechanisms are unknown. Bestrophin-1 (hBest1) has been proposed to be a Cl(-) channel involved in ion and fluid transport by the retinal pigment epithelium (RPE). To date, however, bestrophin currents have only been described in overexpression systems and not in any native cells. To test whether bestrophins function as Ca(2+)-activated Cl(-) (CaC) channels physiologically, we used interfering RNA (RNAi) in the Drosophila S2 cell line. S2 cells express four bestrophins (dbest1-4) and have an endogenous CaC current. The CaC current is abolished by several RNAi constructs to dbest1 and dbest2, but not dbest3 or dbest4. The endogenous CaC current was mimicked by expression of dbest1 in HEK cells, and the rectification and relative permeability of the current were altered by replacing F81 with cysteine. Single channel analysis of the S2 bestrophin currents revealed an approximately 2-pS single channel with fast gating kinetics and linear current-voltage relationship. A similar channel was observed in CHO cells transfected with dbest1, but no such channel was seen in S2 cells treated with RNAi to dbest1. This provides definitive evidence that bestrophins are components of native CaC channels at the plasma membrane.     

Mouse bestrophin-2 is a bona fide Cl(-) channel: identification of a residue important in anion binding and conduction

Bestrophins have recently been proposed to comprise a new family of Cl(-) channels. Our goal was to test whether mouse bestrophin-2 (mBest2) is a bona fide Cl(-) channel. We expressed mBest2 in three different mammalian cell lines. mBest2 was trafficked to the plasma membrane as shown by biotinylation and immunoprecipitation, and induced a Ca(2+)-activated Cl(-) current in all three cell lines (EC(50) for Ca(2+) = 230 nM). The permeability sequence was SCN(-): I(-): Br(-): Cl(-): F(-) (8.2: 1.9: 1.4: 1: 0.5). Although SCN(-) was highly permeant, its conductance was approximately 10% that of Cl(-) and SCN(-) blocked Cl(-) conductance (IC(50) = 12 mM). Therefore, SCN(-) entered the pore more easily than Cl(-), but bound more tightly than Cl(-). Mutations in S79 altered the relative permeability and conductance for SCN(-) as expected if S79 contributed to an anion binding site in the channel. P(SCN)/P(Cl) = 8.2 +/- 1.3 for wild-type and 3.9 +/- 0.4 for S79C. G(SCN)/G(Cl) = 0.14 +/- 0.03 for wild-type and 0.94 +/- 0.04 for S79C. In the S79 mutants, SCN(-) did not block Cl(-) conductance. This suggested that the S79C mutation altered the affinity of an anion binding site for SCN(-). Additional evidence that S79 was located in the conduction pathway was provided by the finding that modification of the sulfhydryl group in S79C with MTSET(+) or MTSES(-) increased conductance significantly. Because the effect of positively and negatively charged MTS reagents was similar, electrostatic interactions between the permeant anion and the channel at this residue were probably not critical in anion selectivity. These data provide strong evidence that mBest2 forms part of the novel Cl(-) conduction pathway in mBest2-transfected cells and that S79 plays an important role in anion binding in the pore of the channel.     

Bestrophin-1 enables Ca2+-activated Cl- conductance in epithelia

Epithelial cells express calcium-activated Cl(-) channels of unknown molecular identity. These Cl(-) channels play a central role in diseases such as secretory diarrhea, polycystic kidney disease, and cystic fibrosis. The family of bestrophins has been suggested to form calcium-activated Cl(-) channels. Here, we demonstrate molecular and functional expression of bestrophin-1 (BEST1) in mouse and human airways, colon, and kidney. Endogenous calcium-activated whole cell Cl(-) currents coincide with endogenous expression of the Vmd2 gene product BEST1 in murine and human epithelial cells, whereas calcium-activated Cl(-) currents are absent in epithelial tissues lacking BEST1 expression. Blocking expression of BEST1 with short interfering RNA or applying an anti-BEST1 antibody to a patch pipette suppressed ATP-induced whole cell Cl(-) currents. Calcium-dependent Cl(-) currents were activated by ATP in HEK293 cells expressing BEST1. Thus, BEST1 may form the Ca2+-activated Cl(-) current, or it may be a component of a Cl(-) channel complex in epithelial tissues.     

