The cardiac type 2 ryanodine receptor (RYR2) is activated by Ca2+-induced Ca2+ release (CICR). The inherent positive feedback of CICR is well controlled in cells, but the nature of this control is debated. Here, we explore how the Ca2+ flux (lumen-to-cytosol) carried by an open RYR2 channel influences its own cytosolic Ca2+ regulatory sites as well as those on a neighboring channel. Both flux-dependent activation and inhibition of single channels were detected when there were super-physiological Ca2+ fluxes (>3 pA). Single-channel results indicate a pore inhibition site distance of 1.2 ± 0.16 nm and that the activation site on an open channel is shielded/protected from its own flux. Our results indicate that the Ca2+ flux mediated by an open RYR2 channel in cells (∼0.5 pA) is too small to substantially regulate (activate or inhibit) the channel carrying it, even though it is sufficient to activate a neighboring RYR2 channel.
In cardiac muscle, surface membrane depolarization activates surface membrane Ca2+ channels (dihydropyridine receptor), which mediate a small Ca2+ influx into a cell. This small Ca2+ influx triggers the opening of type 2 RYR (RYR2) Ca2+ release channels that are located on the membrane of the SR. The opening of RYR2 channels results in a large SR Ca2+ flux that ultimately drives cardiac contractility. The RYR2 activation process is called Ca2+-induced Ca2+ release, or CICR (Fabiato, 1985; Bers, 2001; Fill and Copello, 2002), and represents a positive feedback process that is well controlled in cells. This positive feedback process consists of Ca2+ evoking Ca2+ release that can in turn evoke further Ca2+ release. How this positive feedback is controlled is not well understood. To gain understanding, it is important to define the extent to which the Ca2+ flux carried by an open RYR2 channel can act at its own cytosolic Ca2+ regulatory sites as well as those on neighboring channels.
In cardiac muscle cells, RYR2 channels exist and operate in tightly packed orthogonal arrays with an RYR2-RYR2 center spacing of ∼30 nm (Franzini-Armstrong et al., 1999). The cytoplasmic domain of the RYR2 channel extends ∼12 nm from the SR and has a center-corner distance of ∼14 nm (Samsó et al., 2005; Serysheva et al., 2005). The unit Ca2+ current carried by a single RYR2 channels in cells is thought to be ∼0.5 pA (Mejía-Alvarez et al., 1999; Kettlun et al., 2003; Gillespie and Fill, 2008). This 0.5 pA will elevate (when no buffering is present) local free Ca2+ levels to >15 µM within 25 nm from the open pore (Stern, 1992). The cytosolic Ca2+ EC50 (half-maximal activation) of the RYR2 channel is 1–15 µM depending on the experimental conditions (Meissner, 1994; Sitsapesan and Williams, 1994; Saftenku et al., 2001; Copello et al., 2002; Fill and Copello, 2002). Thus, the physiological Ca2+ flux could substantially influence RYR2 Ca2+ activation status and likely activate neighboring RYR2 channels as well. Self-flux activation is the most fundamental form of Ca2+-induced Ca2+ release and would be the most difficult to control.
Flux regulation of single RYR2 channels has been reported in planar bilayer studies (Sitsapesan and Williams, 1994; Xu and Meissner, 1998; Laver, 2007). Sitsapesan and Williams (1994) reported that sulmazole-activated, not Ca2+-activated, RYR2 channels were sensitive to luminal Ca2+ concentration (consequently, lumen-to-cytosol Ca2+ flux) changes. Xu and Meissner (1998) showed that the Ca2+ flux mediated by caffeine-activated and Ca2+-activated single RYR2 channels can act at both cytosolic Ca2+ activation and/or inhibitory sites. They reported that fluxes ≥0.25 pA can activate and that fluxes ≥8 pA can inhibit. The inhibition is explained by a site ≤3 nm from the open pore. More recently, Laver (2007) proposed that Ca2+ passing through ATP-activated RYR2 channels acts on the cytosolic Ca2+ activation site, but also on a novel high affinity (IC50, 1.2 µM) cytosolic Ca2+ inhibition site. Laver suggests that every pA of Ca2+ current through the channel elevates local Ca2+ 15 µM at the cytosolic activation site (which is 11 nm from the pore) and 0.35 µM at the inhibition site (which is 26 nm from the pore). It is clear that there is no consensus about how the Ca2+ flux carried by an RYR2 channel influences the channel's function.
Here, we define the flux-dependent regulation of single Ca2+-activated RYR2 channels. The channels tested do not have their function and/or cytosolic Ca2+ sensitivity altered by caffeine, sulmazole, or ATP (without Mg2+ present). We find that channels can indeed be regulated by the Ca2+ flux they carry, but only when the flux is substantially larger than the physiological norm. We also more accurately specify the pore-to-inhibition site distance and show that the channel's cytosolic activation machinery is somehow shielded or protected from its own flux.
Heavy SR microsomes enriched in RYR2 channels were isolated from ventricles of adult rat hearts using established procedures (Tate et al., 1985). In brief, tissue was rinsed in saline solution (154 mM NaCl and 10 mM Tris-maleate, pH 6.8) at 4°C and then diced before being homogenized. A heavy SR microsome fraction was then separated from the homogenate by differential centrifugation. Microsomes were resuspended in a saline solution containing 300 mM sucrose before being flash frozen and stored at −80°C. Small aliquots of microsomes were thawed as needed and kept on ice until used.
Planar lipid bilayers were formed across a 100-µm hole in a thin (∼12 µm) Teflon partition separating two aqueous compartments. The bilayer was formed from a mixture of phosphatidylethanolamine, phosphatidylserine, and phosphatidylcholine in a 5:4:1 ratio. One compartment (cytosolic) was virtually grounded, and the cytosolic side of the RYR2 channel was eventually in this compartment (Tu et al., 1994; Fill and Copello, 2002). Initially, the cytosolic compartment was filled with a Tris-HEPES solution (120/250 mM; pH 7.4) and the other compartment (luminal) with Ca-HEPES (50/250 mM; pH 7.4). Microsomes and ∼400 mM CsCH3SO3 were then added to the cytosolic compartment while stirring. After channel incorporation, cytosolic and luminal solutions were changed (10× volume wash) to different test solutions. The composition of these test solutions is specified in the figure legends. Generally, solutions designed to explore the prospect of Ca2+ flux activation have cytosolic free Ca2+ concentrations less than the cytosolic Ca2+ EC50 of the RYR2 channel. Solutions designed to explore Ca2+ flux inactivation have cytosolic Ca2+ levels more than the cytosolic Ca2+ activation EC50 (i.e., activation sites largely occupied). In some cases, the cytosolic solutions contained 1 mM EGTA. The composition of Ca2+ buffer solutions was calculated using the MAXC program (WinMaxC; Stanford University, Palo Alto, CA) and verified using a Ca2+ electrode. All salts and chemicals (unless otherwise specified) were obtained from Sigma-Aldrich or EMD. Lipids were obtained from Avanti Polar Lipids, Inc. All experiments were performed at room temperature (21°C).
The membrane potential across the bilayer was controlled using an Axopatch 200B (MDS Analytical Technologies). Sample rate (unless otherwise specified) was 10 kHz, and recordings were filtered with a Bessel filter at 2 kHz for analysis. Data acquisition and analysis were performed using pClamp 9.0 (MDS Analytical Technologies). Summary results are presented as mean ± SEM, with statistical significance determined using the Student's t test (P < 0.05 or as specified).
In a multi-RYR2 channel cluster, where openings are stochastic, there is always some possibility that all the channels will close simultaneously. This process is often referred to as stochastic attrition. To roughly estimate the time constant of stochastic attrition (τA) within an RYR2 cluster, we used the following equation (Stern and Cheng, 2004):τA=[1−(1−PO)N]·MOTN·(1−PO)N−1·PO(1)In Eq. 1, Po is open probability, N is number of channels present, and MOT is mean open time of individual independently gating RYR2 channels.
In some experiments, the cytosolic solution contained 4 mM DM-nitrophen and 50 µM Rhod-2. Rhod-2 excitation light from a Nd:YAG laser (532 nm; 4 mW) was pulsed (1 ns long at 10 kHz) and focused into a 400-µm diameter multimode optic fiber (n.a. 0.2). Rhod-2 emission was collected through the same fiber and projected onto an avalanche photodiode. High intensity UV flashes (5 ns long) for photolysis from another Nd:YAG laser (355 nm; 2 MW) were applied through the same optic fiber. Intensity of the photolytic flash was controlled by Q-switch triggering and monitored by a joule meter. Instrumentation was synchronized, and single-channel recording done using pClamp software/hardware (MDS Analytical Technologies). The optic fiber was micropositioned 500 µm directly in front of and centered on the bilayer. Thus, DM-nitrophen was photolyzed in the small volume (∼63 nl) between the optic fiber and bilayer. Solution in the photolyzed volume was then exchanged with unphotolyzed solution from the bath (1 ml) by stirring the bath. Rhod-2 emission was used to monitor local Ca2+ levels in the photolyzed volume. Photolysis liberated Ca2+ from DM-nitrophen. This increased the free Ca2+ in the photolyzed volume, and solution exchange (via rapid stirring) reduced it to basal levels with a time constant of 15–19 ms. Note that photolysis can generate a fast (<150 µs), large (>25 µM) transient Ca2+ overshoot that was not detected by our recording system. Identical photolytic Ca2+ stimuli were always applied, regardless of whether such an overshoot was present or not.
To calculate the amplitude and spatial profile of the Ca2+ microdomain around an open RYR2 pore (Stern, 1992), a freeware application (PORE) developed by J. Kenyon (University of Nevada, Reno, NV) was used. PORE is a Visual Basic program that can be run in an Excel spreadsheet.
The Hill equation (Eq. 2) was used to fit (via nonlinear least squares) some results.Po=PoMax·[Ca]HCKD+[Ca]HC(2)Here, [Ca] is Ca2+ concentration, PoMAX is the maximum Po, KD is the dissociation constant, and Hc is the Hill coefficient.
