Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?

Lanthanide gadolinium (Gd3+) blocks CaV1.2 channels at the selectivity filter. Here we investigated whether Gd3+ block interferes with Ca2+-dependent inactivation, which requires Ca2+ entry through the same site. Using brief pulses to 200 mV that relieve Gd3+ block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd3+ block of the Ca2+ influx that enhances inactivation. However, we also found that the extent of Gd3+ block did not change when inactivation was reduced by abolition of Ca2+/calmodulin interaction, showing that Gd3+ does not block the inactivated channel. Thus, Gd3+ block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd3+ site or reduces its affinity, or that Ca2+ occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca2+-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117–11123), and that Ca2+-inactivated channels conduct Na+ when Ca2+ is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709–717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca2+ so that Ca2+ ion blocks the pore. A minimal model, in which the inactivation “gate” is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca2+

Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.

Inflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis

Design, Performance and Calibration of the CMS Forward Calorimeter Wedges

We report on the test beam results and calibration methods using charged particles of the CMS Forward Calorimeter (HF). The HF calorimeter covers a large pseudorapidity region (3\l |\eta| \le 5), and is essential for large number of physics channels with missing transverse energy. It is also expected to play a prominent role in the measurement of forward tagging jets in weak boson fusion channels. The HF calorimeter is based on steel absorber with embedded fused-silica-core optical fibers where Cherenkov radiation forms the basis of signal generation. Thus, the detector is essentially sensitive only to the electromagnetic shower core and is highly non-compensating (e/h \approx 5). This feature is also manifest in narrow and relatively short showers compared to similar calorimeters based on ionization. The choice of fused-silica optical fibers as active material is dictated by its exceptional radiation hardness. The electromagnetic energy resolution is dominated by photoelectron statistics and can be expressed in the customary form as a/\sqrt{E} + b. The stochastic term a is 198% and the constant term b is 9%. The hadronic energy resolution is largely determined by the fluctuations in the neutral pion production in showers, and when it is expressed as in the electromagnetic case, a = 280% and b = 11%

Design, Performance, and Calibration of CMS Hadron Endcap Calorimeters

Detailed measurements have been made with the CMS hadron calorimeter endcaps (HE) in response to beams of muons, electrons, and pions. Readout of HE with custom electronics and hybrid photodiodes (HPDs) shows no change of performance compared to readout with commercial electronics and photomultipliers. When combined with lead-tungstenate crystals, an energy resolution of 8\% is achieved with 300 GeV/c pions. A laser calibration system is used to set the timing and monitor operation of the complete electronics chain. Data taken with radioactive sources in comparison with test beam pions provides an absolute initial calibration of HE to approximately 4\% to 5\%

Design, Performance, and Calibration of the CMS Hadron-Outer Calorimeter

The CMS hadron calorimeter is a sampling calorimeter with brass absorber and plastic scintillator tiles with wavelength shifting fibres for carrying the light to the readout device. The barrel hadron calorimeter is complemented with an outer calorimeter to ensure high energy shower containment in the calorimeter. Fabrication, testing and calibration of the outer hadron calorimeter are carried out keeping in mind its importance in the energy measurement of jets in view of linearity and resolution. It will provide a net improvement in missing \et measurements at LHC energies. The outer hadron calorimeter will also be used for the muon trigger in coincidence with other muon chambers in CMS

The Na⁺/H⁺ Exchanger-3 (NHE3) Activity Requires Ezrin Binding to Phosphoinositide and Its Phosphorylation

<div><p>Na⁺/H⁺ exchanger-3 (NHE3) plays an essential role in maintaining sodium and fluid homeostasis in the intestine and kidney epithelium. Thus, NHE3 is highly regulated and its function depends on binding to multiple regulatory proteins. Ezrin complexed with NHE3 affects its activity via not well-defined mechanisms. This study investigates mechanisms by which ezrin regulates NHE3 activity in epithelial Opossum Kidney cells. Ezrin is activated sequentially by phosphatidylinositol-4,5-bisphosphate (PIP2) binding and phosphorylation of threonine 567. Expression of ezrin lacking PIP2 binding sites inhibited NHE3 activity (-40%) indicating that ezrin binding to PIP2 is required for preserving NHE3 activity. Expression of a phosphomimetic ezrin mutated at the PIP2 binding region was sufficient not only to reverse NHE3 activity to control levels but also to increase its activity (+80%) similar to that of the expression of ezrin carrying the phosphomimetic mutation alone. Calcineurin Homologous Protein-1 (CHP1) is part, with ezrin, of the NHE3 regulatory complex. CHP1-mediated activation of NHE3 activity was blocked by expression of an ezrin variant that could not be phosphorylated but not by an ezrin variant unable to bind PIP2. Thus, for NHE3 activity under baseline conditions not only ezrin phosphorylation, but also ezrin spatial-temporal targeting on the plasma membrane via PIP2 binding is required; however, phosphorylation of ezrin appears to overcome the control of NHE3 transport. CHP1 action on NHE3 activity is not contingent on ezrin binding to PIP2 but rather on ezrin phosphorylation. These findings are important in understanding the interrelation and dynamics of a CHP1-ezrin-NHE3 regulatory complex.</p></div

Effect of ezrin variants in PIP2 binding and 567 phosphorylation sites on NHE3 activity.