Role of the Ca2+ -activated Cl- channels bestrophin and anoctamin in epithelial cells

Two families of proteins, the bestrophins (Best) and the recently cloned TMEM16 proteins (anoctamin, Ano), recapitulate properties of Ca(2+)-activated Cl(-) currents. Best1 is strongly expressed in the retinal pigment epithelium and could have a function as a Ca(2+)-activated Cl(-) channel as well as a regulator of Ca(2+) signaling. It is also present at much lower levels in other cell types including epithelial cells, where it regulates plasma membrane localized Cl(-) channels by controlling intracellular Ca(2+) levels. Best1 interacts with important Ca(2+)-signaling proteins such as STIM1 and can interact directly with other Ca(2+)-activated Cl(-) channels such as TMEM16A. Best1 is detected in the endoplasmic reticulum (ER) where it shapes the dynamic ER structure and regulates cell proliferation, which could be important for renal cystogenesis. Ca(2+)-activated Cl(-) channels of the anoctamin family (TMEM16A) show biophysical and pharmacological properties that are typical for endogenous Ca(2+)-dependent Cl(-) channels. TMEM16 proteins are abundantly expressed and many reports demonstrate their physiological importance in epithelial as well as non-epithelial cells. These channels are also activated by cell swelling and can therefore control cell volume, proliferation and apoptosis. To fully understand the function and regulation of Ca(2+)-activated Cl(-) currents, it is necessary to appreciate that Best1 and TMEM16A are embedded in a protein network and that they probably operate in functional microdomains.     

Biophysics and Structure-Function Relationships of LRRC8-Formed Volume-Regulated Anion Channels

Volume-regulated anion channels (VRACs) are key players in regulatory volume decrease of vertebrate cells by mediating the extrusion of chloride and organic osmolytes. They play additional roles in various physiological processes beyond their role in osmotic volume regulation. VRACs are formed by heteromers of LRRC8 proteins; LRRC8A (also called SWELL1) is an essential subunit that combines with any of its paralogs, LRRC8B–E, to form hexameric VRAC complexes. The subunit composition of VRACs determines electrophysiological characteristics of their anion transport such as single-channel conductance, outward rectification, and depolarization-dependent inactivation kinetics. In addition, differently composed VRACs conduct diverse substrates, such as LRRC8D enhancing VRAC permeability to organic substances like taurine or cisplatin. Here, after a recapitulation of the biophysical properties of VRAC-mediated ion and osmolyte transport, we summarize the insights gathered since the molecular identification of VRACs. We describe the recently solved structures of LRRC8 complexes and discuss them in terms of their structure-function relationships. These studies open up many potential avenues for future research.     

RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells

Cell swelling triggers in most cell typesan outwardly rectifying anion current,I_Cl,swell, via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I_Cl,swell. However, theseexperiments did not allow us to discriminate between a direct activatorrole or a permissive effect. We now show that the Rho pathway did notaffect VRAC activity if this pathway was activated by transfecting CPAEcells with constitutively active isoforms of G (a Rho activatingheterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore,biochemical and morphological analysis failed to demonstrate activationof the Rho pathway during hypotonic cell swelling. Finally,manipulating the Rho pathway with either guanosine5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect onVRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rhopathway exerts a permissive effect on VRACs in CPAE cells, i.e.,swelling-induced opening of VRACs requires a functional Rho pathway,but not an activation of the Rho pathway.

     

Eag1 and Bestrophin 1 are up-regulated in fast-growing colonic cancer cells

Ion channels like voltage-gated ether-á-go-go (Eag1) K(+) channels or Ca(2+)-activated Cl(-) channels have been shown to support cell proliferation. Bestrophin 1 (Best1) has been proposed to form Ca(2+)-activated Cl(-) channels in epithelial cells. Here we show that original T(84) colonic carcinoma cells grow slowly (T(84)-slow) and express low amounts of Eag1 and Best1, whereas spontaneously transformed T(84) cells grow fast (T(84)-fast) and express high levels of both proteins. Both Eag1 and Best1 currents are up-regulated in T(84)-fast cells. Eag1 currents were cell cycle-dependent with up-regulation during G(1)/S transition. T(84)-slow, but not T(84)-fast, cells formed tight monolayers when grown on permeable supports. RNA interference inhibition of Eag1 and Best1 reduced proliferation of T(84)-fast cells, whereas overexpression of Best1 turned T(84)-slow into fast-growing cells. Eag1 and Best1 improve intracellular Ca(2+) signaling and cell volume regulation. These results establish a novel role for bestrophins in cell proliferation.     