The equation relating local [Ca], unit Ca2+ current, and distance from an open pore is well known (Eq. 3).[Ca]=[Ca]∞+i4πFDrexp(−r/λ),where λ=DkON·[B] and [B]=[B]TOT·KDKD+[Ca]∞(3)Here, [Ca] refers to the local free Ca2+ concentration as a function of distance (r) from the pore, [Ca]∞ is the bath free Ca2+ concentration far from the pore, (i) is unit current, and F is the Faraday constant. The diffusion coefficient (D) used for Ca2+ and Mg2+ was 10−9 m2s−1. In Eq. 3, the kON is the on rate for Ca2+ binding to the buffer, [B] is the free buffer concentration, [B]TOT is the total buffer concentration, and KD is the dissociation constant of the Ca buffer complex. The only Ca2+ buffer present in our experiments here was EGTA. The EGTA kON and KD used here were 1.5 × 10−6 M−1s−1 and 1.58 × 10−7 M, respectively.
A function relating Po and Ca2+ current amplitude was generated using the equations above. The logic is that the RYR2 channel has cytosolic Ca2+ regulatory sites that are some distance from the pore. If Ca2+ coming through the pore can act on these sites, Po must be some function of unit current amplitude. In the absence of Ca2+ flux through the pore, Ca2+ action on these cytosolic sites is well described by the Hill function (Eq. 2). Local free Ca2+ concentration as a function of distance and unit current is described by Eq. 3. Thus, Eq. 4 can be generated by substituting [Ca] terms of Eq. 2 with local [Ca] from Eq. 3.Po=PoMax·([Ca]∞+i4πFDrexp(−r/DkON·[B]TOT·KDKD+[Ca]∞))HCKD+([Ca]∞+i4πFDrexp(−r/DkON·[B]TOT·KDKD+[Ca]∞))HC(4)Eq. 4 was used to fit (via nonlinear least squares) Po versus pA results to specify pore site distances when EGTA was present. The exponential terms are unnecessary in the absence of cytosolic buffer.
Ryanodine, caffeine, and ATP sensitivity (not depicted) were used to positively identify the channels studied here as RYR2 channels. To define how (or if) an RYR2 channel's Ca2+ flux feeds back and regulates the channel, the characteristics of its cytosolic Ca2+ regulatory sites need to be defined first. To this end, sample single-channel recordings at three different cytosolic Ca2+ concentrations are shown in Fig. 1 A. The only charge carrier present was Ca2+ (50 mM luminal), and the typical unit current was 3.58 ± 0.03 pA (n = 44) moving in the lumen-to-cytosol direction. These recordings show that the Po increased with cytosolic Ca2+ concentration. Fig. 1 B shows summary Po results (mean ± SEM) collected in three different experimental conditions. Filled circles represent RYR2 cytosolic Ca2+ sensitivity when 50 mM of luminal Ca2+ (50L) is present without cytosolic Mg2+ or ATP. Open squares represent RYR2 cytosolic Ca2+ sensitivity when 5 µM of luminal Ca2+ (0.005L) is present also without cytosolic Mg2+ or ATP. At this lower luminal Ca2+ level, the lumen-to-cytosol current was carried by Cs+ (see legend to Fig. 1). The EC50's for these two datasets are 1.6 ± 0.41 µM (filled circles; Hc = 2.2) and 1.61 ± 0.34 µM (open squares; Hc = 2.7). Open diamonds are mean Po values with 50 mM of luminal Ca2+ (50L), but now with cytosolic Mg-ATP present (see legend to Fig. 1). The thin dashed line is a complete dose–response curve (EC50, 12 ± 2 µM; Hc = 2.6) generated by our group previously in similar experimental conditions (Zoghbi et al., 2004). This latter relationship is likely close to the RYR2's cytosolic Ca2+ EC50 in cells. Because cytosolic Mg2+ inhibits channels by multiple mechanisms (Meissner et al., 1986; Laver et al., 1997) and the RYR2 channel conducts Mg2+ (Fill and Copello, 2002), our RYR2 flux regulation studies described below were done without Mg-ATP present.
Cytosolic calcium sensitivity. (A) Sample single-channel recordings made at three different cytosolic free Ca2+ levels. All recordings were made at 0 mV, and the closed (zero current) level is marked at left margin. Recordings were made with 50 mM of luminal Ca2+ present. The cytosolic solution also contained Tris-HEPES (120/250 mM; pH 7.4). The luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). (B) Cytosolic Ca2+ dependence of Po. All Po measurements were made from >3-min recordings (0 mV) from 8–10 different channels at different steady-state cytosolic free Ca2+ concentrations. Lines are Hill equation fits. Filled circles represent the cytosolic Ca2+ sensitivity of the channel when 50 mM of luminal Ca2+ (50L) is present. Open squares show when 0.005 mM of luminal Ca2+ is present. Open diamonds indicate 50 mM of luminal Ca2+ when cytosolic Mg-ATP is present, and the corresponding dose–response curve (thin dashed line) was reproduced from Zoghbi et al. (2004). In all cases, the cytosolic solution also contained 1 mM EGTA and Tris-HEPES (as above). Mg-ATP refers to 1 mM of free Mg2+ and 5 mM of total ATP. The luminal 0.005 Ca2+ solution contained 100 mM Cs-HEPES, pH 7.4. Thus, Cs+ was the primary charge carrier. (C) Luminal Ca2+ and Mg2+ dependence of Po at 0 mV. Channels were activated by 100 µM of cytosolic free Ca2+. Any endogenous CSQ that was attached to the channel was dissociated from the channel by a high ionic strength wash (see Results). Luminal Mg2+ (triangles) or Ca2+ (inverted triangles) was changed from 50 mM to 5 µM. An analogous luminal Ca2+/Mg2+ sensitivity result was presented previously by Qin et al. (2008). Again, the cytosolic solution also contained Tris-HEPES (as above). The luminal solution contained either Ca-HEPES or Mg-HEPES. When luminal free Ca2+ or Mg2+ was <10 mM, 100 mM Cs-HEPES, pH 7.4, was added to the luminal solution to assure ample charge carrier was always present.
Fig. 1 C shows the luminal Ca2+ and Mg2+ dependence of single RYR2 channels in the presence of 100 µM of cytosolic Ca2+ (no Mg-ATP). In these studies, 100 mM of luminal Cs+ was present when necessary to provide an additional charge carrier. High concentrations of luminal Ca2+ (>5 mM) are known to dissociate calsequestrin (CSQ) from channels in planar bilayers (Beard et al., 2004; Györke et al., 2004; Qin et al., 2008). Because all tested channels here were exposed to >5 mM before recording began, these channels are not likely to have CSQ associated with them. When luminal Ca2+ (Fig. 1 C, inverted triangle) or Mg2+ (triangle) concentration was decreased from 50 mM to 5 µM, the Po also decreased with an IC50 (half-maximal inhibition) of ∼500 µM. This luminal titration explains the different maximum Po's between the 50L and 0.005L curves in Fig. 1 B. This luminal action of Ca2+ and Mg2+ is likely not due to flux feeding through the channel because the cytosolic Ca2+ activation site was essentially saturated (because there was 100 µM of cytosolic Ca2+), and these two ions have opposite action when applied to the cytosolic side of the channel.
We do not believe this CSQ-independent luminal regulation mechanism is of major physiological consequence because it lacks Ca-Mg selectivity. The free Mg2+ inside the SR is very likely equal to that in the cytosol (∼1 mM) because there is no source of energy to hold it out of equilibrium (Somlyo et al., 1985). The process of SR Ca2+ release is thought to lower intra-SR free Ca2+ from ∼1 to ∼0.5 mM (Shannon and Bers, 1997). If so, then the combined intra-SR free Ca2+ and Mg2+ level upon release decreases from ∼2 to ∼1.5 mM. Fig. 1 C indicates that this magnitude change in intra-SR free divalent cation concentration will have very little effect on RYR2 Po via this CSQ-independent luminal regulatory mechanism.
To learn more about the Ca2+ flux dependence of RYR2 gating, we began by calculating the free cytosolic Ca2+ concentration profile around an open RYR2 pore. In Fig. 2 A, the line is the calculated concentration (as a function of distance) when the channel is carrying a 3.58-pA Ca2+ current with no diffusible buffer present. The approximate dimensions of the cytosolic domain of the RYR2 channel (Samsó et al., 2005; Serysheva et al., 2005) are indicated in the inset to help place the distances on the abscissa in context. Note that the free Ca2+ concentrations over the entire cytoplasmic domain of the channel are >100 µM, and the free Ca2+ concentrations within 3 nm of the open pore reach levels >1 mM.
