Journal: bioRxiv

Article Title: Glucocorticoids rapidly modulate Ca V 1.2-mediated calcium signals through Kv2.1 channel clusters in hippocampal neurons

doi: 10.1101/2023.12.24.573220

Figure Lengend Snippet: (A) Left, representative I Ba traces induced by a depolarization pulse from −90 to 0 mV under the vehicle control (0.01% DMSO) condition and subsequently in the presence of 1μM corticosterone in the same hippocampal neuron. Right, statistical analysis of the effect of corticosterone on I Ba using a two-tailed paired t -test (n = 5). ****P = 2.8E-5. (B) The time course of the I Ba inhibition by corticosterone. (C) Left, representative current traces show the inhibitory effect of 1μM corticosterone on I K . Right, statistics for the amplitude of I K from Left using a two-tailed paired t- test (n = 14). ****P = 3.2E-4. (D) Left, representative I K traces induced by a depolarization pulse from −80 to +50 mV under the vehicle control condition and subsequently in the presence of 1μM corticosterone in the same hippocampal neuron. Right, plot of I K activation curves in the DMSO (n = 9 for each data point; blue) and corticosterone-treated (n = 9 for each data point; pink) groups. (E) Left, representative western blot of the Kv2.1 channel surface expression after 10-min corticosterone treatment (1μM) in the extracellular solution in cultured hippocampal neurons. Right, statistics from 14 independent experiments using a two-tailed unpaired t -test. **** P = 2.2E-9. Na + -K + ATPase as a membrane protein loading control. (F) Left, representative western blot of the Ca V 1.2 channel surface expression after 10-min corticosterone treatment (1μM) in the extracellular solution in cultured hippocampal neurons. Right, statistics from 10 independent experiments using a two-tailed unpaired t- test. ****P = 1.7E-7.

Article Snippet: The primary antibodies used were mouse anti-Kv2.1 (1:500, ab192761, Abcam) and rabbit Ca V 1.2 (1:500, ab234438, Abcam), and they were incubated with the cells for 1 day at 4°C in a solution containing 1% horse serum and 0.3% PBST.

Techniques: Two Tailed Test, Inhibition, Activation Assay, Western Blot, Expressing, Cell Culture, Membrane

Journal: bioRxiv

Article Title: Glucocorticoids rapidly modulate Ca V 1.2-mediated calcium signals through Kv2.1 channel clusters in hippocampal neurons

doi: 10.1101/2023.12.24.573220

Figure Lengend Snippet: (A) Left, representative I Ba traces induced by a depolarization pulse from −90 to 0 mV under the control (0.01% DMSO) condition and subsequently in the presence of 1 μM cortisol in the same HEK293 cell. Right, statistics for the amplitude of Kv2.1 current from Left using a two-tailed paired t- test (n = 7). ***P = 8.2E-6. (B) I-V curve of Ca V 1.2 currents in the absence or presence of 1 μM cortisol in HEK293 cell. (C) 1 μM cortisol did not alter the steady-state activation of Ca V 1.2 currents. (D) Left, representative western blot of the Ca V 1.2 channel surface expression after 10-min cortisol treatment (1μM) in the extracellular solution in HEK293 cells. Right, statistics from 4 independent experiments using a two-tailed unpaired t- test. n.s., not significant (P = 0.1143).

Article Snippet: The primary antibodies used were mouse anti-Kv2.1 (1:500, ab192761, Abcam) and rabbit Ca V 1.2 (1:500, ab234438, Abcam), and they were incubated with the cells for 1 day at 4°C in a solution containing 1% horse serum and 0.3% PBST.

Techniques: Two Tailed Test, Activation Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: Glucocorticoids rapidly modulate Ca V 1.2-mediated calcium signals through Kv2.1 channel clusters in hippocampal neurons

doi: 10.1101/2023.12.24.573220

Figure Lengend Snippet: (A) Left, representative western blot of the Ca V 1.2 channel surface expression after 10-min cortisol treatment (1μM) in the extracellular solution in HEK293 cells expressing both Ca V 1.2 and Kv2.1 channels. Right, statistics from 5 independent experiments using a two-tailed unpaired t- test. ***P = 0.0004, **P = 0.00197. (B) Left, representative I Ba traces induced by a depolarization pulse from −90 to 0 mV under the control condition and subsequently in the presence of 1μM cortisol in the same HEK293 cell. Right, statistics for the amplitude of Kv2.1 current from Left using a two-tailed paired t- test (n = 5). ***P = 3.4E-5. (C) Representative western blots (left) and statistical analysis (right) show the effect of cortisol on the Ca V 1.2 channel surface expression when Ca V 1.2 and Kv2.1S586A channels are co-expressed in HEK293 cells. Two-tailed unpaired t- test, n = 6, P = 0.4339.

