Caβ retains its ability to shift GV curves, but maximal conductances are reduced in channels bearing mutations of AID-exposed residues

(A) Macroscopic currents from oocytes coexpressing Caβ with either Ca1.2 E462R or Ca1.2 K465N during the same stimulation protocol used in (shown at the top), with calibration bars corresponding to 20 ms and 200 nA. (B) GV curves in the presence (filled symbol) or absence (open symbol) of Caβ. (C) Plots of tail current amplitudes normalized by Q (I/Q) for Ca1.2 WT (▪), Ca1.2 E462R (•), and Ca1.2 K465N (▴). I/Q (mean ± SEM) versus voltage plots were fitted to the sum of two Boltzmann distributions. The maximal I/Q was 17.8 ± 2.5 nA/pC ( = 24) for Ca1.2 WT + Caβ, 7.4 ± 1.74 nA/pC ( = 11) for Ca1.2 K465N + Caβ, and 3.5 ± 0.8 nA/pC pC ( = 10) for Ca1.2 E462R + Caβ.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation-4

I/Q versus voltage from oocytes expressing Ca1.2 E462R after the injection of purified Caβ protein at the indicated concentrations. Traces correspond to superimposed responses to three 60-ms depolarizing pulses to −30 mV, 0 mV, and +30 mV from a holding voltage of −90 mV. Calibration bars correspond to 20 ms and 200 nA. Experimental I/Q values (8) were fitted to the equation (blue line):Each member of the equation corresponds to templates in absence (−) or presence (+) of saturating concentration of Caβ protein (2.0 μM). Variables defining each template were obtained from the fit to average I/Q plot from each condition. The contribution of +β and −β templates are shown as green and red lines, respectively. (B) As A) but for Ca1.2 K465N. (C) Mean ± SE of β2a-like versus protein concentration ([Caβ]) in μM. Continuous lines show the fit to a standard Hill equation:Where is the apparent dissociation constant and is the Hill coefficient. ranged between 1.4 and 1.6, whereas for WT, E462R, and K465N was 0.20, 0.22, and 0.25 μM, respectively. The number of averaged experiments ranged from three to six for every concentration and calcium channel variant.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Single channel mean currents and I/Q plots from macroscopic currents from different Ca1

2 variants recorded in high Ba and S(-)Bay K8644. (A) Mean current traces for six patches containing single Ca1.2 WT/Caβ channels (black) from seven patches with Ca1.2 E462R/Caβ channels (blue), and from 6 with Ca1.2 K465N/Caβ channels (red). The number of traces averaged in each case was 4,032 for Ca1.2 WT/Caβ, 7,504 for Ca1.2 E462R/Caβ, and 6,104 for Ca1.2 K465N/Caβ. Voltage protocol and recording condition were as described in . Calibration bars correspond to 50 ms and 100 fA. (B) I/Q versus voltage plot for Ca1.2 WT ( = 12), Ca1.2 E462R ( = 12), and Ca1.2 K465N ( = 13) coexpressed with Caβ and recorded in external 76 mM Ba and 0.1 μM of S(-) Bay K 8644 as used for single channel.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Deletion of AID sequence or replacement of the conserved tryptophan abolished modulation by Caβ

(A) Superimposed macroscopic current traces from oocytes coexpressing Caβ either with Ca1.2 W470S or Ca1.2 ΔAID. Each trace was obtained during a 70-ms pulse of increasing amplitude, starting at −40 mV and ending at 150 mV in 10-mV increments. Membrane was held at −80 mV until the beginning of the pulse and returned to −40 mV for the remaining of the trace (shown at the top). Currents were sampled at 2.5 kHz until 3 ms before the end of the pulse, and then at 50 kHz. Traces were filtered at 10 kHz, and a P/−4 prepulse protocol was used to subtract linear components. Calibration bars correspond to 20 ms and 200 nA. (B) Conductance–voltage relationship (GV curve) for the different subunit combinations shown in A. The peak amplitude of the tail currents for each test voltage was normalized by the largest tail current (I/Imax) to generate the GV curves. Open and filled symbols correspond to oocytes recorded with or without Caβ, respectively. The sums of two Boltzmann distributions that best described each set of data are shown as continuous lines.<p><b>Copyright information:</b></p><p>Taken from "Mutations of Nonconserved Residues within the Calcium Channel α-interaction Domain Inhibit β-Subunit Potentiation"</p><p></p><p>The Journal of General Physiology 2008;132(3):383-395.</p><p>Published online Jan 2008</p><p>PMCID:PMC2518731.</p><p></p

Inhibition of platelet procoagulant activity induced by <i>E</i>. <i>coli</i> O111 by αTF MoAb and TFPI.