Flux-dependent inhibition of single RYR2 channel activity. (A) Local diffusion from a point source here was calculated with cytosolic Ca2+ of 100 nM in the presence of different unit Ca2+ currents. The thick line is for 3.58 pA, reflecting the typical unit current we measure when there is 50 mM of luminal Ca2+ present. (Inset) A cartoon depicting the approximate dimensions of a single RYR2 channel. (B) Cytosolic Ca2+ (filled circles) or Mg2+ (open circles) inhibition of single RYR2 channels activated by 100 µM of cytosolic free Ca2+. Experiments here were done at 0 mV. Cytosolic Ca2+ or Mg2+ was changed from 100 µM to 10 mM. Lines are Hill equation fits. The Ca2+ and Mg2+ IC50's were 6.2 ± 0.34 mM (Hc = −1.3) and 5.3 ± 0.69 mM (Hc = −1.5), respectively. Results represent data collected from five to eight different channels. The cytosolic solution also contained Tris-HEPES (120/250 mM; pH 7.4). The luminal solution contained 5 µM of added Ca2+ and 100 mM Cs-HEPES, pH 7.4. (C) Luminal-to-cytosolic Ca2+ flux dependence of Po. These results represent data collected from six to nine RYR2 different channels. The cytosolic free Ca2+ was 7 µM, and unit Ca2+ current was varied by changing the membrane potential. Filled circles are Po values with Ca2+ as sole charge carrier. The thick line is a fit using Eq. 4 (described in Materials and methods) to the filled circles revealing a pore site distance of 1.3 nm. This fit assumes the cytosolic Ca2+ inhibition site properties defined in B, and the pore site distance sensitivity of this relationship is illustrated by the labeled thin lines. Open triangles are Po values when Cs+ (instead of Ca2+) was charge carrier. The cytosolic solution contained 1 mM EGTA and Tris-HEPES (as above). When Ca2+ was charge carrier, the luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). When Cs+ was charge carrier, the luminal solution contained 5 µM of added Ca2+ and 100 mM Cs-HEPES, pH 7.4. (D) Luminal-to-cytosolic Mg2+ flux dependence of Po. These results represent data collected from seven to eight RYR2 channels. Again, cytosolic free Ca2+ was 7 µM, and unit Mg2+ current was varied by changing the membrane potential. The thick line is fit using Eq. 4 and indicates a pore site distance of 1.1 nm. This fit assumes the cytosolic Mg2+ inhibition site properties defined in B, and the IC50 sensitivity of this relationship is illustrated by the labeled thin lines. The cytosolic solution also contained 1 mM EGTA and Tris-HEPES (as above), and the luminal solution contained Mg-HEPES.
Fig. 2 B shows that high concentrations (≥1 mM) of cytosolic Ca2+ (filled circles) or Mg2+ (open circles) inhibit the RYR2 channel. These experiments were done starting with 100 µM of cytosolic Ca2+, and then 1–10 mM Ca2+ or Mg2+ was added to the cytosolic bath. The Ca2+ and Mg2+ IC50's are 6.2 ± 0.34 mM (PoMax = 0.81; Hc = −1.3) and 5.3 ± 0.69 mM (PoMax = 0.81; Hc = −1.5), respectively. These values were not significantly different. In Fig. 2 C, Po was measured as a function of Ca2+ current amplitude when there was 7 µM of cytosolic free Ca2+ present. Current amplitude was modulated by changing membrane potential. Single RYR2 Po decreased as Ca2+ flux amplitude increased (Fig. 2 C, circles). To establish that this Po decrease with unit current amplitude was due to Ca2+ feed-through, we changed the charge carrier to Cs+ (Fig. 2 C, triangles). When Cs+ was charge carrier, the Po did not change with unit current amplitude. The reduced Po associated with the charge carrier change is explained by the applied change in luminal Ca2+ concentration (50 mM for the circles, 5 µM for the triangles; see Fig. 1 B). Fig. 2 D shows an analogous study, but with a lumen-to-cytosol Mg2+ current instead of a Ca2+ current. The Po also decreased as a function of current amplitude. Results in Fig. 2 (C and D) were fit (thick lines) using Eq. 4 (see Materials and methods) and the Hill parameters defined in Fig. 2 B. The fit in Fig. 2 C indicates that the responsible cytosolic inhibition site would need to be 1.3 nm from the pore. The fit in Fig. 2 D indicates that the site would need to be 1.1 nm from the pore.
In our hands, prolonged periods at large nonzero membrane potential reduce bilayer stability, making long steady-state recordings at large membrane potentials impractical. Thus, we used voltage ramps to explore flux inhibition by larger Ca2+ currents. Fig. 3 A shows sample single-channel recordings where the charge carrier was provided by 50 mM of luminal Ca2+. A general decrease in Po is visible as current amplitude increases. In Fig. 3 B, the relationship between Ca2+ current amplitude and Po was determined from ensemble recordings, and one such recording is shown (thick line). This ensemble was generated from 200 single-channel voltage ramp sweeps collected from four different channels. The downward slope of the ensemble recording indicates that Po decreased as Ca2+ current increased. Eq. 4 was used to fit (thin line) the ensemble trace and indicated a pore inhibition site distance of 1.4 nm. The open circles in Fig. 3 B are the flux inhibition points from Fig. 2 C. The second thin line is the fit to another ensemble dataset (not depicted) generated with 10 mM of luminal Ca2+ present. This pore site distance determined from this other dataset was 1.5 nm.
Flux sensitivity of single RYR2 channel activity. (A) Sample single-channel recordings are shown as membrane potential was ramped between −50 and +50 mV. Charge carrier was Ca2+ (50 mM luminal), the cytosolic free Ca2+ was 50 µM, and unit current was in the lumen-to-cytosolic direction. Zero current level is marked by a dotted line. The cytosolic solution also contained Tris-HEPES (120/250 mM; pH 7.4). The luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). (B) Ensemble traces were generated by averaging 200 single-channel sweeps from four different channels with either 50 or 10 mM of Ca2+ luminal solution present. Solutions as above, but the 10-mM Ca2+ luminal solution contained 10 mM Ca-HEPES. Lines are the fits to these ensembles traces (only the 50-mM Ca2+ ensemble trace is shown). The best fit distances were 1.4 and 1.5 nm, respectively. These fits were made assuming a cytosolic Ca2+ inhibition site with IC50 = 6.2 mM (Hc = −1.3). Open circles are data points reproduced from C. (C) Representative all-points histograms when cytosolic free Ca2+ was 0.5 µM. Histograms made at seven different membrane potentials are superimposed (one filled and all others open). Zero current level is centered at 0 pA. Here, the cytosolic solution also contained 1 mM EGTA and Tris-HEPES (as above). The luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). (D) Flux activation of single RYR2 channel activity. Gaussian fitting of all-points histograms compiled from four to six different channels (cytosolic free Ca2+, 0.5 µM) was used to determine how Po varies with Ca2+ current amplitude. Filled circles correspond to when all current was due to lumen-to-cytosol Ca2+ flux (solutions as those described for C). Increasing Ca2+ current from 2 to 5 pA elevated Po. Increasing it further slightly reduced Po. This flux activation and inhibition was fit as described in Materials and methods. Filled squares correspond to when most current was carried by Cs+, instead of Ca2+. To accomplish this, the luminal solution was changed to one containing 50 µM of added Ca2+ and 200 mM Cs-HEPES, pH 7.4. The Po did not change with increasing Cs+ current. (Inset) The cytosolic Ca2+ dependency of MOT of single RYR2 channels (solutions like those described for C).
Calcium flux activation of a single RYR2 channel is demonstrated in Fig. 3 C. Again, the charge carrier was Ca2+ (50 mM luminal), but now the cytosolic bath Ca2+ was 0.5 µM, which is below the cytosolic Ca2+ EC50. This means that the cytosolic activation site is available to respond to local Ca2+ fluctuations. Long recordings were made at different membrane potentials. Sample all-points histograms generated from these recordings are superimposed in Fig. 3 C. The area of the open current peaks (those between 2 and 8 pA) changed as unit current amplitude increased. The Po for each histogram was calculated as the relative area of the open and closed peaks and then plotted as a function of unit Ca2+ current in Fig. 3 D (filled circles). With Ca2+ as charge carrier, the Po increased as unit Ca2+ current changed from 2 to ∼5 pA. It then slightly decreased as current increased further. Fig. 3 D also shows a parallel study done with Cs+ as charge carrier. The current-dependent changes in Po when Ca2+ was the charge carrier (filled circles) were not evident when Cs+ was charge carrier (filled squares). This indicates that the Ca2+ current–dependent Po changes were not a consequence of the membrane potential change.
Eq. 4 was used to fit the rising and falling phases of the Po versus pA data in Fig. 3 D (filled circles). The fit to the falling phase indicated a pore site distance of 0.5 nm. The fit to the activation (rising phase) requires more explanation. Assuming that the responsible activation site operates like the traditional steady-state Ca2+ activation site (EC50 = 1.6 µM; Hc = 2.2), the fit (Fig. 3 D) to the rising phase indicates a pore-activation site distance of 1,700 nm. This is clearly not possible because the cytosolic activation site must be somewhere within ∼18 nm of the pore (Samsó et al., 2005; Serysheva et al., 2005). The local Ca2+ concentration at a distance of 18 nm from this open RYR2 pore, which is carrying a 3.58-pA Ca2+ flux, is 165 µM (Fig. 2 A). This level of cytosolic Ca2+ might be expected to either prolong the opening or immediately reactivate the channel once it closes (Fig. 1 B). If so, the flux would be expected to substantially elevate Po. However, the observed Po change generated by the flux was quite modest. This implies that the RYR2's cytosolic Ca2+ activation machinery may be somehow "shielded" from the channel's own Ca2+ flux. To have some gauge of the extent of this shielding, the pore-activation site distance was fixed at 18 nm and the rising phase (or flux activation) was refit. This refitting indicated that a cytosolic activation site 18 nm from the pore would need to have a Ca2+ EC50 of 440 µM (Hc = 2.97) to explain the results if it were free to respond to the fluxed Ca2+ (Fig. 3 D).
As suggested above, Ca2+ flux could influence activation if the fluxed Ca2+ prolongs channel opening (i.e., extend mean open time [MOT]) and/or if it increases the likelihood of channel reopening. RYR2 MOT and open event frequency were measured when the Ca2+ flux was 1.81 or 3.58 pA. The MOTs (n = 6) were 2.9 ± 2.1 and 12 ± 9 ms, and the frequencies were 3.6 ± 5.4 and 3.4 ± 4.3 Hz, respectively. Although these numbers suggest that the flux activates RYR2 by increasing its MOT (not event frequency), these MOTs were not significantly different. We measured the MOTs of single RYR2 channels exposed to different levels of cytosolic bath Ca2+, and these are plotted in Fig. 3 D (inset, open circles). Note that the MOT here was 71 ± 24 ms at a cytosolic bath Ca2+ of 100 µM. This is significantly larger than the MOT when a 3.58-pA Ca2+ flux elevates local cytosolic Ca2+ to >100 µM (P < 0.05; 71 ± 24 vs. 12 ± 9 ms; n = 6). This supports the idea that the RYR2's cytosolic Ca2+ activation machinery is somehow "shielded" from the channel's own Ca2+ flux.
