rabbit anti ca v 1 2 Search Results


rabbit polyclonal anti-ca v 1.2 (phosphor-ser1928  (Covalab Inc)
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Covalab Inc rabbit polyclonal anti-ca v 1.2 (phosphor-ser1928
Calcium influx through Ca V 1.2 induces PKA-II activity during depolarization. (A) Expression pattern of VGCC α subunits in overnight cultures of rat and mouse DRG determined by RNA-seq . TPM, transcripts per kilobase million. (B) Expression pattern of VGCC α subunits in mouse DRG neuron subgroups determined by single-cell RNA-seq . (C) Time course of pRII intensity in KCl-depolarized rat sensory neurons after pretreatment (10 min) with the NMDA receptor antagonist D-AP5 (10 µM), the Cav3.1-3.3 blocker TTA-P2 (1 µM), and a combination of the Cav2.1/2.2 blocker ω-agatoxin IVA (100 nM), ω-conotoxin MVIIC (200 nM), and ω-conotoxin GVIA (1 µM). (D) Inhibitory effect of verapamil (20 or 200 µM, 10-min pretreatment) on the pRII increase induced by KCl depolarization. (E) Dose–response curve showing the effect of verapamil (0–200 µM; IC 50 = 16 µM) on pRII signals induced by KCl depolarization (3 min). (F) Inhibitory effect of diltiazem (100 µM, 10 min) on the KCl-induced pRII increase. (G) Dose–response curve showing the inhibition of KCl-induced pRII signals by diltiazem (0–200 µM; IC 50 = 37 µM). (H) Reinforcing effect of the Ca V 1 agonist (S)-(-)-Bay K 8644 (2 µM, 10 min) on pRII signals induced by a low dose of KCl (10 mM). (I) Dose–response curve showing the reinforcing effect of Bay K 8644 (0–5 µM; EC 50 = 80 nM) on pRII signals induced by KCl (10 mM, 3 min). (J) Chelation of extracellular calcium with EGTA (2.5 mM, 30-min prestimulation) abolished the pRII response to KCl-depolarization. (K) Effect of the cell-permeable calcium chelator BAPTA-AM (100 µM, 60 min) on pRII signals induced by KCl depolarization (compound effect: F 1,28 = 10.9, P < 0.003). Values in C–K represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; § , P < 0.05; §§ , P < 0.01; §§§ , P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an agonist/antagonist.
Rabbit Polyclonal Anti Ca V 1.2 (Phosphor Ser1928, supplied by Covalab Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Calcium influx through Ca V 1.2 induces PKA-II activity during depolarization. (A) Expression pattern of VGCC α subunits in overnight cultures of rat and mouse DRG determined by RNA-seq . TPM, transcripts per kilobase million. (B) Expression pattern of VGCC α subunits in mouse DRG neuron subgroups determined by single-cell RNA-seq . (C) Time course of pRII intensity in KCl-depolarized rat sensory neurons after pretreatment (10 min) with the NMDA receptor antagonist D-AP5 (10 µM), the Cav3.1-3.3 blocker TTA-P2 (1 µM), and a combination of the Cav2.1/2.2 blocker ω-agatoxin IVA (100 nM), ω-conotoxin MVIIC (200 nM), and ω-conotoxin GVIA (1 µM). (D) Inhibitory effect of verapamil (20 or 200 µM, 10-min pretreatment) on the pRII increase induced by KCl depolarization. (E) Dose–response curve showing the effect of verapamil (0–200 µM; IC 50 = 16 µM) on pRII signals induced by KCl depolarization (3 min). (F) Inhibitory effect of diltiazem (100 µM, 10 min) on the KCl-induced pRII increase. (G) Dose–response curve showing the inhibition of KCl-induced pRII signals by diltiazem (0–200 µM; IC 50 = 37 µM). (H) Reinforcing effect of the Ca V 1 agonist (S)-(-)-Bay K 8644 (2 µM, 10 min) on pRII signals induced by a low dose of KCl (10 mM). (I) Dose–response curve showing the reinforcing effect of Bay K 8644 (0–5 µM; EC 50 = 80 nM) on pRII signals induced by KCl (10 mM, 3 min). (J) Chelation of extracellular calcium with EGTA (2.5 mM, 30-min prestimulation) abolished the pRII response to KCl-depolarization. (K) Effect of the cell-permeable calcium chelator BAPTA-AM (100 µM, 60 min) on pRII signals induced by KCl depolarization (compound effect: F 1,28 = 10.9, P < 0.003). Values in C–K represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; § , P < 0.05; §§ , P < 0.01; §§§ , P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an agonist/antagonist.