Article Snippet: The primary antibodies used were mouse anti-Kv2.1 (1:500, ab192761, Abcam) and rabbit Ca V 1.2 (1:500, ab234438, Abcam), and they were incubated with the cells for 1 day at 4°C in a solution containing 1% horse serum and 0.3% PBST.

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Glucocorticoids rapidly modulate Ca V 1.2-mediated calcium signals through Kv2.1 channel clusters in hippocampal neurons

doi: 10.1101/2023.12.24.573220

Figure Lengend Snippet: (A) Extracellular application of 1 μM corticosterone rapidly reduced cAMP levels in cultured hippocampal neurons (n = 3, ***P < 0.001, one-way ANOVA with Bonferroni post hoc test). (B) Left, representative Western blot of the PKA phosphorylation level after 10-min corticosterone treatment in cultured hippocampal neurons. Right, statistics from 7 independent experiments using a two-tailed unpaired t -test. *P = 0.0101. (C) Representative western blots (left) and statistical analysis (right) showing the effect of PKA inhibitor H89 and agonist Forskolin (Fsk) on the Kv2.1 channel surface expression in cultured hippocampal neurons. n = 6, ****P < 0.0001, **p = 0.0034, n.s., not significant (p = 0.5948), one-way ANOVA with Bonferroni post hoc test. (D) Left, representative Ca V 1.2 current traces induced by a depolarization pulse from −90 to 0 mV under the control condition and subsequently in the presence of 1μM H89 in the same HEK293 cell. Right, statistics for the amplitude of Ca V 1.2 current from Left using a two-tailed paired t- test (n = 10). ****P <0.0001.

Article Snippet: The primary antibodies used were mouse anti-Kv2.1 (1:500, ab192761, Abcam) and rabbit Ca V 1.2 (1:500, ab234438, Abcam), and they were incubated with the cells for 1 day at 4°C in a solution containing 1% horse serum and 0.3% PBST.

Techniques: Cell Culture, Western Blot, Two Tailed Test, Expressing

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Three-dimensional (3D) model structure of the Ca v 1.2 channel with the folded N-terminal structure. b 3D model structure of the Ca v 1.2 channel with the CaM-binding structure, shown in complex with the N-lobe of CaM. c Enlarged view around the alanine 36 (A36) residue of the folded N-terminal structure. The A36 site is highlighted by green dotted circles. The N-terminal spatial Ca 2+ -transforming element (NSCaTE) region (47–68), Ca 2+ , and CaM are indicated in yellow, orange, and magenta, respectively. Molecular graphics were created using UCSF Chimera . d A schematic illustration of the hypothesis that the A36V mutation attenuates Ca 2+ -dependent inactivation (CDI) by conformational equilibrium shift favoring the folded structure.

Article Snippet: After pretreatment, cells were incubated overnight with rabbit polyclonal anti-Ca v 1.2 antibody (1:1000, Alomone Labs) in blocking solution, and then stained for 1 hour with an AlexaFluor 488-conjugated anti-rabbit IgG goat antibody (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) in PBS.

Techniques: Binding Assay, Mutagenesis

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Sanger sequencing results for the de novo variant p.A36V (left) and schematic illustration of the primary structure of the Ca v 1.2 channel (right). The red asterisk indicates the A36V mutation near the N-terminal spatial Ca 2+ -transforming element (NSCaTE). b The N-terminal amino acid sequences for Ca v 1.2 channels (short isoforms). The A36V mutation and the A39V Brugada mutation are indicated in red letters. c Expression of wild-type (WT) and A36V Ca v 1.2 channels in HEK293T cells as detected by anti-Ca v 1.2 antibody. d Membrane localization of WT and A36V Ca v 1.2 channels overexpressed in BHK cells. The plasma membrane was visualized by membrane-tethering red fluorescent protein (RFP-KRasCT). e The fluorescence intensity profiles of the line shown in Fig. 1d. f Plasma membrane to cytoplasm intensity ratio of Ca v 1.2. Statistical comparison was performed by two-tailed Welch’s t test (n.s., not significant). Data are presented as mean ± s.e.m.

