scholarly journals A Store-operated Calcium Channel in Drosophila S2 Cells

2004 ◽  
Vol 123 (2) ◽  
pp. 167-182 ◽  
Author(s):  
Andriy V. Yeromin ◽  
Jack Roos ◽  
Kenneth A. Stauderman ◽  
Michael D. Cahalan
Keyword(s):  
Mammalian Cells ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Reversal Potential ◽  
S2 Cells ◽  
Pipette Solution ◽  
Crac Channels ◽  
Drosophila S2 Cells ◽  
Cell Recording ◽  
Inward Currents

Using whole-cell recording in Drosophila S2 cells, we characterized a Ca2+-selective current that is activated by depletion of intracellular Ca2+ stores. Passive store depletion with a Ca2+-free pipette solution containing 12 mM BAPTA activated an inwardly rectifying Ca2+ current with a reversal potential >60 mV. Inward currents developed with a delay and reached a maximum of 20–50 pA at −110 mV. This current doubled in amplitude upon increasing external Ca2+ from 2 to 20 mM and was not affected by substitution of choline for Na+. A pipette solution containing ∼300 nM free Ca2+ and 10 mM EGTA prevented spontaneous activation, but Ca2+ current activated promptly upon application of ionomycin or thapsigargin, or during dialysis with IP3. Isotonic substitution of 20 mM Ca2+ by test divalent cations revealed a selectivity sequence of Ba2+ > Sr2+ > Ca2+ >> Mg2+. Ba2+ and Sr2+ currents inactivated within seconds of exposure to zero-Ca2+ solution at a holding potential of 10 mV. Inactivation of Ba2+ and Sr2+ currents showed recovery during strong hyperpolarizing pulses. Noise analysis provided an estimate of unitary conductance values in 20 mM Ca2+ and Ba2+ of 36 and 420 fS, respectively. Upon removal of all external divalent ions, a transient monovalent current exhibited strong selectivity for Na+ over Cs+. The Ca2+ current was completely and reversibly blocked by Gd3+, with an IC50 value of ∼50 nM, and was also blocked by 20 μM SKF 96365 and by 20 μM 2-APB. At concentrations between 5 and 14 μM, application of 2-APB increased the magnitude of Ca2+ currents. We conclude that S2 cells express store-operated Ca2+ channels with many of the same biophysical characteristics as CRAC channels in mammalian cells.

Control of descending vasa recta pericyte membrane potential by angiotensin II

2002 ◽  
Vol 282 (6) ◽  
pp. F1064-F1074 ◽  
Author(s):  
Thomas L. Pallone ◽  
James M.-C. Huang
Keyword(s):  
Membrane Potential ◽  
Angiotensin Ii ◽  
Reversal Potential ◽  
Niflumic Acid ◽  
Channel Blockers ◽  
Ang Ii ◽  
Vasa Recta ◽  
Cell Recording ◽  
Inward Currents

Using nystatin perforated-patch whole cell recording, we investigated the role of Cl−conductance in the modulation of outer medullary descending vasa recta (OMDVR) pericyte membrane potential (Ψm) by ANG II. ANG II (10−11 to 10−7 M) consistently depolarized OMDVR and induced Ψm oscillations at lower concentrations. The Cl− channel blockers anthracene-9-decarboxylate (1 mM) and niflumic acid (10 μM) hyperpolarized resting pericytes and repolarized ANG II-treated pericytes. In voltage-clamp experiments, ANG II-treated pericytes exhibited slowly activating currents that were nearly eliminated by treatment with niflumic acid (10 μM) or removal of extracellular Ca2+. Those currents reversed at −31 and −10 mV when extracellular Cl− concentration was 152 and 34 mM, respectively. In pericytes held at −70 mV, oscillating inward currents were sometimes observed; the reversal potential also shifted with extracellular Cl− concentration. We conclude that ANG II activates a Ca2+-dependent Cl− conductance in OMDVR pericytes to induce membrane depolarization and Ψm oscillations.

