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 Ring of Negative Charge in BK Channels Facilitates Block by Intracellular Mg2+ and Polyamines through Electrostatics

2006 ◽  
Vol 128 (2) ◽  
pp. 185-202 ◽  
Author(s):  
Yaxia Zhang ◽  
Xiaowei Niu ◽  
Tinatin I. Brelidze ◽  
Karl L. Magleby
Keyword(s):  
Negative Charge ◽  
Outward Current ◽  
Bk Channels ◽  
Inward Rectification ◽  
Dependent Manner ◽  
Conduction Pathway ◽  
Boltzmann Function ◽  
Voltage Dependent ◽  
Hill Coefficients ◽  
The Hill

Intracellular Mg2+ and natural polyamines block outward currents in BK channels in a highly voltage-dependent manner. Here we investigate the contribution of the ring of eight negatively charged residues (4 x E321/E324) at the entrance to the inner vestibule of BK channels to this block. Channels with or without (E321N/E324N) the ring of negative charge were expressed in oocytes and unitary currents were recorded from inside-out patches over a range of intracellular Mg2+ and polyamine concentrations. Removing the ring of charge greatly decreased the block, increasing KBap (0 mV) for Mg2+ block from 48.3 ± 3.0 to 143 ± 8 mM, and for spermine block from 8.0 ± 1.0 to 721 ± 9 mM (150 mM symmetrical KCl). Polyamines with fewer amine groups blocked less: putrescine < spermidine < spermine. An equation that combined an empirical Hill function for block together with a Boltzmann function for the voltage dependence of KBap described the voltage and concentration dependence of the block for channels with and without the ring of charge. The Hill coefficients for these descriptions were <1 for both Mg2+ and spermine block, and were unchanged by removing the ring of charge. When KCli was increased from 150 mM to 3 M, the ring of charge no longer facilitated block, Mg2+ block was reduced, spermine block became negligible, and the Hill coefficients became ∼1.0. BK channels in cell-attached oocyte patches displayed inward rectification, which was reduced for channels without the ring of charge. Taken together, these observations suggest that the ring of negative charge facilitates block through a preferential electrostatic attraction of Mg2+ and polyamine over K+. This preferential attraction of multivalent blockers over monovalent K+ would decrease the K+ available at the inner vestibule to carry outward current in the presence of Mg2+ or polyamines, while increasing the concentration of blocker available to enter and block the conduction pathway.

scholarly journals Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

1992 ◽  
Vol 100 (5) ◽  
pp. 783-801 ◽  
Author(s):  
L W Haynes
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Single Channel ◽  
Inward Rectification ◽  
Pipette Solution ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Extracellular Side ◽  
Current Voltage Relation ◽  
Cytoplasmic Side

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

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.

Quinidine blocks adenosine 5'-triphosphate-sensitive potassium channels in heart

1990 ◽  
Vol 259 (5) ◽  
pp. H1609-H1612 ◽  
Author(s):  
A. I. Undrovinas ◽  
N. Burnashev ◽  
D. Eroshenko ◽  
I. Fleidervish ◽  
C. F. Starmer ◽  
...  
Keyword(s):  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Pipette Solution ◽  
Channel Current ◽  
Closed Intervals ◽  
Ventricular Cells ◽  
Voltage Dependent ◽  
Inside Out ◽  
Ischemic Arrhythmias

The ATP-sensitive potassium channel current (IK-ATP) was studied in excised inside-out patches from rat ventricular cells at 20-23 degrees C. The bath solution contained 140 mM KF, and the pipette solution contained 140 mM KCl and 1.2 mM MgCl2. ATP (0.5 mM) in the bath inhibited IK-ATP. In the absence of ATP, 10 microM quinidine decreased open probability 67 +/- 1% (n = 6) at -50 mV and 28 +/- 12% at -130 mV (n = 5) without affecting single channel conductance (48-52 pS). The block increased with 25 and 50 microM quinidine and could be reversed on washing quinidine for several minutes. Interburst (closed) intervals were increased by quinidine, whereas open and closed time distributions within bursts were not changed. We conclude that quinidine blocks IK-ATP in a "slow" and voltage-dependent manner in clinically relevant concentrations. Because of the postulated role for IK-ATP in cardiac ischemia, quinidine block of this channel may play a role in ischemic arrhythmias.

scholarly journals Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx

2015 ◽  
Vol 113 (2) ◽  
pp. 440-445 ◽  
Author(s):  
Joseph L. Dynes ◽  
Anna Amcheslavsky ◽  
Michael D. Cahalan
Keyword(s):  
Single Channel ◽  
Calcium Influx ◽  
Optical Recording ◽  
Second Phase ◽  
Channel Output ◽  
Crac Channels ◽  
Cell Recording ◽  
Crac Channel ◽  
Single Channel Currents ◽  
Fluorescence Transients

