Journal: PLoS ONE

Article Title: Fetal Calcium Regulates Branching Morphogenesis in the Developing Human and Mouse Lung: Involvement of Voltage-Gated Calcium Channels

doi: 10.1371/journal.pone.0080294

Figure Lengend Snippet: 5 µm-thick formalin-fixed, paraffin-embedded serial sections of 11 week post-conception human fetal lungs were dewaxed and used for immunohistochemistry. A: Expression of P/Q type, Ca v 2.1, and of T-type, Ca v 3.2, calcium channels could be detected at the basolateral side of epithelial cells and in smooth muscle cells, visualised using DAB (brown staining). Scale bar = 5000 µm. B,C: Higher magnification images (40x and 100x) show little-to-no expression of the N-type calcium channel, Ca v 2.2 or the T-type, Ca v 3.3 in the lung parenchyma. Negative controls were carried out through the substitution of the primary antibody with an isotype control. Sections were counterstained with Harris’ hematoxylin (blue staining). Scale bar = 1000 µm.

Article Snippet: Primary antibody suppliers and dilutions used were : Cav1.2 - rabbit polyclonal (Alomone Labs; mouse: 1/100, human: 1/50) Cav1.3 - rabbit polyclonal (Alomone Labs; mouse: 1/100, human: 1/50) Cav2.1 - rabbit polyclonal (Alomone Labs; mouse: 1/100, human: 1/100) Cav2.2 - rabbit polyclonal (Millipore; mouse: 1/100, human: 1/100) Cav2.3 - rabbit polyclonal (Abcam; mouse: 1/100, human: 1/100) Cav3.2 - goat polyclonal (N-18) (Santa Cruz; mouse: 1/100, human:1/100) Cav3.3 - goat polyclonal (N-20) (Santa Cruz; mouse: 1/100, human:1/100) Secondary antibody suppliers and dilutions were: goat anti-rabbit horse radish peroxidase (HRP) (Cav 1.2, 1.3, 2.1, 2.2, and 2.3; DAKO, Ely, U.K.; 1:200 for both mouse and human) or donkey anti-goat HRP (Cav 3.2 and 3.3; Abcam; 1:200 for both mouse and human).

Techniques: Formalin-fixed Paraffin-Embedded, Immunohistochemistry, Expressing, Staining

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: a Representative confocal image of a nerve terminal arborization. Singly, dually, and innervated by three or more axons NMJs from YFP muscles and also images of the morphologic maturation (S1, the most inmature, and S4, almost fully differentiated, stages) of the postsynaptic clusters from P9 mice. The bar indicates 10 μm. b Confocal immunofluorescence location of α 1D L-, N-, and P/Q-type voltage-dependent calcium channels (VDCCs) at the NMJ. Triple labeling of VDCCs (green fluorescence) with syntaxin (blue fluorescence) and nAChR-α-bungarotoxin (red fluorescence) in merge images. Figure shows the presence of α 1D L-, N-, and P/Q-type-VDCC (in green) in the nerve terminal of P9 Levator auris longus (LAL) muscle endplates. The bar indicates 10 μm

Article Snippet: Muscles were incubated overnight at 4 °C with anti-Ca V 1.3 (CACNA1D) antibody voltage-dependent L-type calcium channel subunit α 1D (1/100; ACC-005, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.1 (CACNA1A) antibody voltage-dependent P/Q-type calcium channel subunit α 1A (1/100; ACC-001, Alomone Labs, Jerusalem, Israel); anti-Ca V 2.2 (CACNA1B) antibody voltage-dependent N-type calcium channel subunit α 1B (1/100; ACC1-002, Alomone Labs, Jerusalem, Israel), and anti-mouse syntaxin (1/1000, S066, Sigma, St Louis, MO, USA).