The local Ca2+ concentration profile near an open Ca2+-conducting pore is established/dissipated quickly (<1 ms) when the channel opens/closes (Stern, 1992). Such fast cytosolic Ca2+ changes (<1 ms) are known to robustly activate RYR2 channels in bilayers (Györke and Fill, 1993; Schiefer et al., 1995; Sitsapesan et al., 1995; Valdivia et al., 1995; Laver and Curtis, 1996). Fig. 4 shows how single RYR2 channels conducting either Cs+ or Ca2+ respond to fast cytosolic Ca2+ changes. Flash photolysis of cytosolic caged Ca2+ was used to rapidly elevate the free Ca2+ level near the channel (see Materials and methods). The steady-state cytosolic free Ca2+ was ∼100 nM, and the resting Po was near zero. Fig. 4 A shows six sample single-channel sweeps when the channel was mediating a Ca2+ flux (3.58 pA; lumen-to-cytosol). The top two recordings (gray) were made when the cytosolic bath solution was not being stirred. This is important because stirring of the cytosolic bath removes local Ca2+ and thus local Ca2+ remained elevated (above basal levels) for several seconds. The channel rapidly activated and remained active for the duration of the recording period. The lower four recordings (black) were made during continuous rapid stirring of the cytosolic bath, and thus the applied local Ca2+ stimuli was transient (rise time, ∼100 µs; decay time constant, ∼15–19 ms). Again, the channel rapidly activated, but this time its activity ceased before the end of the recording period. Shown below the single-channel recordings is an ensemble trace that was generated by averaging >40 single-channel sweeps. Below this ensemble is a recording of the applied Ca2+ stimulus, which was measured via Rhod-2 fluorescence. Fig. 4 B shows a similar experiment, but in this case the channel was mediating a Cs+ flux, not a Ca2+ flux. Again, the channel activated rapidly upon the abrupt Ca2+ elevation and deactivated as the stimulating Ca2+ was washed away.
Kinetics of single RYR2 channel Ca2+ activation and deactivation. Local cytosolic free Ca2+ increases around a channel were generated by flash photolysis of caged Ca2+. Single RYR2 channels were repeatedly activated by photolytic Ca2+ stimuli (>5-s interval). Cytosolic solution contained Tris-HEPES (120/250 mM; pH 7.4), 100 nM of free Ca2+, 4 mM DM-nitrophen, 50 µM Rhod-2, and 2 mM glutathione. (A) Six single RYR2 channel sweeps when the lumen-to-cytosol current (3.58 pA) was carried by Ca2+ are shown at top. The cytosolic solution was as described above, and the luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). Recordings were made at 0 mV. Open events are upward, and zero current level is marked at left. The top two recordings (gray) were made when the cytosolic bath solution was not being stirred. The lower four recordings were made during continuous, vigorous stirring of the cytosolic bath. This stirring efficiently exchanged the local photolyzed solution near the channel with unphotolyzed (basal free Ca2+) solution from the bath, generating transient Ca2+ stimuli (rise time, ∼100 µs; decay time constant, ∼15–19 ms). An ensemble trace generated from >40 single-channel sweeps (while stirring) is shown just below the single-channel recordings. Below the ensemble trace is the corresponding recording of the applied local stimulus (while stirring). (B) Like A, but the lumen-to-cytosol current (7.9 pA) was carried by Cs+. This channel was not the same as shown in A. The cytosolic solution was as described above, and the luminal solution contained 5 µM of added Ca2+ and 250 mM Cs-HEPES, pH 7.4. (C) Calcium activation kinetics. Four ensemble RYR2 activity traces from different RYR2 channels with either Ca2+ (filled circles) or Cs+ (open circles) as charge carrier were averaged and then fit (lines) by a single-exponential function. (D) Calcium deactivation kinetics. The decay phase and exponential fits of the same averaged ensemble traces described in C are shown. Only a small subset of the points fit is actually shown. (E) Time constants of Ca2+ activation and deactivation in response to the applied transient Ca2+ stimulus. Ca-On and Cs-On refer to the activation time constants when Ca2+ or Cs+ was charge carrier, respectively. Ca-Off and Cs-Off refer to the inhibition time constants. Ca-Stim and Cs-Stim refer to Ca2+ stimulus decay time constant when Cs+ or Cs+ was charge carrier, respectively. The asterisk indicates a significant difference (P < 0.02).
Fig. 4 (C and D) show the time courses of RYR2 activation and deactivation on an expanded time scale. Filled circles indicate when the RYR2 was conducting Ca2+, and the open circles are when the RYR2 was conducting Cs+. In Fig. 4 C, the Cs+ rise time points are shifted to avoid overlap. In Fig. 4 D, only a subset of the Po decay points are shown (80 to 5% peak Po). Single exponentials were fit to the Po rise and fall. Fig. 4 E lists the salient time constants. The Ca2+ stimulus decay time constants (Ca-Stim and Cs-Stim) when Ca2+ or Cs+ was charge carrier were not significantly different. The Ca2+ activation time constants (Ca-On and Cs-On) were also not different. However, the Ca2+ deactivation time constants (Ca-Off and Cs-Off) were significantly different. This slower Po decay when Ca2+ was charge carrier may be due to a luminal Ca2+ action (see Fig. 1 C), a feed-through Ca2+ action (see Fig. 3 D), or some combination of both. Note that there was 50 mM of luminal Ca2+ present when Ca2+ was charge carrier and just 5 µM when Cs+ was charge carrier.
Multiple RYR2 channels are frequently incorporated into the bilayer. Occasionally, these channels will open and close in a concerted fashion, as has been reported by others (Marx et al., 2001; Laver, 2007). Fig. 5 shows sample recordings from one such occurrence. All recordings shown here are from the same incorporation, which deposited two channels into the bilayer. Fig. 5 A shows the channel activity when the channels were carrying a lumen-to-cytosol Ca2+ current. The two channels frequently open at the same time, resulting in events with double the usual single-channel current amplitude. Fig. 5 B shows what happens when the charge carrier was changed from Ca2+ to Ba2+. The frequent double amplitude events are no longer evident. Fig. 5 C shows that the double amplitude events return when the charge carrier was changed back to Ca2+. Because cytosolic Ba2+ does not activate the RYR2 channel (Fill and Copello, 2002), these results suggest that Ca2+ passing through the open pore of one channel is activating the second neighboring RYR2 channel. We call this coordinated gating to distinguish it from the flux-independent FK-506 binding protein–mediated coupled gating reported by Marx et al. (2001).
Flux-dependent coordinated RYR2 channel activity. (A) Sample coordinated channel recordings (two channels present) and all-points histogram. The cytosolic solution contained 1 mM EGTA (0.45 µM of free Ca2+) and Tris-HEPES (120/250 mM; pH 7.4). The luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). Recordings (left) were made at 0 mV. Open events are upward, and zero current level is marked for the top recording by a thick solid line. The thin dashed lines (above zero current mark) indicate normal current level for one or two open channels. All-points histograms (right) were made from a long recording from which these sample recordings were taken. (B) Same channel and conditions as in A, but the 50 mM Ca2+ in the luminal solution was exchanged for Ba2+. (C) Same channel and conditions as in B, but the 50 mM Ba2+ in the luminal solution was changed back to Ca2+. (D) Ensemble traces were generated by aligning and summing many individual coordinated events (two channel events). Only the decay phase is shown here. Solutions are those described for A, except that the cytosolic free Ca2+ level was varied as indicated below. Trace marked "a" has a time constant of 47.49 ms and was generated from 2,308 events collected with 1 µM of cytosolic free Ca2+ present. Trace marked "b" has a time constant of 33.65 ms and was generated from 1,252 events collected with 0.7 µM of cytosolic free Ca2+ present. Trace marked "c" has a time constant of 18.62 ms and was generated from 792 events collected with 0.45 µM of cytosolic free Ca2+ present. The inset plots attrition time constants (ms) as a function of cytosolic free Ca2+ concentration (µM). Open circles are the time constants determined by single-exponential fits to the ensemble traces. The connected filled points are the predicted stochastic attrition calculated using Eq. 1, assuming two channels are present. This calculation assumes the single-channel Po's as shown in Fig. 1 B and single-channel MOTs as shown in Fig. 3 D (inset). For example, single-channel Po's were 0.03, 0.09, and 0.19 for 0.45, 0.7, and 1 µM of cytosolic free Ca2+, respectively. Single-channel MOTs for these same Ca2+ levels were 1.04, 6.86, and 11.46 ms, respectively.
The two channels would need to be within ∼1.8 µm of each other for the neighboring channels to "see" each other's fluxed Ca2+ at a level equal to their cytosolic Ca2+ EC50. Peng et al. (2004) showed that single RYR channels in bilayers randomly diffuse an average of 5 µm every 3 s. Thus, it would be very unlikely that two independently diffusing channels would remain within ∼1.8 µm of each other for more than a few seconds. Because we observed consistent Ca2+ flux coordinated gating of neighboring channels for extended periods of time (>1 h), the neighboring channels do not likely diffuse independently of each other. We believe that the neighboring channels are probably physically linked together, perhaps at their normal cellular spacing. If so, then the center spacing between the neighboring channels in the bilayer would be ∼30 nm (Franzini-Armstrong et al., 1999).