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: Calcium influx through Ca V 1.2 induces PKA-II activity during depolarization. (A) Expression pattern of VGCC α subunits in overnight cultures of rat and mouse DRG determined by RNA-seq . TPM, transcripts per kilobase million. (B) Expression pattern of VGCC α subunits in mouse DRG neuron subgroups determined by single-cell RNA-seq . (C) Time course of pRII intensity in KCl-depolarized rat sensory neurons after pretreatment (10 min) with the NMDA receptor antagonist D-AP5 (10 µM), the Cav3.1-3.3 blocker TTA-P2 (1 µM), and a combination of the Cav2.1/2.2 blocker ω-agatoxin IVA (100 nM), ω-conotoxin MVIIC (200 nM), and ω-conotoxin GVIA (1 µM). (D) Inhibitory effect of verapamil (20 or 200 µM, 10-min pretreatment) on the pRII increase induced by KCl depolarization. (E) Dose–response curve showing the effect of verapamil (0–200 µM; IC 50 = 16 µM) on pRII signals induced by KCl depolarization (3 min). (F) Inhibitory effect of diltiazem (100 µM, 10 min) on the KCl-induced pRII increase. (G) Dose–response curve showing the inhibition of KCl-induced pRII signals by diltiazem (0–200 µM; IC 50 = 37 µM). (H) Reinforcing effect of the Ca V 1 agonist (S)-(-)-Bay K 8644 (2 µM, 10 min) on pRII signals induced by a low dose of KCl (10 mM). (I) Dose–response curve showing the reinforcing effect of Bay K 8644 (0–5 µM; EC 50 = 80 nM) on pRII signals induced by KCl (10 mM, 3 min). (J) Chelation of extracellular calcium with EGTA (2.5 mM, 30-min prestimulation) abolished the pRII response to KCl-depolarization. (K) Effect of the cell-permeable calcium chelator BAPTA-AM (100 µM, 60 min) on pRII signals induced by KCl depolarization (compound effect: F 1,28 = 10.9, P < 0.003). Values in C–K represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; § , P < 0.05; §§ , P < 0.01; §§§ , P < 0.001 indicate significance levels between KCl-induced pRII signals in the absence or presence of an agonist/antagonist.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1:2,000; Novus; #NB110-58872), rabbit monoclonal anti-RIIα (phospho-Ser96; 1:1,000, clone 151; Abcam; #ab32390), mouse monoclonal anti-RIIβ (1:2,000; BD Transduction Laboratories; #610625), mouse monoclonal anti-NF200 (1:1,000, clone N52; Sigma; #N0142), mouse monoclonal IgG2a anti-CGRP (1:500, clone 4901; Biorbyt/Biozol; #orb319478), mouse monoclonal anti-NaV1.8 (1:500; NeuroMab; clone N134/12, #75-166), rabbit polyclonal anti-Ca V 1.2 (phosphor-Ser1928; 1:1,000; Covalab; #pab0692), mouse monoclonal anti-Ca V 1 (1:100, clone L57/46; NeuroMab; catalog no. 75–053), mouse monoclonal anti-Ca V 1.2 (1:100, clone N263/31; NeuroMab; catalog no. 73–257), highly cross-adsorbed Alexa Fluor 647–, 555–, and 488–conjugated secondary antibodies (Invitrogen).

