Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: Quantification of Ser-1700 and Ser-1928 phosphorylation in cultured hippocampal neurons. Hippocampal neurons were extracted at 10 and 20 DIV, each sample was split into equal pairs, and Ca V 1.2 was immunoprecipitated. One sample of each pair but not the other was phosphorylated in vitro with purified PKA catalytic subunit under conditions that lead to near complete phosphorylation of the PKA site on Ca V 1.2, as quantified earlier . A, samples of sequential probing of immunoblots with antibodies against pSer-1928, pSer-1700, and finally total Ca V 1.2. B, immunosignals were quantified by film densitometry. pSer-1700 and pSer-1928 signals were corrected for differences in relative amounts of Ca v 1.2. Scatter plots show pSer-1700 and pSer-1928 levels detected in untreated samples as a fraction of the paired maximally phosphorylated samples. Of the Ser-1928 sites 18.4 ± 4.9% were phosphorylated in hippocampal cultures at 10 DIV and 12.1 ± 1.6% at 20 DIV. Of the Ser-1700 sites 21.6 ± 4.9% were phosphorylated at 10 DIV and 28.1 ± 3.8% at 20 DIV. All errors are given as S.D. Statistics: ANOVA: **, p = 0.03; n = 3 obtained in three independent experiments.

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Cell Culture, Immunoprecipitation, In Vitro, Purification, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: Distribution and size of clusters of membrane-expressed phosphomimetic Ca V 1.2-HA constructs. A and B, dendritic segments of neurons transfected with Ca V 1.2-HA, Ca V 1.2-HA-SE, Ca V 1.2-HA-RRQQ, or Ca V 1.2-HA-RRQQ-SE and immunolabeled with anti-HA antibody at DIV 10 ( A ) and DIV 20 ( B ) in non-permeabilized conditions. Co-expressed soluble eGFP outlines the morphology of the dendrites. All channel mutants distribute in clusters along the dendritic shafts ( arrows ) and on the spines ( arrowheads ) similar to the control Ca V 1.2-HA. Scale bar , 5 μm. C, topology of Ca V 1.2-HA indicating the position of the extracellular HA tag, the S1928E exchange (SE, blue circle ), and the double substitution R1696Q,R1697Q (RRQQ, green circle ), which prevents the inhibitory interaction within the C terminus . Yellow represents the Ca V 1.2-HA carrying the RRQQ and SE mutations (Ca V 1.2-HA-RRQQ-SE). D and F , quantification of density (number of clusters per spine or per surface unit of dendritic shaft) and the intensity (average gray values). E and G, Ca V 1.2-HA-SE, Ca V 1.2-HA-RRQQ, and Ca V 1.2-HA-RRQQ-SE expressed as percentage of control Ca V 1.2-HA. Cluster density increased along the shaft at DIV 10 for Ca V 1.2-HA-RRQQ-SE ( D , yellow scatter plot , p = 0.002) and at DIV20 for Ca V 1.2-HA-SE ( F , blue scatter plot , p = 0.008). Cluster fluorescence intensity augmented on the spines and along the dendritic shafts of young neurons expressing Ca V 1.2-HA-RRQQ ( E , green scatter plot , p = 0.004 and p = 0.017, respectively) and Ca V 1.2-HA-RRQQ-SE ( E , yellow scatter plot , p = 0.018 and p = 0.009, respectively). Statistics: Mann-Whitney rank sum test. Analyzed dendritic segments in at least three separate experiments: DIV 10, N CaV1.2-HA = 18–19 and N CaV1.2-HA-SE = 24–26, N CaV1.2-HA = 30 and N CaV1.2-HA-RRQQ = 23, N CaV1.2-HA = 18–19 and N CaV1.2-HA-RRQQ-SE = 15–18; DIV 20, N CaV1.2-HA = 19 and N CaV1.2-HA-SE = 17, N CaV1.2-HA = 26 and N CaV1.2-HA-RRQQ = 23, N CaV1.2-HA = 21–22 and N CaV1.2-HA-RRQQ-SE = 18–20. Scatter plots: mean ± S.D. **, 0.002 < p < 0.009 and *, 0.017 < p < 0.018.

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Construct, Transfection, Immunolabeling, Fluorescence, Expressing, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: FRAP analysis in cultured young and mature hippocampal neurons. Experiments were conducted on neurons overexpressing eGFP-Ca V 1.2 ( red ), eGFP-Ca V 1.2-SE ( blue ), eGFP-Ca V 1.2-RRQQ ( green ), or eGFP-Ca V 1.2-RRQQ-SE ( yellow ). A, C, and E, averaged FRAP curves of dendritic regions in young neurons (DIV 10) were higher for all mutants indicating faster dynamics. B, D , and F, FRAP curves of dendritic regions derived from mature neurons ( DIV 20). B, the lower fluorescence recovery of eGFP-Ca V 1.2-SE ( blue ) than control eGFP-Ca V 1.2 ( red ) indicates a slower dynamics and a reduced fraction of mobile channels. Slower dynamics was also observed at the rising phase of eGFP-Ca V 1.2-RRQQ-SE FRAP curve ( F ). “ A ” indicates the amplitude of curves calculated as mean of the last values of individual curves and then averaged. Two-tailed t test: *, 0.1< p < 0.5; **, 0.01 < p < 0.001; ***, 0.001 < p < 0.0001. Data were obtained from three experiments on at least three different cultures. The number of regions analyzed for each construct derived from at least 11 neurons is indicated in parentheses. Data are presented as mean ± S.E.