Article Snippet: After pretreatment, cells were incubated overnight with rabbit polyclonal anti-Ca v 1.2 antibody (1:1000, Alomone Labs) in blocking solution, and then stained for 1 hour with an AlexaFluor 488-conjugated anti-rabbit IgG goat antibody (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) in PBS.

Techniques: Sequencing, Variant Assay, Mutagenesis, Expressing, Fluorescence, Two Tailed Test

Journal: Translational Psychiatry

Article Title: Identification of ultra-rare disruptive variants in voltage-gated calcium channel-encoding genes in Japanese samples of schizophrenia and autism spectrum disorder

doi: 10.1038/s41398-022-01851-y

Figure Lengend Snippet: a Families of Ba 2+ currents evoked by 30-ms depolarizing pulses from −30 to 60 mV with increments of 10 mV for wild-type (WT) and A36V neuronal Ca v 1.2 channels. b Current density–voltage ( I – V ) relationships. Data are expressed as mean ± s.e.m., WT: n = 18, A36V: n = 12. The values of G, Erev, V 0.5 , and k were −0.40, 63.0 mV, 7.6 mV, and 5.6 mV for WT channels, and −0.50, 61.3 mV, 6.7 mV, and 4.9 mV for A36V Ca v 1.2 channels. c Inactivation curves for WT (○, n = 9) and A36V (●, n = 4) neuronal Ca v 1.2 channels. Data are expressed as mean ± s.e.m. The values of V 0.5 , and k were (respectively) −37.6 mV and 11.5 mV for WT channels, and −41.6 mV and 12.1 mV for A36V Ca v 1.2 channels. d , g Ca 2+ -dependent inactivation (CDI) of neuronal ( d ) and cardiac ( g ) Ca v 1.2 channels. Ba 2+ (blue) and Ca 2+ (black) currents evoked by 350-ms step depolarization to 30 mV were normalized at their peak current amplitudes for WT and A36V Ca v 1.2 channels. e , f, h, i , Ratios of current amplitude to the peak amplitude were plotted against depolarizing time in the Ba 2+ ( e, h ) and the Ca 2+ ( f, i ) external solutions. The numbers of recorded cells were 10 and 15 for WT and A36V neuronal Ca v 1.2 channels ( e , f ), and 8 and 6 for WT and A36V cardiac Ca v 1.2 channels ( h – i ), respectively. Statistical comparison was performed by two-tailed non-paired Student’s t test (* p < 0.05). Data are presented as mean ± s.e.m.

Article Snippet: After pretreatment, cells were incubated overnight with rabbit polyclonal anti-Ca v 1.2 antibody (1:1000, Alomone Labs) in blocking solution, and then stained for 1 hour with an AlexaFluor 488-conjugated anti-rabbit IgG goat antibody (1:1000, Thermo Fisher Scientific) and Hoechst 33342 (Thermo Fisher Scientific) in PBS.

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: Selective posttranslational inhibition of Ca V β 1 -associated voltage-dependent calcium channels with a functionalized nanobody

doi: 10.1038/s41467-022-35025-7

Figure Lengend Snippet: a Cartoon depicting hypothesized association of Ca V 1.2 with β 2b subunits in adult ventricular myocytes (i) and the expected loss of β 2b with gene knockout of this subunit (ii). Bottom, knock out of β 2 in adult heart has only minimal impact on whole-cell Ca 2+ current making it ambiguous whether most Ca V 1.2 are associated with β 2b in adult ventricular cardiomyocytes. Posttranslational inhibition of cardiac Ca V 1.2 using a Ca V β-targeted nanobody fused to Nedd4L HECT (Ca V -aβlator) domain eliminates Ca V 1.2 complexes from the membrane and abolishes current (iii), proving that Ca V 1.2 is stably associated with β 2 in adult cardiomyocytes. b Schematic of a scenario where distinct Ca V β isoforms preferentially associate with particular α 1 -subunit types to mediate different functions (i). Knockdown of one β-isoform may lead to β reshuffling that lessens the functional impact of elimination of the particular Ca V β subunit (ii). By contrast, targeted posttranslational inhibition of the channel complex based on the β isoform would yield a qualitatively different result that more accurately reflects the functional logic of Ca V β molecular diversity in the cell (iii).