Techniques: Activity Assay, Expressing, RNA Sequencing Assay, Inhibition

Deletion of Ca V 1.2 in NaV1.8 + nociceptors reduces PKA-II activity. (A) Expression pattern of Na V 1.8 (Scn10a), Ca V 1.2 (Cacna1c), and RIIβ (Prkar2b) in subgroups of DRG neuron determined by single-cell RNA-seq . (B) Conditional mouse model to delete Ca V 1.2 in Na V 1.8-expressing DRG neurons. (C and D) Distribution of UCHL1 and RIIβ expression levels in DRG neurons of cKO mice and respective controls lacking Cre recombinase (Ctrl). (E) Total numbers of viable DRG neurons after overnight culture determined by HCS microscopy ( n = 3 females and 2 males per genotype). (F) Single-cell data and mean intensities obtained using a Ca V 1.2-specific antibody (clone N263/31) indicating down-regulation of Ca V 1.2 ( n = 10 cultures from three mice per genotype, >6,000 neurons per genotype, Student’s t test). The primary antibody was omitted in respective controls (w/o AB). (G) Basal genotype difference of pRII intensity in all solvent stimulated RIIβ + control neurons ( n = 3 females and 2 males per genotype, >15,000 neurons/condition, Student’s t test). (H) Dose-dependent induction of pRII intensity by KCl (0–80 mM) in cKO and Ctrl mice ( n = 3 females and 2 males per genotype; genotype effect: F 4,62 = 5.6, extra-sum-of-squares F test). (I) Dose-dependent induction of pRII intensity by 10 mM KCl after 10 min preincubation with (S)-(-)-Bay K 8644 (0–5 µM) in cKO and Ctrl mice. ( n = 3 females and 2 males per genotype; genotype effect: F 4,62 = 13.9, extra-sum-of-squares F test). (J) Single-cell data of selected condition shown in I. Values in G–I are means ± SEM; *, P < 0.05; ***, P < 0.001.

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: Deletion of Ca V 1.2 in NaV1.8 + nociceptors reduces PKA-II activity. (A) Expression pattern of Na V 1.8 (Scn10a), Ca V 1.2 (Cacna1c), and RIIβ (Prkar2b) in subgroups of DRG neuron determined by single-cell RNA-seq . (B) Conditional mouse model to delete Ca V 1.2 in Na V 1.8-expressing DRG neurons. (C and D) Distribution of UCHL1 and RIIβ expression levels in DRG neurons of cKO mice and respective controls lacking Cre recombinase (Ctrl). (E) Total numbers of viable DRG neurons after overnight culture determined by HCS microscopy ( n = 3 females and 2 males per genotype). (F) Single-cell data and mean intensities obtained using a Ca V 1.2-specific antibody (clone N263/31) indicating down-regulation of Ca V 1.2 ( n = 10 cultures from three mice per genotype, >6,000 neurons per genotype, Student’s t test). The primary antibody was omitted in respective controls (w/o AB). (G) Basal genotype difference of pRII intensity in all solvent stimulated RIIβ + control neurons ( n = 3 females and 2 males per genotype, >15,000 neurons/condition, Student’s t test). (H) Dose-dependent induction of pRII intensity by KCl (0–80 mM) in cKO and Ctrl mice ( n = 3 females and 2 males per genotype; genotype effect: F 4,62 = 5.6, extra-sum-of-squares F test). (I) Dose-dependent induction of pRII intensity by 10 mM KCl after 10 min preincubation with (S)-(-)-Bay K 8644 (0–5 µM) in cKO and Ctrl mice. ( n = 3 females and 2 males per genotype; genotype effect: F 4,62 = 13.9, extra-sum-of-squares F test). (J) Single-cell data of selected condition shown in I. Values in G–I are means ± SEM; *, P < 0.05; ***, P < 0.001.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1:2,000; Novus; #NB110-58872), rabbit monoclonal anti-RIIα (phospho-Ser96; 1:1,000, clone 151; Abcam; #ab32390), mouse monoclonal anti-RIIβ (1:2,000; BD Transduction Laboratories; #610625), mouse monoclonal anti-NF200 (1:1,000, clone N52; Sigma; #N0142), mouse monoclonal IgG2a anti-CGRP (1:500, clone 4901; Biorbyt/Biozol; #orb319478), mouse monoclonal anti-NaV1.8 (1:500; NeuroMab; clone N134/12, #75-166), rabbit polyclonal anti-Ca V 1.2 (phosphor-Ser1928; 1:1,000; Covalab; #pab0692), mouse monoclonal anti-Ca V 1 (1:100, clone L57/46; NeuroMab; catalog no. 75–053), mouse monoclonal anti-Ca V 1.2 (1:100, clone N263/31; NeuroMab; catalog no. 73–257), highly cross-adsorbed Alexa Fluor 647–, 555–, and 488–conjugated secondary antibodies (Invitrogen).