Peroxynitrite enhances astrocytic volume-sensitive excitatory amino acid release via a src tyrosine kinase-dependent mechanism

Volume-regulated anion channels (VRACs) are critically important for cell volume homeostasis, and under pathological conditions contribute to neuronal damage via excitatory amino (EAA) release. The precise mechanisms by which brain VRACs are activated and/or modulated remain elusive. In the present work we explored the possible involvement of nitric oxide (NO) and NO-related reactive species in the regulation of VRAC activity and EAA release, using primary astrocyte cultures. The NO donors sodium nitroprusside and spermine NONOate did not affect volume-activated d-[3H]aspartate release. In contrast, the peroxynitrite (ONOO-) donor 3-morpholinosydnomine hydrochloride (SIN-1) increased volume-dependent EAA release by approx. 80-110% under identical conditions. Inhibition of ONOO- formation with superoxide dismutase completely abolished the effects of SIN-1. Both the volume- and SIN-1-induced EAA release were sensitive to the VRAC blockers NPPB and ATP. Further pharmacological analysis ruled out the involvement of cGMP-dependent reactions and modification of sulfhydryl groups in the SIN-1-inducedmodulation of EAA release. The src family tyrosine kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2), but not its inactive analog PP3, abolished the effects of SIN-1. A broader spectrum tyrosine kinase inhibitor tyrphostin A51, also completely eliminated the SIN-1-induced EAA release. Our data suggest that ONOO- up-regulates VRAC activity via a src tyrosine kinase-dependent mechanism. This modulation may contribute to EAA-mediated neuronal damage in ischemia and other pathological conditions favoring cell swelling and ONOO- production.     

Bestrophin Cl- channels are highly permeable to HCO3-

Bestrophin-1 (Best1) is a Cl(-) channel that is linked to various retinopathies in both humans and dogs. Dysfunction of the Best1 Cl(-) channel has been proposed to cause retinopathy because of altered Cl(-) transport across the retinal pigment epithelium (RPE). In addition to Cl(-), many Cl(-) channels also transport HCO3(-). Because HCO3(-) is physiologically important in pH regulation and in fluid and ion transport across the RPE, we measured the permeability and conductance of bestrophins to HCO3(-) relative to Cl(-). Four human bestrophin homologs (hBest1, hBest2, hBest3, and hBest4) and mouse Best2 (mBest2) were expressed in HEK cells, and the relative HCO3(-) permeability (P HCO3/PCl) and conductance (G HCO3/GCl) were determined. P HCO3/PCl was calculated from the change in reversal potential (Erev) produced by replacing extracellular Cl(-) with HCO3(-). hBest1 was highly permeable to HCO3(-) (P HCO3)/PCl = approximately 0.44). hBest2, hBest4, and mBest2 had an even higher relative HCO3(-) permeability (P HCO3/PCl = 0.6-0.7). All four bestrophins had HCO3(-) conductances that were nearly the same as Cl(-) (G HCO3/GCl = 0.9-1.1). Extracellular Na+ did not affect the permeation of hBest1 to HCO3(-). At physiological HCO3(-) concentration, HCO3(-) was also highly conductive. The hBest1 disease-causing mutations Y85H, R92C, and W93C abolished both Cl(-) and HCO3(-) currents equally. The V78C mutation changed P HCO3/PCl and G HCO3/GCl of mBest2 channels. These results raise the possibility that disease-causing mutations in hBest1 produce disease by altering HCO3(-) homeostasis as well as Cl(-) transport in the retina.     

2-APB inhibits volume-regulated anion channels independently from intracellular calcium signaling modulation

It has previously been suggested that volume-regulated anion channels (VRACs) and store-operated channels (SOCs) interact with each other according to their expected colocalization in the plasma membrane of LNCaP cells. In order to study interactions between these two channels, we used 2-aminoethoxydiphenyl borate (2-APB) as a regular SOC inhibitor. Surprisingly 2-APB reduced VRAC activity in a dose-dependent manner (IC(50)=122.8 microM), but not 2,2-diphenyltetrahydrofuran (a structural analog of 2-APB). This effect was also present in keratinocytes. We conclude that 2-APB is an inhibitor of the VRAC family, and is also a potent tool to study the SOC-VRAC interaction in LNCaP cells.     