Article Snippet: Cells were then incubated with rabbit anti-Ca V 1.2 primary antibody (Alomone Labs, 1:1000) in PBS containing 1% NGS, 1% BSA, and 0.1% BSA overnight at 4 °C.

Techniques: Gene Knockout, Knock-Out, Inhibition, Stable Transfection, Functional Assay

Journal: Nature Communications

Article Title: Selective posttranslational inhibition of Ca V β 1 -associated voltage-dependent calcium channels with a functionalized nanobody

doi: 10.1038/s41467-022-35025-7

Figure Lengend Snippet: a Schematic of skeletal muscle Cav1.1 complex. b Images of isolated flexor digitorum brevis (FDB) fibers either untransfected (top) or transfected with Chisel-1-P2A-CFP (bottom). c Top, exemplar whole-cell currents from isolated FDB fibers expressing CFP (left) or Chisel-1 (right). Bottom, Population J-V curves from isolated FDB fibers expressing CFP (black squares; n = 13 over 3 independent experiments) or Chisel-1 (red squares; n = 13 over 3 independent experiments). d Top, exemplar gating currents from isolated FDB fibers expressing CFP (left) or Chisel-1 (right). Bottom, Population Q-V curves from isolated FDB fibers expressing CFP (black circles; n = 7 over 2 independent experiments) or Chisel-1 (red circles; n = 8 over 2 independent experiments). * P = 9.32 × 10 −5 compared to CFP control, two-tailed unpaired t test. e Schematic of ventricular cardiomyocyte Ca V 1.2 complex. f Confocal images of cardiomyocytes expressing mCherry ( top ) or Chisel-1-P2A-mCherry (bottom). g Population J-V curves from isolated ventricular myocytes expressing mCherry (black triangles; n = 13 over 3 independent experiments) or Chisel-1 (red triangles; n = 11 over 3 independent experiments). h Top, exemplar whole-cell currents from ventricular myocytes expressing mCherry (left) or Chisel-1 (right) before (black) and after (cyan) application of 1 μM forskolin. Bottom, lack of effect of Chisel-1 on forskolin induced increase in I Ca,L in ventricular myocytes (mCherry, n = 14; Chisel-1, n = 5). Data are means ± SEM. Source data are provided as a Source Data file.

Article Snippet: Cells were then incubated with rabbit anti-Ca V 1.2 primary antibody (Alomone Labs, 1:1000) in PBS containing 1% NGS, 1% BSA, and 0.1% BSA overnight at 4 °C.

Techniques: Isolation, Transfection, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: a CYFIP1 RNA immunoprecipitation (RNA-IP) from DIV 3 WT cortical neurons. Histogram showing relative enrichment of the mRNAs over the non-specific IgG, measured by RT-qPCR of the eluate. The values were normalized for the input and mHprt1 mRNA and expressed as fold change over the non-specific IgG of each mRNA ( n = 4 embryos; mean ± SEM; One-Way ANOVA p < 0.0001; mMap1b mRNA p = 0.0390, mCacna1c mRNA p = 0.0054, mCacna1e mRNA p = 0.0078, mCacna1i mRNA p = 0.0009, mCacng2 mRNA p = 0.9997, mCacnb3 mRNA p = 0.9983). b Total mRNA levels of the Ca 2+ channels in DIV 3 WT and Cyfip1 +/- cortical neurons. Histograms represent mCacna1c , mCacna1e , mCacna1i , mCacng2, mCacnb3 and mCyfip1 mRNA levels, normalized to mH3f3 levels and expressed as a fold change over WT (WT n = 6/7 embryos, Cyfip1 +/- n = 7 embryos; mean ± SEM; Two-tailed Multiple Mann-Whitney test, mCacna1c mRNA p = 0.0766, mCacna1e mRNA p = 0.0435, mCacna1i mRNA p = 0.0202, mCacng2 mRNA p = 0.6282, mCacnb3 mRNA p = 0.5343, mCyfip1 mRNA p = 0.0034). c Left, representative Western Blot showing CYFIP1, Ca V 1.2 (CACNA1C), Ca V 2.3 (CACNA1E), Ca V 3.3 (CACNA1I), Ca V γ2 (CACNG2/Stargazin) and Ca V β3 (CACNB3) in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. The molecular weight of each protein is indicated in kDa. Right, histogram representing Ca V 1.2, Ca V 2.3, Ca V 3.3, Ca V γ2, Ca V β3 and CYFIP1 protein expression levels in membrane-enriched fractions from WT and Cyfip1 +/- DIV 3 cortical neurons. Protein levels were normalized to Coomassie staining (WT n = 4 embryos, Cyfip1 +/- n = 7/8 embryos; mean ± SEM; Two-tailed Multiple unpaired t -test, Ca V 1.2 p = 0.0338, Ca V 2.3 p = 0.0281, Ca V 3.3 p = 0.0129, Ca V γ2 p = 0.2574, Ca V β3 p = 0.6259, CYFIP1 p = 0.0137). d–f Representative images from WT and Cyfip1 +/- DIV 3 cortical neurons stained for Ca V 1.2, Ca V 2.3, Ca V 3.3 (magenta) and βIII-Tubulin (green) (scale bar 20 μm). Histograms show the fluorescence intensity of each calcium channel normalized to βIII-Tubulin in the total neuron (left) and in the axon (right), expressed as a percentage over WT (Ca V 1.2: WT n = 4 embryos, Cyfip1 +/- n = 5 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.1111, axon p = 0.4127; Ca V 2.3: WT n = 5 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0635, axon p = 0.0159; Ca V 3.3: WT n = 4 embryos, Cyfip1 +/- n = 4 embryos; mean ± SEM; Two-tailed Mann-Whitney test, total p = 0.0286, axon p = 0.0286). Source data are provided as a Source Data file.