Techniques: Activity Assay, Expressing, RNA Sequencing Assay, Microscopy

CaV1-expression in neurons of cKO mice or after AAV-mediated knock down of Ca V 1.2. (A) Single-cell data and mean intensities obtained from Ctrl and cKO mice using a Ca V 1-specific antibody (clone L57/46; n = 10 cultures from three mice per genotype, >6,000 neurons per genotype, Student’s t test). (B and C) Single-cell data and mean intensities obtained using a Ca V 1-specific antibody (clone L57/46) at 8 div (B, n = 4, >4,000 neurons per condition) and 15 div (C, n = 4, >1,500 neurons per condition) after transduction with AAV-PHP.S expressing either a scrambled (Scr) or Cacna1c-specific shRNA, indicating down-regulation of Ca V 1 in GFP + neurons. The primary antibody was omitted in respective controls (w/o AB).

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: CaV1-expression in neurons of cKO mice or after AAV-mediated knock down of Ca V 1.2. (A) Single-cell data and mean intensities obtained from Ctrl and cKO mice using a Ca V 1-specific antibody (clone L57/46; n = 10 cultures from three mice per genotype, >6,000 neurons per genotype, Student’s t test). (B and C) Single-cell data and mean intensities obtained using a Ca V 1-specific antibody (clone L57/46) at 8 div (B, n = 4, >4,000 neurons per condition) and 15 div (C, n = 4, >1,500 neurons per condition) after transduction with AAV-PHP.S expressing either a scrambled (Scr) or Cacna1c-specific shRNA, indicating down-regulation of Ca V 1 in GFP + neurons. The primary antibody was omitted in respective controls (w/o AB).

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1:2,000; Novus; #NB110-58872), rabbit monoclonal anti-RIIα (phospho-Ser96; 1:1,000, clone 151; Abcam; #ab32390), mouse monoclonal anti-RIIβ (1:2,000; BD Transduction Laboratories; #610625), mouse monoclonal anti-NF200 (1:1,000, clone N52; Sigma; #N0142), mouse monoclonal IgG2a anti-CGRP (1:500, clone 4901; Biorbyt/Biozol; #orb319478), mouse monoclonal anti-NaV1.8 (1:500; NeuroMab; clone N134/12, #75-166), rabbit polyclonal anti-Ca V 1.2 (phosphor-Ser1928; 1:1,000; Covalab; #pab0692), mouse monoclonal anti-Ca V 1 (1:100, clone L57/46; NeuroMab; catalog no. 75–053), mouse monoclonal anti-Ca V 1.2 (1:100, clone N263/31; NeuroMab; catalog no. 73–257), highly cross-adsorbed Alexa Fluor 647–, 555–, and 488–conjugated secondary antibodies (Invitrogen).