Inhibitory interactions between 5-HT3 and P2X channels in submucosal neurons

2002 ◽  
Vol 283 (6) ◽  
pp. G1238-G1248 ◽  
Author(s):  
Carlos Barajas-López ◽  
Luis M. Montaño ◽  
Rosa Espinosa-Luna
Keyword(s):  
P2x Receptors ◽  
Disulfonic Acid ◽  
Pharmacological Properties ◽  
Pipette Solution ◽  
Outward Currents ◽  
C 23 ◽  
Cell Recording ◽  
Inward Currents ◽  
Inhibitory Interactions ◽  
Subtype 3

Inhibitory interactions between 5-HT subtype 3 (5-HT3) and P2X receptors were characterized using whole cell recording techniques. Currents induced by 5-HT ( I 5-HT) and ATP ( I ATP) were blocked by tropisetron (or ondansetron) and pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid, respectively. Currents induced by 5-HT + ATP ( I 5-HT+ATP) were only as large as the current induced by the most effective transmitter, revealing current occlusion. Occlusion was observed at membrane potentials of −60 and 0 mV (for inward currents), but it was not present at +40 mV (for outward currents). Kinetic and pharmacological properties of I 5-HT+ATP indicate that they are carried through 5-HT3 and P2X channels. Current occlusion occurred as fast as activation of I 5-HT and I ATP, was still present in the absence of Ca2+ or Mg2+, after adding staurosporine, genistein, K-252a, or N-ethylmaleimide to the pipette solution, after substituting ATP with ∝,β-methylene ATP or GTP with GTP-γ-S in the pipette, and was observed at 35°C, 23°C, and 8°C. These results are in agreement with a model that considers that 5-HT3 and P2X channels are in functional clusters and that these channels might directly inhibit each other.

scholarly journals Mobilization of intracellular calcium by intracellular flash photolysis of caged dihydrosphingosine in cultured neonatal rat sensory neurones.

1998 ◽  
Vol 45 (2) ◽  
pp. 311-326 ◽  
Author(s):  
A Ayar ◽  
N M Thatcher ◽  
U Zehavi ◽  
D R Trentham ◽  
R H Scott
Keyword(s):  
Flash Photolysis ◽  
Reversal Potential ◽  
Neonatal Rat ◽  
Xenon Lamp ◽  
Patch Clamp Technique ◽  
Pipette Solution ◽  
Sensory Neurones ◽  
Whole Cell ◽  
Inward Currents ◽  
Current Activation

The ability of dihydrosphingosine to release Ca2+ from intracellular stores in neurones was investigated by combining the whole cell patch clamp technique with intracellular flash photolysis of caged, N-(2-nitrobenzyl)dihydrosphingosine. The caged dihydrosphingosine (100 microM) was applied to the intracellular environment via the CsCl-based patch pipette solution which also contained 0.3% dimethylformamide and 2 mM dithiothreitol. Cultured dorsal root ganglion neurones from neonatal rats were voltage clamped at -90 mV and inward whole cell Ca2+-activated currents were recorded in response to intracellular photorelease of dihydrosphingosine. Intracellular photorelease of dihydrosphingosine (about 5 microM) was achieved using a Xenon flash lamp. Inward Ca2+-activated currents were evoked in 50 out of 57 neurones, the mean delay to current activation following photolysis was 82+/-13 s. The responses were variable with neurones showing transient, oscillating or sustained inward currents. High voltage-activated Ca2+ currents evoked by 100 ms voltage step commands to 0 mV were not attenuated by photorelease of dihydrosphingosine. Controls showed that alone a flash from the Xenon lamp did not activate currents, and that the unphotolysed caged dihydrosphingosine, and intracellular photolysis of 2-(2-nitrobenzylamino) propanediol also did not evoke responses. The dihydrosphingosine current had a reversal potential of -11+/-3 mV (n = 11), and was carried by two distinct Cl- and cation currents which were reduced by 85% and about 20% following replacement of monovalent cations with N-methyl-D-glucamine or application of the Cl- channel blocker niflumic acid (10 microM) respectively. The responses to photoreleased dihydrosphingosine were inhibited by intracellular application of 20 mM EGTA, 10 microM ryanodine or extracellular application of 10 microM dantrolene, but persisted when Ca2+ free saline was applied to the extracellular environment. Intracellular application of uncaged dihydrosphingosine evoked responses which were attenuated by photolysis of the caged Ca2+ chelator Diazo-2. Experiments also suggested that extracellular application of dihydrosphingosine can activate membrane conductances. We conclude that dihydrosphingosine directly or indirectly mobilises Ca2+ from ryanodine-sensitive intracellular stores in cultured sensory neurones.

