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

Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

<div><p>Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent <i>de novo</i> cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.</p></div

Amperometric parameters of exocytotic events induced by 10 µM DMPP in cells transfected with pEGFP, UnR-iRNA, iRNADyn2, Dyn2WT or Dyn2K44A.

<p>Data are means ± SEM of averages, where n is the number of cells. Imax corresponds to the spike amplitude.</p>*<p>p<0.05 compared with pEGFP-transfected cells;</p>†<p>p<0.05 compared with Dyn2 WT-transfected cells (Kruskal-Wallis test).</p

Impaired function or expression of dynamin-2 change F-actin organization pattern.

<p>Cells were transfected with Life-act-ruby (n = 11) or co-transfected with Life-act-ruby and pEGFP (n = 34), iRNA-UnR (n = 9) Dyn2WT (n = 21), Dyn2K44A (n = 31), iRNADyn2 (n = 38) or Eps15ED95/295 (n = 17) plasmids and visualized by TIRF microscopy 48 h later. To evaluate the effects of a pharmacological inhibition of dynamin, cells transfected with Life-act-ruby were treated with 100 µM dynasore (n = 28), or the vehicle DMSO (n = 25) during 1 hr at 37°C. The 81.8% of control cells exhibited a “normal” pattern with clear cortical actin fibers. This value was not significantly different in cells expressing pEGFP (73.6%), iRNA-UnR (88.9%) or Dyn2WT (85.7%) constructs. However, the expression of Dyn2K44A or iRNADyn2, as well as the treatment with dynasore, modified the cortical actin organization and 80.6%, 92.1% and 71.4% of the cells, respectively, exhibited a “punctuate” pattern. The treatment with 4 µM CytoD during 10 minutes at 37°C produced exactly the same effect: 84.6% of the cells displayed a “punctuate” pattern. Eps15ED95/295 expression did not alter actin organization (82.4% of cells exhibited a normal pattern), indicating that dynamin, but not of endocytosis disruption, modified the actin cytoskeleton pattern. Scale bar = 5 µm.</p

Dynamin-2 and actin polymerization regulate the fusion pore expansion and quantal size in BCC.

<p>Chromaffin cells were incubated with 4 µM CytoD during 10 minutes at 37°C. After that the exocytosis was evoked with 10 µM DMPP. <b>A–C:</b> Data show average values ± SEM of Q (A), t_1/2 (B) and foot duration (C) of amperometric spikes induced by 10 µM DMPP in cells transfected with pEGFP (n = 27), Dyn2K44A (n = 13) or iRNADyn2 (n = 16). All amperometric parameter values correspond to the median values of the events from individual cells, which were subsequently averaged per treatment group. Thus, n correspond to the number of cells in each treatment group. Note that the CytoD treatment (grey bars) significantly increased Q, t_1/2 and foot duration of the exocytotic events in cells transfected with pEGFP, without additional effects in cells transfected with Dyn2K44A or iRNADyn2. * p<0.05 compared with the untreated cells (Kruskal-Wallis test).</p

Inhibition of dynamin GTP-ase activity suppresses Ca²⁺-dependent <i>de novo</i> cortical actin polymerization.

<p><b>A:</b> Representative images of F-actin formation in cells permeabilized in the presence of 10 µM free Ca²⁺. Note that no new polymerized cortical actin was observed when the permeabilization was performed in the absence of ATP-Mg (n = 16) or in the presence of 4 µM CytoD (n = 27) or 100 µM dynasore (n = 28) Scale bar = 10 µm <b>B:</b> Quantification of G-actin staining mean intensity at the cell periphery. Data are means of cortical actin fluorescence intensity *p<0.05 compared with cells treated with DMSO (ANOVA).</p

Calcium-dependent cortical actin polymerization in permeabilized chromaffin cells.

<p>Cultured chromaffin cells were permeabilized in buffer KGEP (mM: 139 K⁺-glutamate, 20 Pipes, 5 EGTA, 2 ATP-Mg and 0.01 free calcium, pH 6.6) during 6 minutes with 20 µM digitonin in the presence of 0.3 µM Alexa-Fluor488-G-actin conjugate (AF488-G-actin), fixed and visualized by confocal microscopy. <b>A:</b> Total F-actin was stained using 1 µM phalloidin-rodhamine B (red) and nuclei were stained with 5 µg/ml DAPI (blue). Note that newly synthesized actin was incorporated into pre-existing cortical filaments. <b>B–C:</b> The new formation of cortical actin filaments was assessed by quantifying AF488-G-actin staining mean intensity at the cell periphery in the presence of increasing free Ca²⁺ concentrations. Note that maximal cortical actin polymerization was observed at a range of 1–10 µM of free Ca^2+. Scale = 10 µm. Data are means of cortical actin fluorescence intensity from at least 12 cells per each Ca²⁺ concentration (12 cells for 0.01 µM Ca²⁺, 13 cells for 0.1 µM Ca²⁺, 15 cells for 1 µM Ca²⁺,and 18 cells for 10 µM Ca²⁺).</p