Techniques: Expressing, Transduction, shRNA

Adenoviral knockdown of Ca V 1.2 reduces PKA-II activity. (A) Representative HCS microscopy images of mouse DRG neurons transduced with AAV-PHP.S-U6-shRNA:Scramble-CAG-GFP (Scr) or AAV-PHP.S-U6-shRNA:Cacna1c-CAG-GFP (Cac) to knock down Ca V 1.2. Neurons were transduced after overnight culture (1 div) and fixed 1 wk (8 div) or 2 wk (15 div) later. Cultures were immunolabeled for the neuronal marker UCHL1 and pRII to quantify PKA-II signaling activity. The expression of GFP indicated efficient transduction. Nuclei were stained with Hoechst 34580. Scale bar, 100 µm. (B and C) GFP expression levels in individual neurons (left) and mean numbers of GFP + neurons after 8 div ( n = 6, >6,000 neurons per condition, Student’s t test) as well as after 15 div ( n = 6, total of >1,000 neurons per condition, Student’s t test). (D and E) Single-cell data of all analyzed neurons (left) and mean intensities in GFP − and GFP + neurons (right) obtained using a Ca V 1.2-specific antibody (clone N263/31) at 8 div (D; n = 4, >4,000 neurons) and 15 div (E, n = 3, >1200 neurons) indicating down-regulation of Ca V 1.2 in GFP+ neurons. The primary antibody was omitted in respective controls (w/o AB). (F and G) Effect of AAV-mediated Ca V 1.2 knockdown on pRII intensity levels induced by 3-min stimulation with KCl at 8 div (F; n = 6, >6,000 neurons, two-way ANOVA with Bonferroni’s test) and 15 div (G, n = 6, >1,000 neurons per condition). (H) Cell density plots showing single-cell data of pRII intensities versus GFP expression after 15 div as shown in G. Values in B–G are means ± SEM; *, P < 0.05; ***, P < 0.001.

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: Adenoviral knockdown of Ca V 1.2 reduces PKA-II activity. (A) Representative HCS microscopy images of mouse DRG neurons transduced with AAV-PHP.S-U6-shRNA:Scramble-CAG-GFP (Scr) or AAV-PHP.S-U6-shRNA:Cacna1c-CAG-GFP (Cac) to knock down Ca V 1.2. Neurons were transduced after overnight culture (1 div) and fixed 1 wk (8 div) or 2 wk (15 div) later. Cultures were immunolabeled for the neuronal marker UCHL1 and pRII to quantify PKA-II signaling activity. The expression of GFP indicated efficient transduction. Nuclei were stained with Hoechst 34580. Scale bar, 100 µm. (B and C) GFP expression levels in individual neurons (left) and mean numbers of GFP + neurons after 8 div ( n = 6, >6,000 neurons per condition, Student’s t test) as well as after 15 div ( n = 6, total of >1,000 neurons per condition, Student’s t test). (D and E) Single-cell data of all analyzed neurons (left) and mean intensities in GFP − and GFP + neurons (right) obtained using a Ca V 1.2-specific antibody (clone N263/31) at 8 div (D; n = 4, >4,000 neurons) and 15 div (E, n = 3, >1200 neurons) indicating down-regulation of Ca V 1.2 in GFP+ neurons. The primary antibody was omitted in respective controls (w/o AB). (F and G) Effect of AAV-mediated Ca V 1.2 knockdown on pRII intensity levels induced by 3-min stimulation with KCl at 8 div (F; n = 6, >6,000 neurons, two-way ANOVA with Bonferroni’s test) and 15 div (G, n = 6, >1,000 neurons per condition). (H) Cell density plots showing single-cell data of pRII intensities versus GFP expression after 15 div as shown in G. Values in B–G are means ± SEM; *, P < 0.05; ***, P < 0.001.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1:2,000; Novus; #NB110-58872), rabbit monoclonal anti-RIIα (phospho-Ser96; 1:1,000, clone 151; Abcam; #ab32390), mouse monoclonal anti-RIIβ (1:2,000; BD Transduction Laboratories; #610625), mouse monoclonal anti-NF200 (1:1,000, clone N52; Sigma; #N0142), mouse monoclonal IgG2a anti-CGRP (1:500, clone 4901; Biorbyt/Biozol; #orb319478), mouse monoclonal anti-NaV1.8 (1:500; NeuroMab; clone N134/12, #75-166), rabbit polyclonal anti-Ca V 1.2 (phosphor-Ser1928; 1:1,000; Covalab; #pab0692), mouse monoclonal anti-Ca V 1 (1:100, clone L57/46; NeuroMab; catalog no. 75–053), mouse monoclonal anti-Ca V 1.2 (1:100, clone N263/31; NeuroMab; catalog no. 73–257), highly cross-adsorbed Alexa Fluor 647–, 555–, and 488–conjugated secondary antibodies (Invitrogen).

