scholarly journals Physiological CRAC channel activation and pore properties require STIM1 binding to all six Orai1 subunits

2018 ◽  
Vol 150 (10) ◽  
pp. 1373-1385 ◽  
Author(s):  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Single Channel ◽  
Divalent Cations ◽  
Ion Selectivity ◽  
Functional Coupling ◽  
Open Probability ◽  
Channel Activation ◽  
Channel Current ◽  
Crac Channels ◽  
Crac Channel ◽  
Pore Properties

The binding of STIM1 to Orai1 controls the opening of store-operated CRAC channels as well as their extremely high Ca2+ selectivity. Although STIM1 dimers are known to bind directly to the cytosolic C termini of the six Orai1 subunits (SUs) that form the channel hexamer, the dependence of channel activation and selectivity on the number of occupied binding sites is not well understood. Here we address these questions using dimeric and hexameric Orai1 concatemers in which L273D mutations were introduced to inhibit STIM1 binding to specific Orai1 SUs. By measuring FRET between fluorescently labeled STIM1 and Orai1, we find that homomeric L273D mutant channels fail to bind STIM1 appreciably; however, the L273D SU does bind STIM1 and contribute to channel activation when located adjacent to a WT SU. These results suggest that STIM1 dimers can interact with pairs of neighboring Orai1 SUs. Surprisingly, a single L273D mutation within the Orai1 hexamer reduces channel open probability by ∼90%, triples the size of the single-channel current, weakens the Ca2+ binding affinity of the selectivity filter, and lowers the selectivity for Na+ over Cs+ in the absence of divalent cations. These findings reveal a surprisingly strong functional coupling between STIM1 binding and CRAC channel gating and pore properties. We conclude that under physiological conditions, all six Orai1 SUs of the native CRAC channel bind STIM1 to effectively open the pore and generate the signature properties of extremely low conductance and high ion selectivity.

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.

Permeation and gating properties of a cloned renal K+ channel

1995 ◽  
Vol 268 (2) ◽  
pp. C389-C401 ◽  
Author(s):  
S. Chepilko ◽  
H. Zhou ◽  
H. Sackin ◽  
L. G. Palmer
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Cation Binding ◽  
K Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Membrane Hyperpolarization ◽  
Channel Current ◽  
Inward Currents ◽  
Kinetics Of

The renal K+ channel (ROMK2) was expressed in Xenopus oocytes, and the patch-clamp technique was used to assess its conducting and gating properties. In cell-attached patches with 110 mM K+ in the bath and pipette, the reversal potential was near zero and the inward conductance (36 pS) was larger than the outward conductance (17 pS). In excised inside-out patches the channels showed rectification in the presence of 5 mM Mg2+ on the cytoplasmic side but not in Mg(2+)-free solution. Inward currents were also observed when K+ was replaced in the pipette by Rb+, NH4+, or thallium (Tl+). The reversal potentials under these conditions yielded a selectivity sequence of Tl+ > K+ > Rb+ > NH4+. On the other hand, the slope conductances for inward current gave a selectivity sequence of K+ = NH4+ > Tl+ > Rb+. The differences in the two sequences can be explained by the presence of cation binding sites within the channel, which interact with Rb+ and Tl+ more strongly and with NH4+ less strongly than with K+. Two other ions, Ba2+ and Cs+, blocked the channel from the outside. The effect of Ba2+ (1 mM) was to reduce the open probability of the channels, whereas Cs+ (10 mM) reduced the apparent single-channel current. The effects of both blockers are enhanced by membrane hyperpolarization. The kinetics of the channel were also studied in cell-attached patches. With K+ in the pipette the distribution of open times could be described by a single exponential (tau 0 = 25 ms), whereas two exponentials (tau 1 = 1 ms, tau 2 = 30 ms) were required to describe the closed-time distribution. Hyperpolarization of the oocyte membrane decreased the open probability and tau 0, and increased tau 1, tau 2, and the number of long closures. The presence of Tl+ in the pipette significantly altered the kinetics, reducing tau 0 and eliminating the long-lived closures. These results suggest that the gating of the channel may depend on the nature of the ion in the pore.

Effect of Prozac on whole cell ionic currents in lens and corneal epithelia

1995 ◽  
Vol 269 (1) ◽  
pp. C250-C256 ◽  
Author(s):  
J. L. Rae ◽  
A. Rich ◽  
A. C. Zamudio ◽  
O. A. Candia
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Ionic Currents ◽  
Current Amplitude ◽  
Human Lens ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Ionic Conductances

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.