Many coordinated RYR2 openings were aligned (to t = 0) to generate ensemble events. The on-time constant of these ensemble coordinated events was always ≤0.75 ms, consistent with the cytosolic Ca2+ activation rate measured earlier. Fig. 5 D compares the decay phase of ensemble coordinated events (normalized to peak intra-event nPo) collected at three different resting cytosolic Ca2+ concentrations (a = 1, b = 0.7, and c = 0.45 µM). The decay time constants of coordinated events increased with resting cytosolic Ca2+ concentration. These decay time constants are plotted as a function of cytosolic Ca2+ in the inset in Fig. 5 D (open circles). Also plotted is the predicted stochastic attrition for a cluster of two independently gating channels (line). This stochastic attrition prediction was generated using single RYR2 Po and MOTs results. The decay times of the coordinated events were slower than that predicted for a simple stochastic attrition termination mechanism.
In multichannel studies, the parameter nPo is typically used to describe the probability of channels being open. Fig. 6 A shows how overall (not intra-event) nPo varies with resting cytosolic Ca2+ concentration. Open and filled circles represent nPo without and with cytosolic Mg-ATP present, respectively. The cytosolic EC50 of coordinated events was 1.98 µM (PoMax = 0.86, PoMin = 0.26, and Hc = 1.7). The dotted and dashed lines reflect the Ca2+ sensitivity of individual RYR2 channels with and without cytosolic Mg2+-ATP present (also see Fig. 1 B). In the absence of Mg2+-ATP, the coordinated channels have substantial stationary nPo at low cytosolic Ca2+ levels (0.1 µM). This is most likely due to inter-RYR2 Ca2+ flux activation prolonging activity within the RYR2 cluster. The low nPo with Mg2+-ATP present at 0.1 µM is also consistent with this possibility because Mg2+-ATP reduces the cytosolic RYR2 Ca2+ sensitivity.
Flux inhibition of coordinated RYR2 channel activity. (A) Cytosolic Ca2+ sensitivity of coordinated RYR2 channel activity (nPo). Overall nPo was defined from coordinated channel recordings at 0 mV. The cytosolic solution contained 1 mM EGTA and Tris-HEPES (120/250 mM; pH 7.4). The luminal solution contained Ca-HEPES (50/250 mM; pH 7.4). Open circles summarize the nPo (mean ± SEM; n = 7 different experiments). The solid line is a Hill equation fit to open circles (EC50 = 1.98 µM, PoMax = 0.86, PoMin = 0.26, and Hc = 1.7). Small filled circles are after cytosolic Mg-ATP was added (1 mM of free Mg2+ and 5 mM of total ATP; n = 5 different experiments). Thin dashed and dotted lines are the single RYR2 50L and 50L plus Mg-ATP curves from Fig. 1 B. (B) Sample recording of coordinated events (two channels present). Solutions here are like those described for A (0.1 µM of cytosolic free Ca2+). Sample events at 0, 20, and 40 mV are shown. Open events are upward, zero current level is marked at left, and one open-channel current level is indicated by the dashed lines. All recordings shown are from the same channel incorporation. (C) Inhibition of intra-event nPo by increasing Ca2+ flux. Results collected from five different coordinated channels where only two channels were present. Solutions like those described for A (0.1 or 0.3 µM of cytosolic free Ca2+). Unit current was varied by changing membrane potential. Open circles are individual determinations. Dashed line is reproduced from Fig. 3 C. Thick solid line is a fit to the open circles and indicates a site distance from the pore of 1.5 nm (IC50 = 6.2 mM and Hc = −1.3). The free bath Ca2+ used for this fitting was 134 µM, which is the average predicted free [Ca2+] (for 3.58 and 6.20 pA) at 30 nm from an open pore. This distance (30 nm) is approximately the inter-pore distance of two adjacent RYR2 pores in cells. (D) Calcium diffusion from a point source was calculated with cytosolic Ca2+ of 100 nM and no buffer present (thick lines) for unit Ca2+ currents of 6, 3.58, and 0.5 pA. The dashed line is for 0.5 pA current with 244 µM of cytosolic Ca2+ buffer (Km = 673 nM) present (Bers, 2001). It was assumed that the kON of this buffer was diffusion limited. (Inset) A cartoon depicting salient dimensions of neighboring RYR2 channels.
Fig. 6 B shows selected coordinated events collected at three different membrane potentials. The flicker between the double and single open state (dotted line) increases as unit Ca2+ current gets larger. This increased flicker reflects changing intra-event nPo. Fig. 6 C plots intra-event nPo as a function of unit Ca2+ current and represents results collected from five different channel incorporations. Eq. 4 fit (thick line), assuming the usual cytosolic inhibition site (EC50, 6.2 µM; Hc = −1.3), indicates a pore inhibition site distance of 1.5 nm. This is consistent with our previous single-channel pore inhibition site distance determinations. Fig. 6 D shows predicted local free Ca2+ profiles over a distance covering two neighboring RYR2 channels. Profiles are shown for three different unit currents (6, 3.58, and 0.5 pA). The dotted line is for a 0.5-pA current in the presence of physiological Ca2+ buffering (Bers, 2001).
Our results suggest that single Ca2+-activated RYR2 channels can be modulated by the Ca2+ flux they carry if that flux is large enough (>3 pA). A large lumen-to-cytosol Ca2+ flux can act at both cytosolic activation and inhibition sites. However, the RYR2 carries a Ca2+ flux of <0.5 pA in cells (Mejía-Alvarez et al., 1999; Kettlun et al., 2003; Gillespie and Fill, 2008). Thus, an RYR2 channel is not likely to be regulated by its own flux in cells. Our results also suggest that the single RYR2 cytosolic Ca2+ activation mechanism is much more sensitive to cytosolic bath Ca2+ than to fluxed Ca2+. This leads to the suggestion that the RYR2 cytosolic Ca2+ activation machinery is somehow protected/shielded from its own Ca2+ flux.
Flux-dependent inhibition was measured six ways, and each time the results were fit using Eq. 4 to determine a pore site distance. This fitting assumed the responsible site was the same that operates under steady-state conditions (IC50 = 6.2 mM; Hc = −1.3). The six individual pore site determinations were 1.3, 1.1, 1.4, 1.5, 0.5, and 1.5 nm, yielding an average pore inhibition site distance of 1.2 ± 0.16 nm. This is consistent with Xu and Meissner (1998), who predicted an RYR2 pore inhibition site distance of <3 nm assuming steady-state parameters similar to those we used. For the RYR1 channel, a pore inhibition site distance of 3–6 nm was reported, also assuming traditional steady-state inhibition parameters (Tripathy and Meissner, 1996). Laver (2007) more recently suggested that the RYR2 channel is regulated by a cytosolic inhibition site with an IC50 of 1.2 µM with a pore inhibition site distance of 26 nm. Because the maximum cytosolic pore site distance is ∼20 nm, Laver (2007) suggests that this site may be on the very periphery of the channel or possibly on an adjacent, closely associated regulatory protein. The difference between our pore inhibition site determination and that of Laver (2007) could be explained if our channels, and those of Xu and Meissner (1998), did not have this adjacent, closely associated regulatory protein.
Xu and Meissner (1998) also reported that RYR2 Ca2+ fluxes ≥0.25 pA can act at cytosolic activation sites, and those ≥8 pA can act on inhibitory sites. Our results are generally consistent with this, except that we found detectable Ca2+ flux activation and inhibition only when Ca2+ fluxes were >3 pA. Our lower flux sensitivity may be explained by the absence of caffeine in our studies. Xu and Meissner (1998) also reported a paradoxical RYR2 activation upon 20 mM BAPTA application and proposed that RYR2 has "BAPTA-inaccessible" activation and "BAPTA-accessible" inhibition sites. In other words, they suggested that the cytosolic Ca2+ activation site is protected somehow. We make a similar prediction here, but ours is not based on BAPTA buffering.
Flux-dependent RYR2 activation was observed here (Fig. 3, C and D). It was observed when the cytosolic resting free Ca2+ was low (0.5 µM). It increased Po from near zero to ∼0.08 as Ca2+ flux amplitude increased from 2 to ∼5 pA. This level of Ca2+ current will elevate local Ca2+ concentration over the entire cytosolic domain of the RYR2, carrying it to levels >100 µM. When free Ca2+ in the cytosolic bath is elevated to 100 µM (see Fig. 1 B), the Po of the RYR2 channels is ∼0.8 (i.e., 10 times greater than the maximal Ca2+ flux activation observed). Thus, the relatively small degree of RYR2 Ca2+ flux activation suggests that the channel's cytosolic Ca2+ activation mechanism is somehow shielded from its own Ca2+ flux.
Our results also show that the function of two neighboring RYR2 channels can be coordinated when the channels were carrying a lumen-to-cytosol Ca2+ flux, consistent with the findings of Laver (2007). Like Laver (2007), our results indicate that this coordinated function is due to inter-RYR2 Ca2+ flux activation. Thus, single RYR2 channels are not very sensitive to their own Ca2+ flux, but that very same flux is sufficient to robustly/consistently activate a neighboring channel. Why might a single RYR2 channel be insensitive to its own flux? Some possible flux "protection" mechanisms might include: (1) Ca2+ occupancy rendering cytosolic activation site(s) flux insensitive, and/or (2) a protein conformational-dependent mechanism where channel opening physically alters the activation machinery somehow.
The Ca2+ occupancy possibility can be explained as follows. The channel opens when cytosolic bath Ca2+ binds to the activation site(s). Once open, Ca2+ flux through the channel elevates the local Ca2+ concentration. The fluxed Ca2+, however, cannot bind to the activation site(s) if the initial activating Ca2+ is already bound. The bound Ca2+ provides some inherent "protection" of the activation site(s). If the initial activating Ca2+ remains bound after the channel closes, this protection would last until the Ca2+ unbinds. Standard Ca2+-binding kinetics is consistent with this possibility because local fluxed Ca2+ levels theoretically dissipate faster than the channels close (Schiefer et al., 1995; Vélez et al., 1997; Soeller and Cannell, 2002). If the channel does indeed close with Ca2+ still bound, however, then open event duration (MOT) would be independent of cytosolic Ca2+ concentration, and this is clearly not the case. Fig. 3 D (inset) as well as Xu and Meissner (1998) show that RYR2 MOT varies with steady-state cytosolic Ca2+ levels. This implies that Ca2+ unbinds before the channel closes, giving Ca2+ a chance to rebind and prolong opening. Thus, a Ca2+ occupation protection mechanism may be kinetically possible but is not consistent with experimental observation. The conformational-dependent mechanism can be explained as follows. Protein movements associated with channel activation may determine (fatefully link) opening with channel closing. This type of mechanism was first proposed by Stern et al. (1997), and they called it fateful inactivation. In this case, the cytosolic Ca2+ sensitivity of MOT would not be generated by Ca2+ rebinding to the open channel but, instead, would be a consequence of how the channel was activated. This could explain our MOT results.