Article Snippet: Membranes were incubated with the following antibodies rabbit anti-CYFIP1 (1:1000; Sigma-Aldrich, #AB6046), rabbit anti-Ca V 1.2 (CACNA1C) (1:500, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:500, Alomone Labs, #ACC006), rabbit anti-Ca V 3.3 (CACNA1I) (1:500, Alomone Labs, #ACC009), rabbit anti-Stargazin (Ca V γ2/CACNG2) (1:500, Alomone Labs, #ACC012) and rabbit anti-Ca V β3 (CACNB3) (1:500, Alomone Labs, #ACC008), HuD/Elavl4 (1:500, Santa Cruz, #sc-48421) and HuR/Elavl1 (1:500, Santa Cruz, #sc-5261).

Techniques: RNA Immunoprecipitation, Quantitative RT-PCR, Two Tailed Test, MANN-WHITNEY, Western Blot, Membrane, Molecular Weight, Expressing, Staining, Fluorescence

Journal: Nature Communications

Article Title: CYFIP1 governs the development of cortical axons by modulating calcium availability

doi: 10.1038/s41467-025-65801-0

Figure Lengend Snippet: CYFIP1, potentially interacting with the RNA binding proteins HuD and HuR (previously identified as CYFIP1 interactors), is implicated in regulating the mRNA stability of calcium channel subunits Cacna1c , Cacna1e and Cacna1i . In Cyfip1 +/- neurons, reduced CYFIP1 levels result in a decrease protein abundance of the regulated calcium channel subunits, consequently leading to a decrease in intracellular and mitochondrial calcium concentration. Low levels of calcium ions may affect mitochondria polarity and motility, both of which we found impaired in Cyfip1 +/- axons. The decreased calcium concentration and the mitochondrial defects concur in reducing axonal growth observed in Cyfip1 +/- neurons. By restoring the intracellular calcium homeostasis, both the axonal growth and mitochondrial defects are rescued. Created in BioRender. https://BioRender.com/xquv8cy .

Article Snippet: Membranes were incubated with the following antibodies rabbit anti-CYFIP1 (1:1000; Sigma-Aldrich, #AB6046), rabbit anti-Ca V 1.2 (CACNA1C) (1:500, Alomone Labs, #ACC003), rabbit anti-Ca V 2.3 (CACNA1E) (1:500, Alomone Labs, #ACC006), rabbit anti-Ca V 3.3 (CACNA1I) (1:500, Alomone Labs, #ACC009), rabbit anti-Stargazin (Ca V γ2/CACNG2) (1:500, Alomone Labs, #ACC012) and rabbit anti-Ca V β3 (CACNB3) (1:500, Alomone Labs, #ACC008), HuD/Elavl4 (1:500, Santa Cruz, #sc-48421) and HuR/Elavl1 (1:500, Santa Cruz, #sc-5261).

Techniques: RNA Binding Assay, Quantitative Proteomics, Concentration Assay