Eicosanoids modulate apical Ca(2+)-dependent K+ channels in cultured rabbit principal cells

1992 ◽  
Vol 263 (1) ◽  
pp. F116-F126 ◽  
Author(s):  
B. N. Ling ◽  
C. L. Webster ◽  
D. C. Eaton
Keyword(s):  
Single Channel ◽  
Primary Cultures ◽  
Cell Volume Regulation ◽  
Principal Cell ◽  
Resting Membrane Potential ◽  
Open Probability ◽  
Channel Activation ◽  
K Channels ◽  
Pg E2

Patch clamp technology was utilized to study the effects of apical phospholipase A2 (PLA2) metabolites on “maxi K” channels in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures (B. N. Ling, C. F. Hinton, and D. C. Eaton. Kidney Int. 40: 441–452, 1991). At resting membrane potential, this channel is quiescent in the cell-attached configuration. Apical application of the PLA2 agonist melittin (1 microgram/ml) for 10 min increased single-channel open probability (Po) from 0.0004 +/- 0.0010 to 0.11 +/- 0.05. Similarly, apical exposure to 50 microM arachidonic acid (AA) or 0.5 microM prostaglandin (PG) E2, but not 0.5 microM PGF2 alpha, also increased channel activity. Conversely, 10 microM of the PLA2 antagonist quinacrine applied apically decreased Po. Removal of apical bath Ca2+ did not prevent melittin-, AA-, or PGE2-induced channel activation. We then examined the role of maxi K channels and eicosanoids in principal cell volume regulation. Within seconds of reducing basolateral bath tonicity (285 to 214 mosmol/kgH2O), NPo (i.e., no. of channels x Po) initially increased approximately 200%, followed by a delayed but prolonged activation phase that was attenuated by removal of apical bath Ca2+. Pretreatment with 10 microM quinacrine, 100 microM indomethacin (cyclooxygenase inhibitor), or 0.25 microM thapsigargin (to deplete intracellular Ca2+ stores) abolished the initial phase of swelling-induced channel activation.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Endogenous Protease Activation of ENaC

2005 ◽  
Vol 126 (4) ◽  
pp. 339-352 ◽  
Author(s):  
Adedotun Adebamiro ◽  
Yi Cheng ◽  
John P. Johnson ◽  
Robert J. Bridges
Keyword(s):  
Single Channel ◽  
Apical Membrane ◽  
Open Channels ◽  
Na Channel ◽  
Fluctuation Analysis ◽  
Protease Inhibition ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Channel Properties

Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further investigation but does suggest a potential compensatory regulatory mechanism to maintain INa at some minimal threshold value.

scholarly journals Orai1 pore residues control CRAC channel inactivation independently of calmodulin

2016 ◽  
Vol 147 (2) ◽  
pp. 137-152 ◽  
Author(s):  
Franklin M. Mullins ◽  
Michelle Yen ◽  
Richard S. Lewis
Keyword(s):  
Crystal Structure ◽  
Conformational Changes ◽  
Negative Charge ◽  
Noise Analysis ◽  
Dominant Negative ◽  
Side Chain ◽  
Open Probability ◽  
Crac Channels ◽  
Channel Inactivation ◽  
Crac Channel

Ca2+ entry through CRAC channels causes fast Ca2+-dependent inactivation (CDI). Previous mutagenesis studies have implicated Orai1 residues W76 and Y80 in CDI through their role in binding calmodulin (CaM), in agreement with the crystal structure of Ca2+–CaM bound to an Orai1 N-terminal peptide. However, a subsequent Drosophila melanogaster Orai crystal structure raises concerns about this model, as the side chains of W76 and Y80 are predicted to face the pore lumen and create a steric clash between bound CaM and other Orai1 pore helices. We further tested the functional role of CaM using several dominant-negative CaM mutants, none of which affected CDI. Given this evidence against a role for pretethered CaM, we altered side-chain volume and charge at the Y80 and W76 positions to better understand their roles in CDI. Small side chain volume had different effects at the two positions: it accelerated CDI at position Y80 but reduced the extent of CDI at position W76. Positive charges at Y80 and W76 permitted partial CDI with accelerated kinetics, whereas introducing negative charge at any of five consecutive pore-lining residues (W76, Y80, R83, K87, or R91) completely eliminated CDI. Noise analysis of Orai1 Y80E and Y80K currents indicated that reductions in CDI for these mutations could not be accounted for by changes in unitary current or open probability. The sensitivity of CDI to negative charge introduced into the pore suggested a possible role for anion binding in the pore. However, although Cl− modulated the kinetics and extent of CDI, we found no evidence that CDI requires any single diffusible cytosolic anion. Together, our results argue against a CDI mechanism involving CaM binding to W76 and Y80, and instead support a model in which Orai1 residues Y80 and W76 enable conformational changes within the pore, leading to CRAC channel inactivation.

scholarly journals Close Functional Coupling Between Ca2+Release-Activated Ca2+Channels and Reactive Oxygen Species Production in Murine Macrophages

2006 ◽  
Vol 2006 ◽  
pp. 1-8 ◽  
Author(s):  
Sheng-Wei Jin ◽  
Li Zhang ◽  
Qin-Quan Lian ◽  
Shang-Long Yao ◽  
Ping Wu ◽  
...  
Keyword(s):  
Confocal Laser ◽  
Functional Coupling ◽  
Ros Production ◽  
Murine Macrophages ◽  
Crac Channels ◽  
Transport Chain ◽  
Transient Rise ◽  
Carbonyl Cyanide ◽  
Crac Channel ◽  
Species Production