Techniques: Immunofluorescence, Labeling, Fluorescence

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7α accelerates the onset of facilitation of Ca v 2.1 channels. Inset top, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV preconditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. A , Effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. B , in tsA-201 cells co-expressing Ca v 2.1 channel with Syt-7α, the slope is significantly increased compared with control cells. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 at physiological Ca 2+ levels. Inset top, Pulse protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Inset, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effect of Syt-7α on facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α on the decay from facilitation of Ca v 2.1 channels. Inset top, Pulse protocol for measuring decay of facilitation. Ca 2+ currents are elicited by test pulses to +10 mV before (P1) and after (P2) a conditioning prepulse to +10 mV for 5 ms. Inset bottom left, Decay from facilitation measured by comparing τ between control and Syt-7α transfected cells. Inset bottom right, Comparison of P2/P1 facilitation ratio at Δ t = 0 between control and Syt-7α-expressing cells. Main panel. Effect of Syt-7α on the decay from facilitation. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1 and was plotted against the interval between the conditioning prepulse and P2. Shown are results obtained with 50-ms conditioning prepulse. Graph shows the effect of Syt-7α on decay of facilitation as a function of interpulse duration. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7α potentiates Ca v 2.1 facilitation in a paired-pulse protocol following change in prepulse voltage. Inset top, Pulse protocol shown represents paired pulse protocol. Ca 2+ current was recorded using 10 m m Ca 2+ and 0.5 m m EGTA in the external and internal solutions, respectively. Pulse 1 (P1; depolarization from −80 to +10 mV) elicits the first Ca 2+ current. A second 5-ms pulse (P2) generating a second I Ca is applied 2 ms after a 50-ms conditioning prepulse with variable voltages (−40 to 60 mV). Inset bottom, Example traces from control and Syt-7α transfected tsA cells following P1 and P2 pulses. Main panel, Graph shows the effects of Syt-7α isoform on facilitation as a function of prepulse voltage. The facilitation ratio was obtained by normalizing the peak current from P2 to that from P1. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Transfection

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α on prepulse facilitation of Ca v 2.1 channel. Facilitation of voltage-dependent activation of Ca 2+ currents. Inset, Pulse protocol to study the voltage dependence of activation before (open circle or squares; P1) and after (closed circle or squares; P2) a depolarizing prepulse from −80 to +10 mV. Tail currents were measured by holding potential at −40 mV for 5 ms after test pulses (P1, P2) to variable voltages (−40 to +80 mV). Peak tail currents were normalized to the largest tail current measured during the nonfacilitated prepulses (P1) and plotted against the test pulse voltage. A , In control tsA cells, the protocol shows an increase in facilitation P2 normalized to P1. B , Syt-7α potentiated facilitation amplitude of Ca v 2.1 and induced a right shift in prepulse facilitation curve. C , Overlaying the two graphs in A , B shows the increase in amplitude of facilitation and the right shift in voltage dependency of activation. D , Difference in voltage shift in P1 and P2 between cells co-expressing Ca v 2.1 and Syt-7α and control cells. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques: Activation Assay, Expressing

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Effect of Syt-7α prepulse facilitation of Ca v 2.1 channel at physiological levels. Inset, Voltage protocol. Currents recorded with 2 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A - C , Effect of Syt-7α on facilitation as a function of prepulse voltage. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7β and Syt-7γ differentially modulate prepulse facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. A , Ca v 2.1 alone. B , Ca v 2.1 with Syt-7γ. C , Overlay of panels A , B . D , V 50 values for results in panel C . E , Ca v 2.1 alone. F , Ca v 2.1 with Syt-7γ. G , overlay of panels D , E . H , V 50 values from panel G . Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on the voltage dependence of facilitation of Ca v 2.1 channels (Extended Data ).

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7 isoforms differentially modulate Ca 2+ -dependent inactivation of Ca v 2.1 channels. Ca v 2.1 currents were elicited by depolarizing from a holding potential of −80 mV to a test potential of +10 mV. A , Time courses (200 ms) of I Ba with 10 m m Ba 2+ as a permeant cation. B , Time courses (1000 ms) of I Ca with 10 m m Ca 2+ as permeant ion. Syt-7α and Syt-7β significantly slowed inactivation of the Ca v 2.1 channel in the presence of 10 m m Ca 2+ , whereas Syt-7γ had no effect. Data are represented as mean ± SEM.

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: eNeuro

Article Title: Synaptotagmin-7 Enhances Facilitation of Ca v 2.1 Calcium Channels

doi: 10.1523/ENEURO.0081-22.2022

Figure Lengend Snippet: Syt-7β and Syt-7γ differentially modulate facilitation of Ca v 2.1 channels. Inset, Pulse protocol. Currents recorded with 10 m m extracellular Ca 2+ and 0.5 m m EGTA in the intracellular recording solution were elicited by test pulses to +10 mV before (P1) and 5 ms after (P2) 10-mV conditioning prepulses of the indicated durations. A , Left, Syt-7β increases the facilitation ratio with increasing prepulse duration. Right, Syt-7β accelerates the onset of facilitation as a function of prepulse duration. B , Left, Syt-7γ increases the facilitation ratio with increasing prepulse duration. Right, Syt-7γ does not accelerate the onset of facilitation as a function of prepulse duration. Facilitation was obtained by normalizing the peak current from P2 to that from P1. Single-exponential fits of the data are shown. Data are represented as mean ± SEM additional experiments with different pulse protocols provide additional information on the effects of Syt-7β and Syt-7γ on facilitation of Ca v 2.1 channels (Extended Data ).