scholarly journals Use of Drosophila S2 Cells as a Model for Studying Ehrlichia chaffeensis Infections

2008 ◽  
Vol 74 (6) ◽  
pp. 1886-1891 ◽  
Author(s):  
Alison Luce-Fedrow ◽  
Tonia Von Ohlen ◽  
Daniel Boyle ◽  
Roman R. Ganta ◽  
Stephen K. Chapes
Keyword(s):  
Mammalian Cells ◽  
Genetic Manipulation ◽  
Rrna Gene ◽  
Ehrlichia Chaffeensis ◽  
S2 Cells ◽  
Obligate Intracellular Bacterium ◽  
Obligate Intracellular ◽  
Mammalian Cell Lines ◽  
Drosophila S2 Cells ◽  
Human Monocytic Ehrlichiosis

ABSTRACT Ehrlichia chaffeensis is an obligate intracellular bacterium and the causative agent of human monocytic ehrlichiosis. Although this pathogen grows in several mammalian cell lines, no general model for eukaryotic cellular requirements for bacteria replication has yet been proposed. We found that Drosophila S2 cells are permissive for the growth of E. chaffeensis. We saw morulae (aggregates of bacteria) by microscopy, detected the E. chaffeensis 16S rRNA gene by reverse transcriptase PCR, and used immunocytochemistry to detect E. chaffeensis in S2 and mammalian cells. Bacteria grown in S2 cells reinfected mammalian macrophages. S2 cells were made nonpermissive for E. chaffeensis through incubation with lipopolysaccharide. Our results demonstrate that S2 cells are an appropriate system for studying the pathogenesis of E. chaffeensis. The use of a Drosophila system has the potential to serve as a model system for studying Ehrlichia due to its completed genome, ease of genetic manipulation, and the availability of mutants.

scholarly journals Transport of polyamines in Drosophila S2 cells: kinetics, pharmacology and dependence on the plasma membrane proton gradient

2005 ◽  
Vol 393 (2) ◽  
pp. 583-589 ◽  
Author(s):  
Rafael Romero-Calderón ◽  
David E. Krantz
Keyword(s):  
Mammalian Cells ◽  
Organic Anion ◽  
S2 Cells ◽  
Transport Activity ◽  
Molecular Identity ◽  
Polyamine Transport ◽  
Drosophila S2 Cells ◽  
Solute Carrier ◽  
Polyamine Uptake

Polyamine transport activities have been described in diverse multicellular systems, but their bioenergetic mechanisms and molecular identity remain unclear. In the present paper, we describe a high-affinity spermine/spermidine transport activity expressed in Drosophila S2 cells. Ion-replacement experiments indicate that polyamine uptake across the cell membrane is Na+-, K+-, Cl−- and Ca2+-independent, but pH-sensitive. Additional experiments using ionophores suggest that polyamine uptake may be H+-coupled. Pharmacological experiments show that polyamine uptake in S2 cells is selectively blocked by MGBG {methylglyoxal bis(guanylhydrazone) or 1,1′-[(methylethanediylidine)-dinitrilo]diguanidine} and paraquat (N,N-dimethyl-4,4′-bipyridylium), two known inhibitors of polyamine uptake in mammalian cells. In addition, inhibitors known to block the Slc22 (solute carrier 22) family of organic anion/cation transporters inhibit spermine uptake in S2 cells. These data and the genetic tools available in Drosophila will facilitate the molecular identification and further characterization of this activity.

scholarly journals Monovalent Permeability, Rectification, and Ionic Block of Store-operated Calcium Channels in Jurkat T Lymphocytes

1998 ◽  
Vol 111 (4) ◽  
pp. 521-537 ◽  
Author(s):  
Hubert H. Kerschbaum ◽  
Michael D. Cahalan
Keyword(s):  
T Lymphocytes ◽  
Intracellular Ph ◽  
Calcium Release ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Pipette Solution ◽  
Crac Channels ◽  
Cell Recording ◽  
Voltage Dependent ◽  
Crac Channel