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Cell Culture, Derivative Assay, Fluorescence, Two Tailed Test, Construct

Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: Single particle tracking analysis of Ca V 1.2-HA, Ca V 1.2-HA-SE, Ca V 1.2-HA-RRQQ, and Ca V 1.2-HA-RRQQ-SE. A and D, representative reconstructed trajectories of QDs bound to channel mutants and superimposed to dendritic segments outlined with soluble eGFP. Bar , 5 μm. B and E, MSD curves of Ca V 1.2-HA ( red ), Ca V 1.2-HA-SE ( blue ), Ca V 1.2-HA-RRQQ ( green ), and Ca V 1.2-HA-RRQQ-SE ( yellow ) represented as mean ± S.E.; *, 0.01 < p < 0.04; **, 0.001 < p < 0.009; ***, p < 0.001. C and F, box plots of the diffusion coefficients (median ± interquartile range). Numerical and n values are provided in . Statistics: Shapiro-Wilk normality test failed with p < 0.05. Mann-Whitney rank sum test: *, p = 0.010; ***, p < 0.001.

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Single-particle Tracking, Diffusion-based Assay, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: Diffusion coefficients (Diff. Coeff.) of dendritic Ca V 1.2-HA, Ca V 1.2-HA-SE, Ca V 1.2-HA-RRQQ, and Ca V 1.2-HA-RRQQ-SE

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Diffusion-based Assay

Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: Proteolytic processing of the Ca V 1.2 C terminus upon activation of NMDA receptors. A and B , immunoblots of endogenous Ca V 1.2 in whole cell lysates from cultured neurons at DIV 10 ( A ) and 20 ( B ). Drug treatments are indicated at the top of each lane and were used previously ( , ). All scatter plots show the ratio between densitometric quantification of cleaved ( lower bands ) and full-length Ca V 1.2 ( upper bands ) in the experimental conditions corresponding to the lanes above. A, at DIV 10 the amount of cleaved Ca V 1.2 increased upon 30 min of 50 μ m NMDA and was inhibited by concomitant administration of dl -APV 100 μ m (APV in the figure). B, at DIV 20 a 5-min exposure to 50 μ m NMDA followed by return to drug-free medium for 30 min augmented the fraction of truncated Ca V 1.2. Co-administration of dl -APV abolished this result. C , immunoblot of biotinylated surface-exposed Ca V 1.2 extracted from mature cultured neurons treated for 1 h with vehicle (mock), bicuculline (40 μ m ), and bicuculline plus dl -APV (40 and 100 μ m , respectively). Bicuculline increased the fraction of cleaved Ca V 1.2 and dl -APV prevented this process. Scatter plots, mean ± S.D. n = 3 ( B and C ) to 4 ( A ), on 3–4 different cultures. Statistics: one-way ANOVA followed by Dunnett's multiple comparison test, * p < 0.05.

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Activation Assay, Western Blot, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: Molecular mimicking of C-terminal phosphorylation tunes the surface dynamics of Ca V 1.2 calcium channels in hippocampal neurons

doi: 10.1074/jbc.M117.799585

Figure Lengend Snippet: Model of Ca V 1.2 channels dynamics in dendrites of cultured hippocampal neurons. Full-length and truncated channels are present in neurons. Activation of NMDA receptors (NMDAR, yellow rectangle ) causes the cleavage of the Ca V 1.2 C terminus ( A ), which allows the PKA to phosphorylate Ser-1700 ( B ). Ser-1928 is also a substrate for PKA ( B ). The majority of channels is clustered in signaling complexes. Outside of clusters, mobile channels are diffusely distributed . In young neurons pSer-1700 and pSer-1928 promote the diffusive behavior of extra-clustered channels ( C ). In mature neurons only pSer-1700 favors the Ca V 1.2 diffusive state, whereas pSer-1928 stabilizes the channels at the clusters ( D ).

Article Snippet: After 1 h incubation with blocking buffer (20 m m Tris, pH 7.4, 150 m m NaCl, 0.1% Tween 20, and 4% dried nonfat milk (Regilait)) at room temperature, membranes were exposed to rabbit anti-Ca V 1.2 (Alomone, ACC-3300 ( )) (1:500) in blocking buffer overnight at 4 °C.

Techniques: Cell Culture, Activation Assay