Article Snippet: Proteins were blotted with antibodies against Syt-7 (mouse monoclonal antibody N275/14, Product Number MABN665, Millipore Sigma) or Ca v 2.1 (rabbit polyclonal antibody catalog #ACC-001, Alomone Labs).

Techniques:

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: Western blots and histograms of α1D-L-, N-, and P/Q VGCC proteins in the LAL muscle of mice during development (P5-P7-P30). The developmental change in the P/Q VGCC protein level is parallel with the changes observed in other presynaptic molecules (nPKCε isoform and Munc18-1). Data are mean value ± SD, * p < 0.05, ** p < 0.01, *** p < 0.005 ( n = 5; 3 repeats)

Article Snippet: The specificity of anti-α1D L, P/Q, and N VGCC antibodies used in this study has been tested using KO mice by Alomone Labs and validated also by some researchers.

Techniques: Western Blot

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: In (a) we show the percentage of singly- and polyinnervated NMJ after 4 applications over the LAL surface (one application every day between P5–P8 (observation at P9) of one of the following VGCC inhibitor substances: nitrendipine (NT 1 μM, an L-type channel blocker), ω-conotoxin-GVIA (ω-CON 1 μM, N-type channel blocker), and ω-agatoxin-IVA (ω-AGA 100 nM, P/Q-type blocker). Also, the L activator Bay-K8644 (5 μM), the P/Q- and N-type activator GV-58 (20 μM), and the intracellular calcium chelator BAPTA-AM (5 μM). The histogram in ( b ) shows the percentage of S1-S4 clusters in the untreated control mice (PBS) and after the 4 applications of the aforesaid substances. Data were presented as percentages of NMJ ± SD. Fisher’s test: * p < 0.05, ** p < 0.01, *** p < 0.005. The confocal images in (c) show examples of representative NMJ areas with singly, dually, and innervated by three or more axons (the corresponding number of asterisks) from YFP muscles. At the left, the L-type channel blocker nitrendipine (NT) delays axon loss because many multi-innervated NMJs persist. By the contrary, at the right, the L activator Bay-K8644 increases the number of monoinnervated junctions. The bar indicates 10 μm

Article Snippet: The specificity of anti-α1D L, P/Q, and N VGCC antibodies used in this study has been tested using KO mice by Alomone Labs and validated also by some researchers.

Techniques:

Journal: Molecular Neurobiology

Article Title: Involvement of the Voltage-Gated Calcium Channels L- P/Q- and N-Types in Synapse Elimination During Neuromuscular Junction Development

doi: 10.1007/s12035-022-02818-2

Figure Lengend Snippet: Graphic representation of the results. The activity-dependent signaling between the nerve terminals that are in competition through several metabotropic receptors can result in the modulation of the downstream effector kinases, specifically cPKCβI, nPKCε, and PKA. Changes in kinases activity can allow the coordinate phosphorylation of the L-type CaV1.3 and P/Q-type VGCC. The high calcium entry through these operative channels present in immature nerve endings can result in their final loss. Also, muscle CaV1.1 and contractile activity can contribute to the synapse elimination. A component of this mechanism may be mediated by a retrograde influence from the postsynaptic site, via the BDNF-TrkB pathway, on the presynaptic calcium channels

Article Snippet: The specificity of anti-α1D L, P/Q, and N VGCC antibodies used in this study has been tested using KO mice by Alomone Labs and validated also by some researchers.

Techniques: Activity Assay

Journal: Neuron

Article Title: Precision calcium imaging of dense neural populations via a cell body-targeted calcium indicator

doi: 10.1016/j.neuron.2020.05.029

Figure Lengend Snippet: Highlights

Article Snippet: Anti-Cav2.1 , Alomone , ACC-001, RRID:AB_2039764.

Techniques: Recombinant, Transfection, Plasmid Preparation, Clone Assay, Sequencing, Software

Journal: Neuron

Article Title: Precision calcium imaging of dense neural populations via a cell body-targeted calcium indicator

doi: 10.1016/j.neuron.2020.05.029

Figure Lengend Snippet: Highlights

Article Snippet: Anti-Cav2.1, Alomone (ACC-001) at 1:250; anti-rabbit IgG (H+L) Alexa 647 (A-21245) at 1:1000.

Techniques: Recombinant, Transfection, Plasmid Preparation, Clone Assay, Sequencing, Software