ATP regulates anion channel-mediated organic osmolyte release from cultured rat astrocytes via multiple Ca2+-sensitive mechanisms

Ubiquitously expressed volume-regulated anion channels (VRACs) are activated in response to cell swelling but may also show limited activity in nonswollen cells. VRACs are permeable to inorganic anions and small organic osmolytes, including the amino acids aspartate, glutamate, and taurine. Several recent reports have demonstrated that neurotransmitters or hormones, such as ATP and vasopressin, induce or strongly potentiate astrocytic whole cell Cl^– currents and amino acid release, which are inhibited by VRAC blockers. In the present study, we explored the intracellular signaling mechanisms mediating the effects of ATP on D-[³H]aspartate release via the putative VRAC pathway in rat primary astrocyte cultures. Cells were exposed to moderate (5%) or substantial (30%) reductions in medium osmolarity. ATP strongly potentiated D-[³H]aspartate release in both moderately swollen and substantially swollen cells. These ATP effects were blocked (80% inhibition) by intracellular Ca²⁺ chelation with BAPTA-AM, calmodulin inhibitors, or a combination of the inhibitors of protein kinase C (PKC) and calmodulin-dependent kinase II (CaMK II). In contrast, control D-[³H]aspartate release activated by the substantial hyposmotic swelling showed little (25% inhibition) sensitivity to the same pharmacological agents. These data indicate that ATP regulates VRAC activity via two separate Ca²⁺-sensitive signaling cascades involving PKC and CaMK II and that cell swelling per se activates VRACs via a separate Ca²⁺/calmodulin-independent signaling mechanism. Ca²⁺-dependent organic osmolyte release via VRACs may contribute to the physiological functions of these channels in the brain, including astrocyte-to-neuron intercellular communication. volume-regulated anion channels; protein kinase C; calcium/calmodulin-dependent kinase II; glutamate release; neuron-glia communication     

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt‐based anti‐cancer drugs

Although platinum‐based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume‐regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8‐dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug‐induced apoptosis independently from drug uptake, possibly by impairing VRAC‐dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D‐containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.     

Anion permeation in Ca(2+)-activated Cl(-) channels

Ca(2+)-activated Cl channels (Cl(Ca)Cs) are an important class of anion channels that are opened by increases in cytosolic [Ca(2+)]. Here, we examine the mechanisms of anion permeation through Cl(Ca)Cs from Xenopus oocytes in excised inside-out and outside-out patches. Cl(Ca)Cs exhibited moderate selectivity for Cl over Na: P(Na)/P(Cl) = 0.1. The apparent affinity of Cl(Ca)Cs for Cl was low: K(d) = 73 mM. The channel had an estimated pore diameter >0.6 nm. The relative permeabilities measured under bi-ionic conditions by changes in E(rev) were as follows: C(CN)(3) > SCN > N(CN)(2) > ClO(4) > I > N(3) > Br > Cl > formate > HCO(3) > acetate = F > gluconate. The conductance sequence was as follows: N(3) > Br > Cl > N(CN)(2) > I > SCN > COOH > ClO(4) > acetate > HCO(3) = C(CN)(3) > gluconate. Permeant anions block in a voltage-dependent manner with the following affinities: C(CN)(3) > SCN = ClO(4) > N(CN)(2) > I > N(3) > Br > HCO(3) > Cl > gluconate > formate > acetate. Although these data suggest that anionic selectivity is determined by ionic hydration energy, other factors contribute, because the energy barrier for permeation is exponentially related to anion hydration energy. Cl(Ca)Cs exhibit weak anomalous mole fraction behavior, implying that the channel may be a multi-ion pore, but that ions interact weakly in the pore. The affinity of the channel for Ca(2+) depended on the permeant anion at low [Ca(2+)] (100-500 nM). Apparently, occupancy of the pore by a permeant anion increased the affinity of the channel for Ca(2+). The current was strongly dependent on pH. Increasing pH on the cytoplasmic side decreased the inward current, whereas increasing pH on the external side decreased the outward current. In both cases, the apparent pKa was voltage-dependent with apparent pKa at 0 mV = approximately 9.2. The channel may be blocked by OH(-) ions, or protons may titrate a site in the pore necessary for ion permeation. These data demonstrate that the permeation properties of Cl(Ca)Cs are different from those of CFTR or ClC-1, and provide insights into the nature of the Cl(Ca)C pore.     