We used whole-cell recording to characterize ion permeation, rectification, and block of monovalent current through calcium release-activated calcium (CRAC) channels in Jurkat T lymphocytes. Under physiological conditions, CRAC channels exhibit a high degree of selectivity for Ca2+, but can be induced to carry a slowly declining Na+ current when external divalent ions are reduced to micromolar levels. Using a series of organic cations as probes of varying size, we measured reversal potentials and calculated permeability ratios relative to Na+, PX/PNa, in order to estimate the diameter of the conducting pore. Ammonium (NH4+) exhibited the highest relative permeability (PNH4/PNa = 1.37). The largest permeant ion, tetramethylammonium with a diameter of 0.55 nm, had PTMA/PNa of 0.09. N-methyl-d-glucamine (0.50 × 0.64 × 1.20 nm) was not measurably permeant. In addition to carrying monovalent current, NH4+ reduced the slow decline of monovalent current (“inactivation”) upon lowering [Ca2+]o. This kinetic effect of extracellular NH4+ can be accounted for by an increase in intracellular pH (pHi), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pHi reduced monovalent and divalent current amplitudes through CRAC channels with a pKa of 6.8. In several channel types, Mg2+ has been shown to produce rectification by a voltage-dependent block mechanism. Mg2+ removal from the pipette solution permitted large outward monovalent currents to flow through CRAC channels while also increasing the channel's relative Cs+ conductance and eliminating the inactivation of monovalent current. Boltzmann fits indicate that intracellular Mg2+ contributes to inward rectification by blocking in a voltage-dependent manner, with a zδ product of 1.88. Ca2+ block from the outside was also found to be voltage dependent with zδ of 1.62. These experiments indicate that the CRAC channel, like voltage-gated Ca2+ channels, achieves selectivity for Ca2+ by selective binding in a large pore with current–voltage characteristics shaped by internal Mg2+.

scholarly journals Induction of focal adhesions and motility in Drosophila S2 cells

2014 ◽  
Vol 25 (24) ◽  
pp. 3861-3869 ◽  
Author(s):  
Susana A. Ribeiro ◽  
Michael V. D'Ambrosio ◽  
Ronald D. Vale
Keyword(s):  
Focal Adhesion ◽  
Mammalian Cells ◽  
Focal Adhesions ◽  
S2 Cells ◽  
Cell Interior ◽  
Drosophila S2 Cells ◽  
Cytoskeletal Dynamics ◽  
A Cell ◽  
P21 Activated Kinase ◽  
Dynamic Structures

Focal adhesions are dynamic structures that interact with the extracellular matrix on the cell exterior and actin filaments on the cell interior, enabling cells to adhere and crawl along surfaces. We describe a system for inducing the formation of focal adhesions in normally non–ECM-adherent, nonmotile Drosophila S2 cells. These focal adhesions contain the expected molecular markers such as talin, vinculin, and p130Cas, and they require talin for their formation. The S2 cells with induced focal adhesions also display a nonpolarized form of motility on vitronectin-coated substrates. Consistent with findings in mammalian cells, the degree of motility can be tuned by changing the stiffness of the substrate and was increased after the depletion of PAK3, a p21-activated kinase. A subset of nonmotile, nonpolarized cells also exhibited focal adhesions that rapidly assembled and disassembled around the cell perimeter. Such cooperative and dynamic fluctuations of focal adhesions were decreased by RNA interference (RNAi) depletion of myosin II and focal adhesion kinase, suggesting that this behavior requires force and focal adhesion maturation. These results demonstrate that S2 cells, a cell line that is well studied for cytoskeletal dynamics and readily amenable to protein manipulation by RNAi, can be used to study the assembly and dynamics of focal adhesions and mechanosensitive cell motility.