<p>NHE3 activity was measured as the rate of Na⁺-dependent intracellular pH recovery. Results are expressed as percentage of change in NHE3 activity. Bars and error bars represent the means and SE, respectively (4 experiments per condition). **P < 0.01 ANOVA compare to control (empty-vector-transfected cells). NS: Statistically insignificant compare to control. Effect of expression of <b>A.</b> Ezrin-WT, Ezrin-PIP2^-, Ezrin-T/D and Ezrin-PIP2^- T/D. <b>B.</b> Ezrin-WT, Ezrin-PIP2^-, Ezrin-T/A and Ezrin-PIP2^- T/A on NHE3 activity.</p

Effect of CHP1 expression on Ezrin-WT or Ezrin-PIP2^-distribution in sub-cellular fraction.

<p>Cellular fractions were prepared as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.g002" target="_blank">Fig 2</a>. <b>A:</b> One of the immunoblots is shown. GFP = transfected mEGFP tagged ezrin (Ezrin-GFP) and Endo = endogenous ezrin. <b>B:</b> Statistical analysis of signal intensity ratios relative to the immuno-detected band in S and P is reported. The signal of total ezrin in S plus P fractions was defined as equal to 100% for each condition and results are expressed as percentage of total ezrin in the P vs. S fraction. Antigen signals were normalized to β-actin. Bars and error bars represent the means ± SE, respectively (4 experiments per condition). *P<0.05/**P < 0.01 ANOVA, membrane fraction of tagged ezrin in the presented groups compared to the membrane fraction of tagged ezrin in cells transfected with Ezrin-WT. ^■P<0.05 ANOVA, membrane fraction of endogenous ezrin in the presented groups compared to the membrane fraction of endogenous ezrin in cells transfected with Ezrin-WT. ^$P<0.05 ANOVA, membrane fraction of tagged ezrin in Ezrin-PIP2^- compared to Ezrin-PIP2^- + CHP1 transfected cells.</p

Distribution of ezrin and its variants in sub-cellular fraction.

<p>Cellular fractions were prepared from OK cells and equal amounts of proteins (50 μg/lane) from a membrane-enriched fraction (P, 100,000 x g pellet) and cytosol (S, 100,000 x g supernatant) were blotted and probed with anti-total ezrin. <b>A:</b> One of the immunoblots is shown. GFP = transfected mEGFP tagged ezrin (Ezrin-GFP) and Endo = endogenous ezrin. <b>B:</b> Statistical analysis of signal intensity ratios relative to the immuno-detected band in S and P is reported. The signal of total ezrin in S plus P fractions was defined as equal to 100% for each condition and results are expressed as percentage of total ezrin in the P vs. S fraction. Antigen signals were normalized to β-actin. Bars and error bars represent the means ± standard errors (SE), respectively (4 experiments per condition). *P<0.05/** P<0.01 ANOVA, the membrane fraction of tagged ezrin in the presented groups compared to the membrane fraction of tagged ezrin in cells transfected with Ezrin-WT. ^■P<0.05 ANOVA, membrane fraction of endogenous ezrin in the presented groups compared to the membrane fraction of endogenous ezrin in cells transfected with Ezrin-WT. ^$$P<0.01 ANOVA membrane fraction of tagged ezrin in Ezrin-PIP2^- compared to Ezrin-PIP2^- T/D transfected cells.</p

Effect of CHP1 expression on the ezrin-mediated control of NHE3 activity.

<p>NHE3 activity was measured as described in the legend of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129306#pone.0129306.g003" target="_blank">Fig 3</a>. Bars and error bars represent the means and SE, respectively (8 experiments per condition). **P < 0.01 ANOVA compare to control (empty-vector-transfected cells), NS: Statistically insignificant compare to control. ^$$$P<0.001 ANOVA, CHP1 expression compared to CHP1 and Ezrin-T/A co-expression. Effect of expression of <b>A.</b> Ezrin-WT, Ezrin-PIP2^-, CHP1, CHP1 + Ezrin-WT or CHP1 + Ezrin-PIP2^-. <b>B.</b> Ezrin-T/A, Ezrin-PIP2^- T/A, CHP1, CHP1 + Ezrin-T/A or CHP1 + Ezrin-PIP2^- T/A on NHE3 activity.</p