There is a prevalent contention that the effects of luminal Ca2+ (including flux-dependent regulation) require the presence of a cytosolic RYR2 agonist like ATP or caffeine. Consequently, some previous studies of Ca2+ flux regulation have been done in the presence of such agonists (Tripathy and Meissner, 1996; Xu and Meissner, 1998; Laver, 2007). The use of ATP is occasionally justified by arguing that ATP is a "physiological" agonist. This is certainly true, but the agonistic action of ATP in cells is normally offset by the antagonistic action of Mg2+. Applying ATP alone shifts RYR2 cytosolic Ca2+ sensitivity, making the channel more sensitive to Ca2+ flux regulation. For example, Laver (2007) reports an EC50 of 5.4 µM if RYR2 channels are activated by Ca2+ alone and an EC50 of 0.5 µM when they are coactivated by Ca2+ and ATP (no Mg2+). When ATP and Mg2+ are added together, the cytosolic Ca2+ sensitivity (EC50) of the RYR2 channel shifts from ∼2 to ∼12 µM (Fig. 1 B), making the channels less prone to Ca2+ flux activation. Here, studies of RYR2 flux regulation were done with Ca2+-activated channels.
In cells, the RYR2 channel carries a unit Ca2+ current of ∼0.5 pA (Mejía-Alvarez et al., 1999; Kettlun et al., 2003; Gillespie and Fill, 2008). The cytosol is thought to contain ∼244 µM of Ca2+ buffer (average Km = 673 nM) (Bers, 2001). Using these values and the known dimensions of a single RYR2 channel (Samsó et al., 2005; Serysheva et al., 2005), some predictions about Ca2+ flux regulation of RYR2 channels in cells can be made.
Fig. 6 D shows the Ca2+ profile around an open pore conducting a 0.5-pA Ca2+ current with and without physiological buffers present. We assume here that the cytosolic buffer has a diffusion-limited kON. Because there are clearly some slow and fixed buffers in cells, the Ca2+ profile with the buffer present likely overestimates the action of the buffer. Our results indicate that the cytosolic Ca2+ inhibition site sits at a point 1.2 nm from the pore and has an IC50 of 6.2 mM. Fig. 6 D suggests that there will be ∼0.4 mM Ca2+ at this site when the channel opens in cells, and thus little (if any) Ca2+ flux–dependent inhibition should occur. Because cytosolic Ca2+ and Mg2+ inhibit the RYR2 channel with similar IC50's (Fig. 2 B), the action of fluxed Ca2+ may well depend on how much Mg2+ is present. Assuming the cytosol contains ∼1 mM of free Mg2+, the combined free Ca2+ and Mg2+ concentration at the cytosolic inhibition site would increase from 1 and 1.4 mM when the channel opens. Our results indicate that this would only minimally reduce Po (0.73 to 0.69) or generate a relatively minor degree of flux-dependent inhibition.
The cytosolic Ca2+ activation site must be somewhere within 18 nm of the pore. The RYR2 Ca2+ EC50 in cells (with Mg-ATP present) is 12 µM (Fig. 1 B) (Zoghbi et al., 2004). With a physiological Ca2+ current (0.5 pA), the entire cytoplasmic domain around an open single RYR2 would "see" local Ca2+ levels >20 µM. Thus, it is possible for the Ca2+ flux to feedback and influence the activation status of the channel carrying it. Our results, however, suggest that channels are largely insensitive to their own Ca2+ flux, even when that flux is >2 pA. Thus, the flux carried by an RYR2 channel in cells is unlikely to influence its own activation status.
The RYR2-RYR2 center spacing in cells is ∼30 nm (Franzini-Armstrong et al., 1999). Fig. 6 D indicates that local Ca2+ will be 7–15 µM at a distance of 30 nm from an open RYR2 pore conducting a 0.5-pA Ca2+ current. Thus, the opening of an RYR channel in a cell will elevate local Ca2+ levels around neighboring channels to about the RYR2 Ca2+ activation EC50 (∼12 µM). This likely explains the highly concerted channel activation within an RYR2 cluster that underlies the Ca2+ spark in cells. Release sites (i.e., RYR2 clusters) in cells are separated by ∼760 nm radially and by ∼1,800 nm longitudinally (Parker et al., 1996). With normal physiological Ca2+ buffering, the local Ca2+ level falls to basal levels (100 nM) at a distance of ∼300 nm from an open RYR2 channel conducting a 0.5-pA Ca2+ current. Thus, RYR2 Ca2+ release at one cluster would not be likely to activate RYR2 channels in a neighboring cluster, consistent with sparks being localized at non-propagating events in cells.
We thank Dr. Ariel Escobar for his help and imaging hardware/software expertise. We also thank Dr. Julio Copello for his technical expertise and Dr. Eduardo Rios for his suggestion to generate Eq. 4 to better specify pore site distances.
This work was supported by National Institutes of Health Grants R01HL57832 and R01AR054098 to M. Fill.
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2001. Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer Academic Press, London. 427 pp.
. 1996. Ca2+ sparks involving multiple Ca2+ release sites along Z-lines in rat heart cells. J. Physiol. 497:31–38.
. 1995. Ca2+ activation and Ca2+ inactivation of canine reconstituted cardiac sarcoplasmic reticulum Ca(2+)-release channels. J. Physiol. 489:337–348.
. 1994. Regulation of the gating of the sheep cardiac sarcoplasmic reticulum Ca(2+)-release channel by luminal Ca2+. J. Membr. Biol. 137:215–226.
. 1995. New insights into the gating mechanisms of cardiac ryanodine receptors revealed by rapid changes in ligand concentration. Circ. Res. 77:765–772.
. 1985. Electron probe X-ray microanalysis of post-tetanic Ca2+ and Mg2+ movements across the sarcoplasmic reticulum in situ. J. Biol. Chem. 260:6801–6807.
. 1985. Nucleotide specificity of cardiac sarcoplasmic reticulum. GTP-induced calcium accumulation and GTPase activity. J. Biol. Chem. 260:9618–9623.
© 2009 Liu et al. This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jgp.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).


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标准图书馆 > 资源分类 > Internation > ISO > BS ISO 22197-3-2011
BS ISO 22197-3-2011
BSI Standards Publication BS ISO 22197-3:2011 Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for air-purification performance of semiconducting photocatalytic materials Part 3: Removal of tolueneBS ISO 22197-3:2011 BRITISH STANDARD National foreword This British Standard is the UK implementation of ISO 22197-3:2011. The UK participation in its preparation was entrusted to Technical Committee RPI/13, Advanced technical ceramics. A list of organizations represented on this committee can be obtained on request to its secretary. This publication does not purport to include all the necessary provisions of a contract. Users are responsible for its correct application. © BSI 2011 ISBN 978 0 580 62883 2 ICS 81.060.30 Compliance with a British Standard cannot confer immunity from legal obligations. This British Standard was published under the authority of the Standards Policy and Strategy Committee on 30 April 2011. Amendments issued since publication Date Text affectedBS ISO 22197-3:2011Reference number ISO 22197-3:2011(E) ©ISO 2011INTERNATIONAL STANDARD ISO 22197-3 First edition 2011-04-01 Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for air-purification performance of semiconducting photocatalytic materials — Part 3: Removal of toluene Céramiques techniques — Méthodes d essai relatives à la performance des matériaux photocatalytiques semi-conducteurs pour la purification de l air — Partie 3: Élimination du toluène BS ISO 22197-3:2011 ISO 22197-3:2011(E) PDF disclaimer This PDF file may contain embedded typefaces. In accordance with Adobe s licensing policy, this file may be printed or viewed but shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In downloading this file, parties accept therein the responsibility of not infringing Adobe s licensing policy. The ISO Central Secretariat accepts no liability in this area. Adobe is a trademark of Adobe Systems Incorporated. Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below. COPYRIGHT PROTECTED DOCUMENT © ISO 2011 All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or ISO s member body in the country of the requester. ISO copyright office Case postale 56 • CH-1211 Geneva 20 Tel. + 41 22 749 01 11 Fax + 41 22 749 09 47 E-mail [email protected] Web www.iso.org Published in Switzerland ii © ISO 2011 – All rights reservedBS ISO 22197-3:2011 ISO 22197-3:2011(E) © ISO 2011 – All rights reserved iiiContents Page Foreword iv 1 Scope1 2 Normative references1 3 Terms and definitions .2 4 Symbols3 5 Principle .3 6 Apparatus.3 6.1 Test equipment3 6.2 Test gas supply .4 6.3 Photoreactor 5 6.4 Light source .6 6.5 Analytical system 6 7 Test piece .7 8 Procedure.7 8.1 General aspects.7 8.2 Pretreatment of test piece 7 8.3 Toluene removal test.8 9 Calculation .8 10 Test method for test pieces with lower performance9 11 Test report9 Annex A (informative) Results of round-robin test .10 Bibliography11 BS ISO 22197-3:2011 ISO 22197-3:2011(E) iv © ISO 2011 – All rights reservedForeword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies). The work of preparing International Standards is normally carried out through ISO technical committees. Each member body interested in a subject for which a technical committee has been established has the right to be represented on that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization. International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2. The main task of technical committees is to prepare International Standards. Draft International Standards adopted by the technical committees are circulated to the member bodies for voting. Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote. Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. ISO shall not be held responsible for identifying any or all such patent rights. ISO 22197-3 was prepared by Technical Committee ISO/TC 206, Fine ceramics. ISO 22197 consists of the following parts, under the general title Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for air-purification performance of semiconducting photocatalytic materials: ⎯ Part 1: Removal of nitric oxide ⎯ Part 2: Removal of acetaldehyde ⎯ Part 3: Removal of toluene ⎯ Part 4: Removal of formaldehyde ⎯ Part 5: Removal of methyl mercaptan BS ISO 22197-3:2011 INTERNATIONAL STANDARD ISO 22197-3:2011(E)© ISO 2011 – All rights reserved 1Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for air-purification performance of semiconducting photocatalytic materials — Part 3: Removal of toluene 1 Scope This part of ISO 22197 specifies a test method for the determination of the air-purification performance of materials that contain a photocatalyst or have photocatalytic films on the surface, usually made from semiconducting metal oxides, such as titanium dioxide or other ceramic materials, by continuous exposure of a test piece to the model air pollutant under illumination with ultraviolet light (UV-A). This part of ISO 22197 is intended for use with different kinds of materials, such as construction materials in flat sheet, board or plate shape, that are the basic forms of materials for various applications. This part of ISO 22197 also applies to structured filter materials including honeycomb-form, woven and non-woven fabrics, and to plastic or paper materials if they contain ceramic microcrystals and composites. This part of ISO 22197 does not apply to powder or granular photocatalytic materials. This test method is usually applicable to photocatalytic materials produced for air purification. This method is not suitable for the determination of other performance attributes of photocatalytic materials, i.e. decomposition of water contaminants, self-cleaning, antifogging and antibacterial actions. It concerns the removal of toluene. 2 Normative references The following referenced documents are indispensable for the application of this document. For dated references, only the edition cited applies. For undated references, the latest edition of the referenced document (including any amendments) applies. ISO 80000-1:2009, Quantities and units — Part 1: General ISO 2718:1974, Standard layout for a method of chemical analysis by gas chromatography ISO 4677-1:1985, Atmospheres for conditioning and testing — Determination of relative humidity — Part 1: Aspirated psychrometer method ISO 4892-3:2006, Plastics — Methods of exposure to laboratory light sources — Part 3: Fluorescent UV lamps ISO 5725-2:1994, Accuracy (trueness and precision) of measurement methods and results — Part 2: Basic method for the determination of repeatability and reproducibility of a standard measurement method ISO 6145-7:2001, Gas analysis — Preparation of calibration gas mixtures using dynamic volumetric methods — Part 7: Thermal mass-flow controllers BS ISO 22197-3:2011 ISO 22197-3:2011(E) 2 © ISO 2011 – All rights reservedISO 10677:— 1) , Fine ceramics (advanced ceramics, advanced technical ceramics) — Ultraviolet light source for testing semiconducting photocatalytic materials ISO/IEC 17025:2005, General requirements for the competence of testing and calibration laboratories ISO 22197-1:2007, Fine ceramics (advanced ceramics, advanced technical ceramics) — Test method for air-purification performance of semiconducting photocatalytic materials — Part 1: Removal of nitric oxide 3 Terms and definitions For the purposes of this document, the following terms and definitions apply. 3.1 photocatalyst substance that performs one or more functions based on oxidization and reduction reactions under photoirradiation, including decomposition and removal of air and water contaminants, deodorization, and antibacterial, self-cleaning and antifogging actions 3.2 photocatalytic materials materials in which or on which the photocatalyst is added by coating, impregnation, mixing, etc. NOTE Such photocatalytic materials are intended primarily for use as building and road construction materials to obtain the above-mentioned functions. 3.3 zero-calibration gas air that does not contain pollutants (i.e. in which common pollutants are below 0,01 µl/l) NOTE The zero-calibration gas is prepared from indoor air using a laboratory air-purification system, or supplied as synthetic air in a gas cylinder. 3.4 standard gas diluted gases of known concentrations supplied in cylinders and certified by an accredited laboratory 3.5 test gas mixture of air and pollutant(s) of known concentration prepared from a standard gas or a zero-calibration gas, to be used for the performance test of a photocatalytic material NOTE The flow rate, concentration, etc., are expressed at the standard state (0 °C, 101,3 kPa) and dry-gas basis (exclusion of water vapour). 3.6 dark condition test condition with no light illumination by the light source for testing and room lighting NOTE Usually the test gas is supplied for comparison with the illuminated reaction. 1) To be published. BS ISO 22197-3:2011 ISO 22197-3:2011(E) © ISO 2011 – All rights reserved 34 Symbols For the purposes of this document, the following symbols apply. f the flow rate of test gas converted into that at the standard state (0 °C, 101,3 kPa, and dry-gas basis) (l/min) φ Tthe volume fraction of toluene at the reactor exit (µl/l) φ T0the supply volume fraction of toluene (µl/l) n Tthe quantity of toluene removed by the test piece (µmol) R the removal percentage, by test piece, of toluene (%) 5 Principle This part of ISO 22197 concerns the development, comparison, quality assurance, characterization, reliability, and design data generation of photocatalytic materials (Reference [3] in the Bibliography). The method described is intended to obtain the air-purification performance of photocatalytic materials by exposing a test piece to model polluted air under illumination by ultraviolet (UV) light (Reference [4] in the Bibliography). Toluene (C 7 H 8 ) is chosen as a typical aromatic volatile organic compound (VOC) with offensive odour. The test piece, placed in a flow-type photoreactor, is activated by UV illumination, and adsorbs and oxidizes gas-phase toluene to form carbon dioxide (CO 2 ) and other oxidation products (References [5] to [7] in the Bibliography). The air-purification performance is determined from the amount of toluene removed by the test piece, in micromoles ( μmol). The simple adsorption by the test piece (not due to photocatalysis) is evaluated by tests in the dark. However, some test pieces adsorb toluene very strongly, and a stable concentration of toluene may not be attained in the designated time of test. The photocatalytic activity may depend on physical and chemical properties of pollutants, mainly due to the adsorption process involved. For a better evaluation of air-purification performance of photocatalytic materials, it is recommended to combine one or more suitable test methods as described in other parts of ISO 22197. 6 Apparatus 6.1 Test equipment The test equipment enables a photocatalytic material to be examined for its pollutant-removal capability by supplying the test gas continuously, while providing photoirradiation to activate the photocatalyst. It is the same as that used in the test method for the removal of nitric oxide (ISO 22197-1) and consists of a test gas supply, a photoreactor, a light source, and pollutant-measurement equipment. Since low concentrations of pollutants are to be tested, the system shall be constructed with materials of low adsorption and resistant to ultraviolet (UV) radiation (e.g. acrylic resin, borosilicate glass). An example of a testing system is shown in Figure 1. BS ISO 22197-3:2011 ISO 22197-3:2011(E) 4 © ISO 2011 – All rights reservedDimensions in millimetres Key 1 light source 7 mass-flow controller 2 optical window 8 gas mixers 3 test piece 9 humidifier 4 standard gas (pollutant) 10 analyser 5 air-purification system 11 vent 6 compressor Figure 1 — Schematic diagram of test equipment 6.2 Test gas supply The test gas supply provides air polluted with the model contaminant at a predetermined concentration, temperature and humidity, and supplies it continuously to the photoreactor. It consists of flow regulators, a humidifier, gas mixers, etc. The flow rate of each gas should be within 5 % of the designated value, which is easily attained by using thermal mass-flow controllers, with knowledge of the temperature and gas type at calibration in accordance with ISO 6145-7. The expression of gas flow rate in this part of ISO 22197 is that converted to the standard state (0 °C, 101,3 kPa, and dry-gas basis). Typical capacities of flow controller for pollutant gas, dry air and wet air are 10 ml/min, 500 ml/min and 500 ml/min, respectively. The standard toluene gas before dilution, normally balanced with nitrogen in a cylinder, shall have a volume fraction of 10 µl/l to 50 µl/l. BS ISO 22197-3:2011 ISO 22197-3:2011(E) © ISO 2011 – All rights reserved 56.3 Photoreactor The photoreactor holds a planar test piece within a 50 mm wide trough, with its surface parallel to an optical window for photoirradiation. The reactor shall be fabricated from materials that adsorb little test gas and withstand irradiation of near-UV light. The test piece shall be separated from the window by a 5,0 mm ± 0,5 mm thick air layer. The test gas shall pass only through the space between the test piece and the window. This gap shall be accurately set up according to the thickness of the test piece, for example, by using height-adjusting plates with different thicknesses, as shown in Figure 2 a). When a filter-type material is tested, an alternative type of test-piece holder shall be used, which holds the test piece while allowing the test gas to pass through the cells of the filter under illumination [Figure 2 b)]. Quartz or borosilicate glass that absorbs minimal light at wavelengths longer than 300 nm shall be used for the window. Dimensions in milli
BSISO22197 2011
本文标题:BS ISO 22197-3-2011

redpajama_set_name: RedPajamaCommonCrawl


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Happy New Year 2020: Wishes, Messages, SMS, Quotes, Images.
Shahrukh Khan Videos Cinecurry; 110 videos; 1,399 views; Last updated on Apr 10, 2015; Play all Share. Loading. Save. Sign in to YouTube. Sign in. Compilation: Cutest Pictures Of AbRam by Cinecurry. 1:02. India Poochega-Sabse Shaana Kaun Starts Tonight by Cinecurry. 3:02 (Private video) Shah Rukh Khan Is Back On TV, Turns Game Show Host by Cinecurry. 3:24. Shah Rukh And Kajol Celebrate 1000.
Shah Rukh Khan is all set for Farah Khan's Happy New Year Chennai Express is unstoppable,reaches Rs 216.86 crore Shah Rukh Khan: I taught Rohit love,he taught me comic timing.