Techniques: Activity Assay, Microscopy, Transduction, shRNA, Immunolabeling, Marker, Expressing, Staining

PKA-dependent phosphorylation of Ser1928 regulates Ca V 1.2 gating. (A) Representative images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were labeled for UCHL1 to identify the neurons, phospho-Ser1928 of Ca V 1.2 (pCa V 1.2), and Ca V 1 channels (Ca V 1, clone L57/46). Green or red encircled objects indicate automatically selected or rejected objects, respectively. Scale bar, 100 µm. (B) Enlarged section demonstrating the modified image analysis to quantify in nuclear (orange) and cytoplasmic (blue) regions of neurons (green). (C) Cell density plots of single-cell data of pCa V 1.2/Ca V 1-labeled neurons stimulated with solvent control (Ctrl) or KCl (40 mM) for 1 min. The Spearman’s rank correlation coefficient (ρ) and its P value are shown. (D) Depolarization (40 mM KCl) induces Ca V 1.2 phosphorylation, which is inhibited by verapamil (VP; 200 µM, 10 min). (E) The Ca V 1 intensity is unchanged after depolarization. (F and G) KCl-induced increase of pCa V 1.2 intensity in cytoplasmic versus nuclear regions. (H) Effect of the PKA inhibitor H89 on the pCa V 1.2 increase induced by depolarization. (I) Effect of the calcineurin inhibitor FK506 on the of pCa V 1.2 increase induced by depolarization. (J) Inhibitory effect of the PKA inhibitor H89 (25 µM, 30 min) on the pRII increase induced by depolarization. (K) Dose–response of KCl (0–80 mM) in the absence or presence of H89 (25 µM, 30 min). Data in D–J represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions; § , P < 0.05; §§ , P < 0.01; §§§ , P < 0.001 indicate significance levels between the absence and presence of an agonist/antagonist.

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: PKA-dependent phosphorylation of Ser1928 regulates Ca V 1.2 gating. (A) Representative images of rat DRG neurons stimulated with solvent (Ctrl) or KCl (40 mM) for 1 min. Cultures were labeled for UCHL1 to identify the neurons, phospho-Ser1928 of Ca V 1.2 (pCa V 1.2), and Ca V 1 channels (Ca V 1, clone L57/46). Green or red encircled objects indicate automatically selected or rejected objects, respectively. Scale bar, 100 µm. (B) Enlarged section demonstrating the modified image analysis to quantify in nuclear (orange) and cytoplasmic (blue) regions of neurons (green). (C) Cell density plots of single-cell data of pCa V 1.2/Ca V 1-labeled neurons stimulated with solvent control (Ctrl) or KCl (40 mM) for 1 min. The Spearman’s rank correlation coefficient (ρ) and its P value are shown. (D) Depolarization (40 mM KCl) induces Ca V 1.2 phosphorylation, which is inhibited by verapamil (VP; 200 µM, 10 min). (E) The Ca V 1 intensity is unchanged after depolarization. (F and G) KCl-induced increase of pCa V 1.2 intensity in cytoplasmic versus nuclear regions. (H) Effect of the PKA inhibitor H89 on the pCa V 1.2 increase induced by depolarization. (I) Effect of the calcineurin inhibitor FK506 on the of pCa V 1.2 increase induced by depolarization. (J) Inhibitory effect of the PKA inhibitor H89 (25 µM, 30 min) on the pRII increase induced by depolarization. (K) Dose–response of KCl (0–80 mM) in the absence or presence of H89 (25 µM, 30 min). Data in D–J represent means ± SEM; n = 3–4 experiments; >2,000 neurons/condition; two-way ANOVA with Bonferroni’s test; **, P < 0.01; ***, P < 0.001 indicate significance levels between baseline and stimulated conditions; § , P < 0.05; §§ , P < 0.01; §§§ , P < 0.001 indicate significance levels between the absence and presence of an agonist/antagonist.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1:2,000; Novus; #NB110-58872), rabbit monoclonal anti-RIIα (phospho-Ser96; 1:1,000, clone 151; Abcam; #ab32390), mouse monoclonal anti-RIIβ (1:2,000; BD Transduction Laboratories; #610625), mouse monoclonal anti-NF200 (1:1,000, clone N52; Sigma; #N0142), mouse monoclonal IgG2a anti-CGRP (1:500, clone 4901; Biorbyt/Biozol; #orb319478), mouse monoclonal anti-NaV1.8 (1:500; NeuroMab; clone N134/12, #75-166), rabbit polyclonal anti-Ca V 1.2 (phosphor-Ser1928; 1:1,000; Covalab; #pab0692), mouse monoclonal anti-Ca V 1 (1:100, clone L57/46; NeuroMab; catalog no. 75–053), mouse monoclonal anti-Ca V 1.2 (1:100, clone N263/31; NeuroMab; catalog no. 73–257), highly cross-adsorbed Alexa Fluor 647–, 555–, and 488–conjugated secondary antibodies (Invitrogen).