Two bestrophins cloned from Xenopus laevis oocytes express Ca(2+)-activated Cl(-) currents

Ca2+-activated Cl- channels play important diverse roles from fast block to polyspermy to olfactory transduction, but their molecular identity has not been firmly established. By searching sequence databases with the M2 pore domain of ligand-gated anion channels, we identified potential Ca2+-activated Cl- channels, which included members of the bestrophin family. We cloned two bestrophins from Xenopus oocytes, which express high levels of Ca2+-activated Cl- channels. The Xenopus bestrophins were expressed in a variety of tissues. We predict that bestrophin has six transmembrane domains with the conserved RFP domain playing an integral part in ionic selectivity. When Xenopus bestrophins were heterologously expressed in human embryonic kidney-293 cells, large Ca2+-activated Cl- currents were observed. The currents are voltage- and time-independent, do not rectify, have a Kd for Ca2+ of approximately 210 nm, and exhibit a permeability ratio of I- > Br- > Cl- > aspartate. The W93C and G299E mutations produce non-functional channels that exert a dominant negative effect on wild type channels. We conclude that bestrophins are the first molecularly identified Cl- channels that are dependent on intracellular Ca2+ in a physiological range.     

Hydrogen peroxide potentiates volume-sensitive excitatory amino acid release via a mechanism involving Ca2+/calmodulin-dependent protein kinase II

Excessive excitatory amino acid (EAA) release in cerebral ischemia is a major mechanism responsible for neuronal damage and death. A substantial fraction of ischemic EAA release occurs via volume-regulated anion channels (VRACs). Hydrogen peroxide (H2O2), which is abundantly produced during ischemia and reperfusion, activates a number of protein kinases critical for VRAC functioning and has recently been reported to activate VRACs. In the present study, we explored the effects of H2O2 on volume-dependent EAA release in cultured astrocytes, measured as the release of preloaded D-[3H]aspartate. 100-1,000 microm H2O2 enhanced swelling-induced EAA release by approximately 2.5-3-fold (EC50 approximately 10 microM). The VRAC blockers ATP, phloretin, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) potently inhibited both control swelling-induced and the H2O2-potentiated release, suggesting a role for VRACs. The H2O2-induced component of EAA release was attenuated by the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) and completely eliminated by the calmodulin antagonists trifluoperazine and W-7 and the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN-93. Inhibitors of tyrosine kinases, protein kinase C, and the myosin light chain kinase were ineffective in blocking the H2O2 response. H2O2 treatment of swollen astrocytes, but not swelling alone, resulted in CaMKII activation that was inhibited by KN-93, as determined by a phospho-Thr286 CaMKII antibody. These data demonstrate that H2O2 strongly up-regulates astrocytic volume-sensitive EAA release via a CaMKII-dependent mechanism and in this way may potently promote pathological EAA release and brain damage in ischemia.     

Rescue of HIV-1 broad neutralizing antibody-expressing B cells in 2F5 VH x VL knockin mice reveals multiple tolerance controls

The HIV-1 broadly neutralizing Ab (bnAb) 2F5 has been shown to be poly-/self-reactive in vitro, and we previously demonstrated that targeted expression of its VDJ rearrangement alone was sufficient to trigger a profound B cell developmental blockade in 2F5 V(H) knockin (KI) mice, consistent with central deletion of 2F5 H chain-expressing B cells. In this study, we generate a strain expressing the entire 2F5 bnAb specificity, 2F5 V(H) × V(L) KI mice, and find an even higher degree of tolerance control than observed in the 2F5 V(H) KI strain. Although B cell development was severely impaired in 2F5 V(H) × V(L) KI animals, we demonstrate rescue of their B cells when cultured in IL-7/BAFF. Intriguingly, even under these conditions, most rescued B cell hybridomas produced mAbs that lacked HIV-1 Envelope (Env) reactivity due to editing of the 2F5 L chain, and the majority of rescued B cells retained an anergic phenotype. Thus, when clonal deletion is circumvented, κ editing and anergy are additional safeguards preventing 2F5 V(H)/V(L) expression by immature/transitional B cells. Importantly, 7% of rescued B cells retained 2F5 V(H)/V(L) expression and secreted Env-specific mAbs with HIV-1-neutralizing activity. This partial rescue was further corroborated in vivo, as reflected by the anergic phenotype of most rescued B cells in 2F5 V(H) × V(L) KI × Eμ-Bcl-2 transgenic mice and significant (yet modest) enrichment of Env-specific B cells and serum Igs. The rescued 2F5 mAb-producing B cell clones in this study are the first examples, to our knowledge, of in vivo-derived bone marrow precursors specifying HIV-1 bnAbs and provide a starting point for design of strategies aimed at rescuing such B cells.     