Portrait Shahrukh Khan Indian Bollywood Stock Photos.
A new picture of Shah Rukh Khan's daughter, Suhana Khan, apparently posing in a restaurant, has been shared online.Suhana is currently in New York City, where she recently enrolled at the New.Happy New Year (sometimes abbreviated as HNY) is a 2014 Indian Hindi-language action comedy film directed by Farah Khan and produced by Gauri Khan under the banner of Red Chillies Entertainment.The film features an ensemble cast of Shah Rukh Khan, Deepika Padukone, Abhishek Bachchan, Sonu Sood, Boman Irani, Vivaan Shah and Jackie Shroff.It was distributed worldwide by Yash Raj Films.Apr 20, 2014 - This Pin was discovered by muskan rathi. Discover (and save!) your own Pins on Pinterest.
Available for viewing 15 pictures shahrukh khan happy new year clipart, all in different sizes. Find what you need using our navigation and search. Ask other users about Shahrukh khan happy new year clipart.Shahrukh Khan and Deepika Padukone during the promotions Happy New Year on the sets of Comedy Nights With Kapil, in Mumbai.
Dedicated to Bollywood's King, the one and only Shah Rukh Khan. Here, you will find all his gorgeous photographs ranging from the 90s-00's to the present, from his memorable movies which have made him into the superstar he is today.
Shahrukh Khan and Katrina Kaif might soon be coming together for the second time after Jab Tak Hai Jaan for Farah Khan's next project, Happy New Year.
Indiawaale happy new year official song (2014). Happy new year movie songs 2014 shahrukh khan, deepika padukone music launch. Airina medina mena on. Happy new shahrukh khan and deepika rock in film promo. Watch online full hindi movie happy new year (2014) on putlocker blu ray rip. Happy new year hindi movie trailer 2014 deepika sonu - youtube.
Shah Rukh Khan (born 2 November 1965), also known by the initialism SRK, is an Indian actor, film producer, and television personality.Referred to in the media as the Baadshah of Bollywood (in reference to his 1999 film Baadshah), King of Bollywood and King Khan, he has appeared in more than 80 Bollywood films, and earned numerous accolades, including 14 Filmfare Awards.
A: Shahrukh's much awaited Happy New Year released recently on 24th October 2014. The movie released with Venus in the Zodiac Sign Libra. There was a Stellium of Venus, Saturn, Moon and Sun in the Zodiac Sign Libra on the release date. Mercury and Rahu were retrograde in Virgo Sign. The transiting Jupiter was exalted, and was aspecting Shahrukh's Natal Moon.
Happy New Year (2014) cast and crew credits, including actors, actresses, directors, writers and more.
New Delhi: Superstar Shah Rukh Khan can practically do anything. The actor has always managed to shut his critics every time his age was made an issue. He always proves that he can push his.
Happy New Year is the first film to change the rules of the game by taking Marketing and Promotions to a whole new level. The grand success of the SLAM tour, touched billions of people's hearts worldwide. The movie promotions were massive not only nationally, but also internationally. The whole team toured the US continent and the musical extravaganza became an instant hit.
Happy New Year is an upcoming 2014 Bollywood action comedy-drama film directed by Farah Khan and produced by Gauri Khan under the banner of Red Chillies Entertainment. The film has an ensemble cast which includes, Shahrukh Khan, Deepika Padukone, Abhishek Bachchan, Boman Irani, Vivaan Shah, Sonu Sood and Jackie Shroff. The film will be distributed worldwide by Yash Raj Films.
Happy New Year 2020: Check out Video Jukebox of Bhojpuri party songs for New Year's Eve 2020. The Bhojpuri Songs of the Jukebox are sung by top Bhojpuri singers Pawan Singh, Akshara Singh, Gunjan.
Shahrukh Khan Alone This Eid 2012 August 20, 2012 News anushka, eid, eid mubarak, katrina kaif, shahrukh khan Admin Shahrukh Khan, who usually spends time with family and close friends and associates during Eid, is celebrating the festival all alone this year.
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Rajiname Lyrics by Palwinder Tohra ft. Afsana Khan is the Latest Punjabi song written by Gill Talwara and music of this new song is given by Ronn Sandhu.
Happy New Year 2020: Wishes, Messages, SMS, Quotes, Images, Status, Greetings, Wallpaper, Photos and Pics; Solar Eclipse on 26th December 2019: When, Where, How to watch the last Surya Grahan of.


YarnArt Jeans yarn, 2.5 mm crochet hook.
Make chains as long as you need (put it on your lamb gathering at the waist to determine the size).
R 1: from the 2nd ch from hook inc in every st to the end of row, turn.
R 2: inc in every st to the end of row, turn.
R 3: inc in every st to the end of row, turn.
R 4: inc in every st to the end of row.
Cut out circle from beige felt. Embroider eyes and nose with black floss. Tint cheeks using dry pastel crayon.
Sew the face on the head.


Oh, damn it! Another soul could escape the hell and reincarnate to experience life once again. Go, get up on Earth to track your escapee. As soon as you got your reincarny you must find something to kill him. Make sure your target is dead before you get back to the underworld to your dark master Lucifer himself.


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Home/News/New Smartwatch Application For Accurate Signature.../Full Text
New Smartwatch Application For Accurate Signature Verification Developed
By Ben-Gurion niversity of the Negev (Israel)
Researchers say their new system uses the motion sensors in smartwatches to verify handwritten signatures.
Credit: Mirchu.net
Researchers at Ben-Gurion University of the Negev (BGU) and Tel Aviv University (TAU) in Israel say they have developed a system that uses smartwatch devices and software to verify handwritten signatures and detect even the most skilled forgeries.
The new method for online signature verification utilizes motion sensors found in conventional devices.
The software employs motion data gathered from a user's wrist to identify the writer during the signing process. The system, using data compiled from accelerometer and gyroscope sensors, analyzes changes in rotational motion and orientation, and trains a machine-learning algorithm to distinguish between genuine or forged signatures.
The researchers recruited 66 TAU students to record 15 samples of their genuine signature using a digital pen and a tablet while wearing a smartwatch on their writing hand. Each student then studied trace recordings of other people's genuine signatures and was asked to forge five of them.
"The results for both random and skilled forgery tests were encouraging, and confirmed our system is able to successfully distinguish between genuine and forged signatures with a high degree of accuracy," says BGU's Ben Nassi.
From Ben-Gurion University of the Negev (Israel)
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north-rhine-westphalia
General information: First Jewish presence: 1575; peak Jewish population: 43 in 1849; Jewish population in 1933: unknown (22 in 1932)
Summary: Two Jewish families were expelled from Raesfeld in 1683. A Jewish community was later established there without the required permission of the authorities, an offense for which the entire community was imprisoned in 1740. In 1750, however, a Jewish man named Israel Jost received a letter of protection from the authorities, which was renewed when he died in 1807; his descendants would form the bulk of Raesfeld's future Jewish community. The Jewish population (most local Jews were cattle dealers) grew while Raesfeld was under French rule. In 1812, the community converted a barn into a prayer hall and classroom. Later, in 1863, a synagogue—it housed a mikveh and classrooms—was built in Raesfeld. Local Jews operated an elementary school from 1835 until 1880, after which (until 1900) children received religious instruction from teachers who served Raesfeld and its surrounding Jewish communities. Rabbis from Borken or Recklinghausen officiated at weddings and funerals. Few Jews lived in Raesfeld in 1933, and the local authorities, probably out of regard for their amiable relationship with the town's Jews, did not enforce the boycott. The synagogue was nevertheless vandalized and looted by local SA men on Pogrom Night. The cemetery was desecrated in 1940, and the remaining Jews were deported in 1941/1942. Approximately 36 Raesfeld Jews were murdered in the Shoah. A memorial plaque was unveiled in the town after the war.
Photo: The synagogue of Raesfeld. Courtesy of: City Archive of Raesfeld.
Author / Sources: Harold Slutzkin
Sources: LIG, SIA
Located in: north-rhine-westphalia


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A consistent, harmonized appearance helps assure your potential audience that they can be confident in what you have to offer.


The tenets of Sean McVay's coaching philosophy are never far from sight at the Los Angeles Rams' training complex.
They're the last surviving members of the Never Miss a Super Bowl Club, the only known fans with a perfect attendance record for the NFL's biggest game.
While he won't put all the blame on current receiving superstar Antonio Brown for his current situation with the Steelers, retired receiver Hines Ward says that he needs to be held "accountable".


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Aquaman Superstar, Jason Momoa And Lisa Bonet Announce Split After 16 Year Relationship
Published by Swiftloaded | Thursday, 2 February 2023, 10:23 am
Star couple, Jason Momoa and Lisa Bonet are splitting up after 16 years together – 12 years of partnership and 4 years of marriage.
The former couple released a joint statement on Jason's Instagram, writing: 'We have all felt the squeeze and changes of these transformational times… A revolution is unfolding ~and our family is of no exception… feeling and growing from the seismic shifts occurring. And so~ We share our Family news~ That we are parting ways in marriage.
We share this not because we think it's newsworthy ~ But so that~ as we go about our lives we may do so with dignity and honesty. The love between us carries on, evolving in ways it wishes to be known and lived. We free each other ~to be who we are learning to become… Our devotion unwavering to this sacred life & our Children. Teaching our Children What's possible ~ Living the Prayer May Love Prevail ✨ J & L. [sic]'
A post shared by Jason Momoa (@prideofgypsies)
Lisa and Jason got together all the way back in 2005 after meeting at a jazz club.
Jason told James Corden that he had 'fireworks going off inside' and said 'I convinced her to take me home because I was living in a hotel and I didn't have a car. We went to [a café] and we sat down. She ordered a Guinness and that was it. I love Guinness. And then I ordered grits, and we had Guinness and grits, and the rest is history.'
The pair married over 10 years later in 2017. They have two children together, Lola and Wolf.
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