<p>Leukocyte-free platelets (L-FP) were pre-incubated with either αTF MoAb or TFPI, before stimulation with <i>E</i>. <i>coli</i> O111 or VWF+Ris (positive control). Inhibition of TF reduce the generation of FXa by the strain O111 around 40% (*p = 0.031, n = 6) and, when platelets were activated with VWF+Ris, the inhibition was about 30% (*p = 0.031, n = 5) (A). Data analyzed with Wilcoxon signed rank test, two tails. Similar results were observed in L-FPs incubated with TFPI before the stimulation with <i>E</i>. <i>coli</i> or VWF+Ris, reaching levels of inhibition of 50% (*p 0.03, n = 4) and 60% (*p 0.04, n = 5), respectively (B). The data were analyzed by Mann-Whitney test.</p

<i>E</i>. <i>coli</i> O111 induced platelet-dependent TF procoagulant activity, involving TLR4 activation.

<p><i>E</i>. <i>coli</i> O111 increased FXa generation compared to N-S platelets (***p = 0.0005, n = 8) in assays without external source of TF and phospholipids. The amount of FXa generated was not significantly different than that of platelets stimulated with VWF+Ris (**p = 0.004, n = 8). TLR4 inhibition resulted in a ±-50% reduction in the generated FXa with strain O111 (**p = 0.004, n = 8), but did not influence the FXa generated by VWF+Ris activation of platelets (A). TRAP stimulated platelets did not generated FXa (B). Data analyzed by Paired t test, two-tailed. Pre-incubation of platelets with an inhibitory α-GPIbα MoAb (AP1) did not affect the FXa generated by <i>E</i>. <i>coli</i> but instead, reduce significantly the FXa triggered in platelets with VWF+Ris (*p = 0.03, n = 6) (C). Pre-incubation of platelets with a non-immune irrelevant IgG did not induced significant changes in FXa generation when stimulated with bacteria or VWF+Ris (n = 4) (C). Data were analyzed with Wilcoxon signed rank test, two tails.</p

<i>E</i>. <i>coli</i> O111 induces platelet TF-dependent thrombin generation, involving TLR4 activation.

<p>This thrombin generation assay did not use an external source of TF and phospholipids, and FcγRIIA was previously blocked with FcR blocking reagent. The interaction of platelets with <i>E</i>. <i>coli</i> O111 shortened the Lag Time (*p < 0.016, n = 7) (A) and increased the Velocity index of TG-PRP (* p< 0.016, n = 7) (B) compared with N-S platelets. This effect was similar to that obtained after PRP stimulation with ristocetin (used as positive control) or the unrelated strain <i>E</i>. <i>coli</i> DH5α, which also shortened Lag Time (* p <0.01, n = 3) and increased the Velocity index of TG-PRP (* p < 0.03, n = 3) (right panels figures A and B). TLR4 inhibition (along with FcγR previously blocked) prolonged the Lag Time (*p = 0.016, n = 7) and reduced the Velocity Index (*p = 0.016, n = 7) of TG in PRP incubated with the strain O111. Nevertheless, this inhibition did not have effect in Lag Time nor Velocity Index when PRP was stimulated with ristocetin or the strain DH5α (right panels figures A and B). Figure 2C shows no significant changes in TG-PRP generated by the strain O111 when a non-immune IgG was used (n = 4). The data was analyzed by Wilcoxon signed rank test, two-tailed.</p

P-selectin expression in platelets stimulated by <i>E</i>. <i>coli</i> O111.

<p>Human platelets were activated with <i>E</i>. <i>coli</i> for 30 min at 37°C, in a ratio 1:10 (platelet: bacteria). The CD62P expression induced by the strain O111 was significantly higher compared with non-stimulated platelets (*p = 0.03, n = 6), but lower than the response to TRAP (**p = 0.004, n = 5). Previous inhibition of platelet TLR4, reduced significantly the exposure of P-selectin induced by the bacteria (*p = 0.03, n = 6). TLR4 inhibition had no effect on P-selectin expression in TRAP-stimulated platelets (A). Platelets in PRP, stimulated with LPS (10μg/mL) did not show signs of activation (n = 5) (B). The data was analyzed by Wilcoxon signed rank test, two-tailed.</p