Two conventional protein kinase C isoforms, alpha and beta I, are involved in the ATP-induced activation of volume-regulated anion channel and glutamate release in cultured astrocytes

Volume-regulated anion channels (VRACs) are activated by cell swelling and are permeable to inorganic and small organic anions, including the excitatory amino acids glutamate and aspartate. In astrocytes, ATP potently enhances VRAC activity and glutamate release via a P2Y receptor-dependent mechanism. Our previous pharmacological study identified protein kinase C (PKC) as a major signaling enzyme in VRAC regulation by ATP. However, conflicting results obtained with potent PKC blockers prompted us to re-evaluate the involvement of PKC in regulation of astrocytic VRACs by using small interfering RNA (siRNA) and pharmacological inhibitors that selectively target individual PKC isoforms. In primary rat astrocyte cultures, application of hypoosmotic medium (30% reduction in osmolarity) and 20 μM ATP synergistically increased the release of excitatory amino acids, measured with a non-metabolized analog of l -glutamate, d -[³H]aspartate. Both Go6976, the selective inhibitor of Ca²⁺-sensitive PKCα, βI/II, and γ, and MP-20-28, a cell permeable pseudosubstrate inhibitory peptide of PKCα and βI/II, reduced the effects of ATP on d -[³H]aspartate release by ∼45–55%. Similar results were obtained with a mixture of siRNAs targeting rat PKCα and βI. Surprisingly, down-regulation of individual α and βI PKC isozymes by siRNA was completely ineffective. These data suggest that ATP regulates VRAC activity and volume-sensitive excitatory amino acid release via cooperative activation of PKCα and βI.     

ATP potently modulates anion channel-mediated excitatory amino acid release from cultured astrocytes

Volume-dependent ATP release andsubsequent activation of purinergic P2Y receptors have been implicatedas an autocrine mechanism triggering activation of volume-regulatedanion channels (VRACs) in hepatoma cells. In the brain ATP is releasedby both neurons and astrocytes and participates in intercellularcommunication. We explored whether ATP triggers or modulates therelease of excitatory amino acid (EAAs) via VRACs in astrocytes inprimary culture. Under basal conditions exogenous ATP (10 µM)activated a small EAA release in 70-80% of the cultures tested.In both moderately (5% reduction of medium osmolarity) andsubstantially (35% reduction of medium osmolarity) swollen astrocytes,exogenous ATP greatly potentiated EAA release. The effects of ATP weremimicked by P2Y agonists and eliminated by P2Y antagonists or the ATPscavenger apyrase. In contrast, the same pharmacological maneuvers didnot inhibit volume-dependent EAA release in the absence of exogenous ATP, ruling out a requirement of autocrine ATP release for VRAC activation. The ATP effect in nonswollen and moderately swollen cellswas eliminated by a 5-10% increase in medium osmolarity or byanion channel blockers but was insensitive to tetanus toxin pretreatment, further supporting VRAC involvement. Our data suggest that in astrocytes ATP does not trigger EAA release itself but actssynergistically with cell swelling. Moderate cell swelling and ATP mayserve as two cooperative signals in bidirectional neuron-astrocytecommunication in vivo.

     

Inhibition of volume-regulated anion channels by dominant-negative caveolin-1

Caveolae are flask-shaped invaginations of the plasma membrane formed by the association of caveolin proteins with lipid rafts. In endothelial cells, caveolae function as signal transduction centers controlling NO synthesis and mechanotransduction. We now provide evidence that the endothelial volume-regulated anion channel (VRAC) is also under the control of the caveolar system. When calf pulmonary artery endothelial (CPAE) cells were transfected with caveolin-1 Delta1-81 (deletion of amino acids 1 to 81), activation of VRAC by hypotonic cell swelling was strongly impaired. Concomitantly, caveolin-1 Delta1-81 disturbed the formation of caveolin-1 containing lipid rafts as evidenced by sucrose density gradient centrifugation. In nontransfected cells, endogenous caveolin-1 typically associated with low-density, detergent-resistant lipid rafts. However, transient expression of caveolin-1 Delta1-81 caused a redistribution of endogenous caveolin-1 to high-density, detergent-soluble membrane fractions. We therefore conclude that the interaction between caveolin-1 and detergent-resistant lipid rafts is an important prerequisite for endothelial VRAC activity.