Antagonistic Modulation of a Hyperpolarization-Activated Cl– Current in Aplysia Sensory Neurons by SCPB and FMRFamide

2003 ◽  
Vol 90 (2) ◽  
pp. 586-598 ◽  
Author(s):  
Ned Buttner ◽  
Steven A. Siegelbaum
Keyword(s):  
Sensory Neurons ◽  
Reversal Potential ◽  
Outward Current ◽  
Cell Voltage ◽  
High Concentration ◽  
Pipette Solution ◽  
Whole Cell ◽  
Whole Cell Recording ◽  
Type K ◽  
Cell Recording

Whole cell voltage-clamp recordings from Aplysia mechanosensory neurons obtained from the pleural ganglion were used to investigate the actions on membrane currents of the neuropeptides SCPB and FMRFamide. At the start of whole cell recording, SCPB typically evoked an inward current at a holding potential of –40 mV, due to the cAMP-mediated closure of the S-type K+ channel, whereas FMRFamide evoked an outward current, due to the opening of the S-type K+ channels mediated by 12-lipoxygenase metabolites of arachidonic acid. However, after several minutes of whole cell recording with a high concentration of chloride in the whole cell patch pipette solution, the responses to SCPB and FMRF-amide at –40 mV were inverted; SCPB evoked an outward current, whereas FMRFamide and YGGFMRFamide evoked inward currents. Ion substitution experiments and reversal potential measurements revealed that these responses were due to the opposing regulation of a Cl– current, whose magnitude was greatly enhanced by dialysis with the high Cl–-containing pipette solution. SCPB inhibited this Cl– current through production of cAMP and activation of PKA. YGGFMRFamide activated this Cl– current by stimulating a cGMP-activated phosphodiesterase that hydrolyzed cAMP. Thus a cAMP-dependent Cl– current undergoes antagonistic modulation by two neuropeptides in Aplysia sensory neurons.

scholarly journals Separation and Characterization of Currents through Store-operated CRAC Channels and Mg2+-inhibited Cation (MIC) Channels

2002 ◽  
Vol 119 (5) ◽  
pp. 487-508 ◽  
Author(s):  
Murali Prakriya ◽  
Richard S. Lewis
Keyword(s):  
Calcium Release ◽  
Divalent Cations ◽  
Noise Analysis ◽  
Crac Channels ◽  
Unitary Conductance ◽  
New Information ◽  
Crac Channel ◽  
High Doses ◽  
Whole Cell Recordings

Although store-operated calcium release–activated Ca2+(CRAC) channels are highly Ca2+-selective under physiological ionic conditions, removal of extracellular divalent cations makes them freely permeable to monovalent cations. Several past studies have concluded that under these conditions CRAC channels conduct Na+and Cs+with a unitary conductance of ∼40 pS, and that intracellular Mg2+modulates their activity and selectivity. These results have important implications for understanding ion permeation through CRAC channels and for screening potential CRAC channel genes. We find that the observed 40-pS channels are not CRAC channels, but are instead Mg2+-inhibited cation (MIC) channels that open as Mg2+is washed out of the cytosol. MIC channels differ from CRAC channels in several critical respects. Store depletion does not activate MIC channels, nor does store refilling deactivate them. Unlike CRAC channels, MIC channels are not blocked by SKF 96365, are not potentiated by low doses of 2-APB, and are less sensitive to block by high doses of the drug. By applying 8–10 mM intracellular Mg2+to inhibit MIC channels, we examined monovalent permeation through CRAC channels in isolation. A rapid switch from 20 mM Ca2+to divalent-free extracellular solution evokes Na+current through open CRAC channels (Na+-ICRAC) that is initially eightfold larger than the preceding Ca2+current and declines by ∼80% over 20 s. Unlike MIC channels, CRAC channels are largely impermeable to Cs+(PCs/PNa= 0.13 vs. 1.2 for MIC). Neither the decline in Na+-ICRACnor its low Cs+permeability are affected by intracellular Mg2+(90 μM to 10 mM). Single openings of monovalent CRAC channels were not detectable in whole-cell recordings, but a unitary conductance of 0.2 pS was estimated from noise analysis. This new information about the selectivity, conductance, and regulation of CRAC channels forces a revision of the biophysical fingerprint of CRAC channels, and reveals intriguing similarities and differences in permeation mechanisms of voltage-gated and store-operated Ca2+channels.

Sign in / Sign up

Export Citation Format

Share Document