Techniques: Labeling, Modification

Model of Ca V 1.2 regulation in DRG neurons. Depolarization of DRG neurons results in Ca V 1.2 and calcium-dependent activation of PKA-II. The induction of PKA-II activity is not sensitive to opioids or inhibitors of calcium-stimulated ACs, phosphodiesterases (PDEs), and cAMP antagonists such as Rp-cAMPS, indicating a cAMP-independent activation mechanism (red shadow). In addition, inhibition of CaMKII or ERK1/2 is not affecting the induction of PKA-II activity after depolarization, excluding that PKA-II is downstream of these kinases. Depolarization sensitizes Ca V 1.2 by PKA-dependent phosphorylation of Ser1928. Dephosphorylation of Ca V 1.2 by the phosphatase calcineurin (CaN) inactivates the channel. CsA, cyclosporine A; MOR, µ opioid receptor.

Journal: The Journal of Cell Biology

Article Title: Depolarization induces nociceptor sensitization by Ca V 1.2-mediated PKA-II activation

doi: 10.1083/jcb.202002083

Figure Lengend Snippet: Model of Ca V 1.2 regulation in DRG neurons. Depolarization of DRG neurons results in Ca V 1.2 and calcium-dependent activation of PKA-II. The induction of PKA-II activity is not sensitive to opioids or inhibitors of calcium-stimulated ACs, phosphodiesterases (PDEs), and cAMP antagonists such as Rp-cAMPS, indicating a cAMP-independent activation mechanism (red shadow). In addition, inhibition of CaMKII or ERK1/2 is not affecting the induction of PKA-II activity after depolarization, excluding that PKA-II is downstream of these kinases. Depolarization sensitizes Ca V 1.2 by PKA-dependent phosphorylation of Ser1928. Dephosphorylation of Ca V 1.2 by the phosphatase calcineurin (CaN) inactivates the channel. CsA, cyclosporine A; MOR, µ opioid receptor.

Article Snippet: The following antibodies were used in this study: chicken polyclonal anti-UCHL1 (1:2,000; Novus; #NB110-58872), rabbit monoclonal anti-RIIα (phospho-Ser96; 1:1,000, clone 151; Abcam; #ab32390), mouse monoclonal anti-RIIβ (1:2,000; BD Transduction Laboratories; #610625), mouse monoclonal anti-NF200 (1:1,000, clone N52; Sigma; #N0142), mouse monoclonal IgG2a anti-CGRP (1:500, clone 4901; Biorbyt/Biozol; #orb319478), mouse monoclonal anti-NaV1.8 (1:500; NeuroMab; clone N134/12, #75-166), rabbit polyclonal anti-Ca V 1.2 (phosphor-Ser1928; 1:1,000; Covalab; #pab0692), mouse monoclonal anti-Ca V 1 (1:100, clone L57/46; NeuroMab; catalog no. 75–053), mouse monoclonal anti-Ca V 1.2 (1:100, clone N263/31; NeuroMab; catalog no. 73–257), highly cross-adsorbed Alexa Fluor 647–, 555–, and 488–conjugated secondary antibodies (Invitrogen).

Techniques: Activation Assay, Activity Assay, Inhibition, De-Phosphorylation Assay