Orai1 comprises the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel. When bound and activated by stromal interacting molecule 1 (STIM1), an endoplasmic reticulum (ER)-resident calcium sensor, Orai1 channels possess high selectivity for calcium but extremely small conductance that has precluded direct recording of single-channel currents. We have developed an approach to visualize Orai1 activity by fusing Orai1 to fluorescent, genetically encoded calcium indicators (GECIs). The GECI–Orai1 probes reveal local Ca2+ influx at STIM1–Orai1 puncta. By whole cell recording, these fusions are fully functional as CRAC channels. When GECI–Orai1 and the CRAC-activating domain (CAD) of STIM1 were coexpressed at low levels and imaged using a total internal reflectance fluorescence microscope, cells exhibited sporadic fluorescence transients the size of diffraction-limited spots and the brightness of a few activated GECI proteins. Transients typically rose rapidly and fell into two classes according to duration: briefer “flickers” lasting only a few hundred milliseconds, and longer “pulses” lasting one to several seconds. The size, intensity, trace shape, frequency, distribution, physiological characteristics, and association with CAD binding together demonstrate that GECI–Orai1 fluorescence transients correspond to single-channel Orai1 responses. Single Orai1 channels gated by CAD, and small Orai1 puncta gated by STIM1, exhibit repetitive fluctuations in single-channel output. CAD binding supports a role in open state maintenance and reveals a second phase of CAD/STIM1 binding after channel opening. These first recordings of single-channel Orai1 currents reveal unexpected dynamics, and when paired with CAD association, support multiple single-channel states.

scholarly journals Rapid inactivation of depletion-activated calcium current (ICRAC) due to local calcium feedback.

1995 ◽  
Vol 105 (2) ◽  
pp. 209-226 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
Time Course ◽  
Measurement Techniques ◽  
Fast Inactivation ◽  
Patch Clamp Recording ◽  
Crac Channels ◽  
Whole Cell Patch Clamp ◽  
Close Proximity ◽  
Rapid Inactivation ◽  
Voltage Dependent ◽  
Crac Channel

Rapid inactivation of Ca2+ release-activated Ca2+ (CRAC) channels was studied in Jurkat leukemic T lymphocytes using whole-cell patch clamp recording and [Ca2+]i measurement techniques. In the presence of 22 mM extracellular Ca2+, the Ca2+ current declined with a biexponential time course (time constants of 8-30 ms and 50-150 ms) during hyperpolarizing pulses to potentials more negative than -40 mV. Several lines of evidence suggest that the fast inactivation process is Ca2+ but not voltage dependent. First, the speed and extent of inactivation are enhanced by conditions that increase the rate of Ca2+ entry through open channels. Second, inactivation is substantially reduced when Ba2+ is present as the charge carrier. Third, inactivation is slowed by intracellular dialysis with BAPTA (12 mM), a rapid Ca2+ buffer, but not by raising the cytoplasmic concentration of EGTA, a slower chelator, from 1.2 to 12 mM. Recovery from fast inactivation is complete within 200 ms after repolarization to -12 mV. Rapid inactivation is unaffected by changes in the number of open CRAC channels or global [Ca2+]i. These results demonstrate that rapid inactivation of ICRAC results from the action of Ca2+ in close proximity to the intracellular mouths of individual channels, and that Ca2+ entry through one CRAC channel does not affect neighboring channels. A simple model for Ca2+ diffusion in the presence of a mobile buffer predicts multiple Ca2+ inactivation sites situated 3-4 nm from the intracellular mouth of the pore, consistent with a location on the CRAC channel itself.

scholarly journals An essential and NSF independent role for α-SNAP in store-operated calcium entry

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Yong Miao ◽  
Cathrine Miner ◽  
Lei Zhang ◽  
Phyllis I Hanson ◽  
Adish Dani ◽  
...  
Keyword(s):  
Essential Role ◽  
Calcium Release ◽  
Calcium Entry ◽  
Snare Complex ◽  
Functional Coupling ◽  
Crac Channels ◽  
Store Depletion ◽  
Store Operated Calcium Entry ◽  
Crac Channel

Store-operated calcium entry (SOCE) by calcium release activated calcium (CRAC) channels constitutes a primary route of calcium entry in most cells. Orai1 forms the pore subunit of CRAC channels and Stim1 is the endoplasmic reticulum (ER) resident Ca2+ sensor. Upon store-depletion, Stim1 translocates to domains of ER adjacent to the plasma membrane where it interacts with and clusters Orai1 hexamers to form the CRAC channel complex. Molecular steps enabling activation of SOCE via CRAC channel clusters remain incompletely defined. Here we identify an essential role of α-SNAP in mediating functional coupling of Stim1 and Orai1 molecules to activate SOCE. This role for α-SNAP is direct and independent of its known activity in NSF dependent SNARE complex disassembly. Importantly, Stim1-Orai1 clustering still occurs in the absence of α-SNAP but its inability to support SOCE reveals that a previously unsuspected molecular re-arrangement within CRAC channel clusters is necessary for SOCE.