Aim. To investigate the role ofCa2+release-activatedCa2+(CRAC) channels in the ROS production in macrophages.Methods. The intracellular[Ca2+]iwas analyzed by confocal laser microscopy. The production of ROS was assayed by flow cytometry.Results. Both LPS and thapsigargin induced an increase in intracellular[Ca2+]i, either in the presence or absence of extracellularCa2+in murine macrophages. TheCa2+signal was sustained in the presence of externalCa2+and only initiated a mild and transient rise in the absence of externalCa2+. CRAC channel inhibitor 2-APB completely suppressed theCa2+entry signal evoked by thapsigargin, and suppressed approximately93%of theCa2+entry signal evoked by LPS. The increase in intracellular[Ca2+]iwas associated with increased ROS production, which was completely abolished in the absence of extracellularCa2+or in the presence of CRAC channel inhibitors 2-APB andGd3+. The mitochondrial uncoupler carbonyl cyanidep-trifluoromethoxy-phenylhydrazone and the inhibitor of the electron transport chain, antimycin, evoked a marked increase in ROS production and completely inhibited thapsigargin and LPS-evoked responses.Conclusions. These findings indicate that the LPS-induced intracellular[Ca2+]iincrease depends on theCa2+entry through CRAC channels, and close functional coupling between CRAC and ROS production in murine macrophages.

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.

BAY K 8644 and ClO4- potentiate caffeine contracture without Ca2+ release channel activation

1997 ◽  
Vol 272 (1) ◽  
pp. C41-C47 ◽  
Author(s):  
T. Oba ◽  
M. Koshita ◽  
T. Aoki ◽  
M. Yamaguchi
Keyword(s):  
Time Constant ◽  
Bay K 8644 ◽  
Open Probability ◽  
Release Channel ◽  
Channel Activation ◽  
Channel Current ◽  
Caffeine Contracture ◽  
Dihydropyridine Receptors ◽  
Open Time ◽  
External K

Effects of perchlorate (ClO4-) and BAY K 8644 on caffeine contracture and Ca2+ release channel current were studied in frog skeletal muscle. Single fibers produced a small transient contracture on addition of 2.2 mM caffeine. ClO4 at 10 mM enhanced caffeine contracture 3.7-fold. This effect was inhibited by 10 microM nifedipine pretreatment. An increase in caffeine contracture was also obtained after exposure to 0.1 microM BAY K 8644 for 1 h. At 20 mM, external K+ potentiated caffeine contracture 2.2-fold. ClO4- (< 10 mM) and BAY K 8644 (0.1-1 microM) did not affect open probability (Po), unitary conductance, and open and closed time constants of the Ca2+ release channel current. BAY K 8644 at 0.1 microM did not further enhance the channel that had been activated by 2 mM caffeine. However, 20-30 mM ClO4 increased Po significantly and led the channel to a long open state by increasing the slow open time constant and decreasing the fast closed time constant. These results suggest that binding of ClO4 and BAY K 8644 to dihydropyridine receptors elicits a further increase in Ca2+ release from the sarcoplasmic reticulum.

A novel GABAergic action mediated by functional coupling between GABAB-like receptor and two different high-conductance K+ channels in cricket Kenyon cells

2013 ◽  
Vol 109 (7) ◽  
pp. 1735-1745 ◽  
Author(s):  
Atsunao Nakamura ◽  
Masami Yoshino
Keyword(s):  
Single Channel ◽  
Firing Frequency ◽  
Resting Membrane Potential ◽  
Functional Coupling ◽  
Coupling Mechanism ◽  
Open Probability ◽  
Kenyon Cells ◽  
Voltage Dependent ◽  
Patch Configuration ◽  
High Conductance

The γ-aminobutyric acid type B (GABAB) receptor has been shown to attenuate high-voltage-activated Ca2+ currents and enhance voltage-dependent or inwardly rectifying K+ currents in a variety of neurons. In this study, we report a novel coupling of GABAB-like receptor with two different high-conductance K+ channels, Na+-activated K+ (KNa) channel and Ca2+-activated K+ (KCa) channel, in Kenyon cells isolated from the mushroom body of the cricket brain. Single-channel activities of KNa and KCa channels in response to bath applications of GABA and the GABAB-specific agonist SKF97541 were recorded with the cell-attached patch configuration. The open probability ( Po) of both KNa and KCa channels was found to be increased by bath application of GABA, and this increase in Po was antagonized by coapplication of the GABAB antagonist CGP54626, suggesting that GABAB-like receptors mediate these actions. Similarly, GABAB-specific agonist SKF97541 increased the Po of both KNa and KCa channels. Perforated-patch recordings using β-escin further revealed that SKF97541 increased the amplitude of the outward currents elicited by step depolarizations. Under current-clamp conditions, SKF97541 decreased the firing frequency of spontaneous action potential (AP) and changed the AP waveform. The amplitude and duration of AP were decreased, whereas the afterhyperpolarization of AP was increased. Resting membrane potential, however, was not significantly altered by SKF97541. Taken together, these results suggest that GABAB-like receptor is functionally coupled with both KNa and KCa channels and this coupling mechanism may serve to prevent AP formation and limit excitatory synaptic input.

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