Abstract TP272: Calcium Release-activated Calcium (CRAC) Channel Inhibition Protects Against Brain Injury and Neuronal Death Induced by Activated Microglia

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Rachid Kacimi ◽  
Jong Youl Kim ◽  
Ken Stauderman ◽  
Michael Dunn ◽  
Sudarshan Hebbar ◽  
...  
Keyword(s):  
Brain Injury ◽  
Calcium Signaling ◽  
Calcium Release ◽  
Neuronal Cell ◽  
Western Blots ◽  
Activated Microglia ◽  
Crac Channels ◽  
Channel Inhibition ◽  
Bv2 Cells ◽  
Crac Channel

Inflammatory responses following ischemia can worsen neurological outcome, and represent a potential target for therapeutic intervention. Store-operated Ca 2+ entry (SOCE) mediated by CRAC channels contribute to calcium signaling in immune cells. CRAC channels consist of the endoplasmic reticulum resident Ca 2+ -sensing protein stromal interaction molecule 1 (STIM1) and the calcium channel protein ORAI1 located in the plasma membrane. Prolonged Ca 2+ entry through CRAC channels activates, via calcineurin, nuclear factor of activated T cells (NFAT), involved in T cell proliferation and cytokine expression. Microglia mediate inflammation in the injured brain, but little is known about the role of CRAC channels in this process. We studied novel CRAC channel inhibitors to explore their therapeutic potential in microglia-mediated injury. A neuron cell line (Neuro-2A, N-2A) was cultured alone or with microglial BV2 cells then exposed to 2h oxygen glucose deprivation (OGD). Some cultures were treated with a novel CRAC channel inhibitor. Toll-like receptor (TLR) -3, -4 agonists or IFNγ were also used to activate microglia. Western blots revealed the presence of CRAC channel proteins STIM1 and ORAI1 in microglia. CRAC channel inhibition decreased NO release and inflammatory proteins iNOS and COX-2 expression in activated microglia, but did not affect STIM1 or ORAI1 expression. CRAC channel inhibitors also reduced agonist induced intracellular calcium accumulation in BV2 cells. Agonists also activated JNK1/2 kinase, NFAT, NF-κB, CREB & STAT1 in microglia, but only JNK1/2 kinase & NFAT were attenuated by inhibitor. OGD decreased N2A neuronal cell viability, further exacerbated by BV2 cells, but neuronal cells were protected by CRAC channel inhibition (n=5, *p<0.05). We then treated male C57/BL6 mice exposed to experimental brain trauma (TBI) and found that CRAC channel inhibition led to decreased lesion size, brain hemorrhage and improved neurological deficits (n=6-7/grp, *p<0.05). We suggest a novel anti-inflammatory approach for treating acute brain injury. Our observations also shed light on new calcium signaling pathways, not previously described in brain injury models.

Bidirectional regulation of calcium release–activated calcium (CRAC) channel by SARAF

2021 ◽  
Vol 220 (12) ◽  
Author(s):  
Elia Zomot ◽  
Hadas Achildiev Cohen ◽  
Inbal Dagan ◽  
Ruslana Militsin ◽  
Raz Palty
Keyword(s):  
Gene Expression ◽  
Calcium Release ◽  
Cell Receptor ◽  
Crac Channels ◽  
Bidirectional Regulation ◽  
Dependent Inactivation ◽  
Store Operated Calcium Entry ◽  
Crac Channel ◽  
Initial Activation ◽  
Downstream Gene Expression

Store-operated calcium entry (SOCE) through the Ca2+ release–activated Ca2+ (CRAC) channel is a central mechanism by which cells generate Ca2+ signals and mediate Ca2+-dependent gene expression. The molecular basis for CRAC channel regulation by the SOCE-associated regulatory factor (SARAF) remained insufficiently understood. Here we found that following ER Ca2+ depletion, SARAF facilitates a conformational change in the ER Ca2+ sensor STIM1 that relieves an activation constraint enforced by the STIM1 inactivation domain (ID; aa 475–483) and promotes initial activation of STIM1, its translocation to ER–plasma membrane junctions, and coupling to Orai1 channels. Following intracellular Ca2+ rise, cooperation between SARAF and the STIM1 ID controls CRAC channel slow Ca2+-dependent inactivation. We further show that in T lymphocytes, SARAF is required for proper T cell receptor evoked transcription. Taking all these data together, we uncover a dual regulatory role for SARAF during both activation and inactivation of CRAC channels and show that SARAF fine-tunes intracellular Ca2+ responses and downstream gene expression in cells.

scholarly journals Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes.

1996 ◽  
Vol 107 (5) ◽  
pp. 597-610 ◽  
Author(s):  
A Zweifach ◽  
R S Lewis
Keyword(s):  
T Lymphocytes ◽  
Voltage Dependence ◽  
Single Channel ◽  
Current Amplitude ◽  
Second Phase ◽  
Channel Activation ◽  
Calcium Dependent ◽  
Crac Channels ◽  
Crac Channel ◽  
Two Phases

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.

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