ca v 2 1  (Addgene inc)

Journal: Science Advances

Article Title: α-Synuclein expression is required for somatodendritic dopamine release and immediate early gene induction

doi: 10.1126/sciadv.ady6978

Figure Lengend Snippet: ( A ) Representative single-channel currents recorded in a cell-attached configuration from cultured WT and SKO SN DA neurons in the presence of N-, P/Q-, and R-type channel blockers show unitary currents (downward deflections) due to LTCC openings elicited by a voltage ramp (bottom trace). ( B ) Single-channel current amplitude was identical for LTCC in WT and SKO mice (ns by two-way ANOVA; n = 8 WT and 9 SKO). ( C ) Ensemble average P O - V relationships show no change in the probability of channel openings between WT and SKO neurons (ns by two-way ANOVA). ( D ) Population data confirm no change in maximal open channel probability (ns by t test). ( E and G ) Representative confocal images of cultured hippocampal neurons (14 days postplating) from WT and SKO mice immunostained for α1C (E) or α1D (G) subunits of the LTCC, as well as αSyn and pan-neuronal microtubule-associated protein 2 (MAP2). Note that staining intensity of the α1D subunit was low, making the quantification of the signal less reliable. Scale bar, 10 μm. ( F and H ) Analysis of average cytosolic and membrane α1C (F) and α1D (H) staining intensity ( n = 70 to 78 cells in each group from three independent experiments. * P < 0.05, ** P < 0.01, or **** P < 0.0001 by t test).

Article Snippet: Primary antibodies include anti-actin (mouse monoclonal, Sigma-Aldrich, #A5441, RRID: AB_476744; 1:1000), anti–glyceraldehyde-3-phosphate dehydrogenase (mouse monoclonal, Proteintech, #60004-1-Ig, RRID: AB_2107436; 1:1000), anti–pCREB (Ser 133 , rabbit monoclonal, Cell Signaling Technology, #9198S; 1:500), anti-CREB1 (rabbit polyclonal, ABclonal, #A11064, RRID: AB_2758389; 1:500), anti–c-Fos (rabbit monoclonal, Cell Signaling Technology, #2250S, RRID: AB_2247211; 1:1000), anti-Ca v 1.2 α1C (rabbit polyclonal, Proteintech, #21774-1-AP, RRID: AB_2878918; 1:500), and anti-Ca v 1.3 α1D (rabbit polyclonal, Alomone Labs, #ACC-005, RRID: AB_2039775; 1:100).

Techniques: Cell Culture, Staining, Membrane

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Structural representation of DI–DII PD interface in Ca V 2.1-WT and Ca V 2.1-T698F mutant. Possible π-H and π-π quadrangle interaction in Ca V 2.1-T698F mutant shown as dotted lines. (B) Aligned amino acid sequences of DIIS6, DIIP2, and DIP1 PD helices from human (h) and mouse (m) L-type (Ca V 1.1–1.4) and Ca V 2 (2.1–2.3) channels. Conserved and unique residues participating in π-H and π-π quadrangle interactions are highlighted. (C) Representative whole-cell current traces of Ca V 2.1-WT, Ca V 2.1-T698F, and Ca V 1.2-F737T mutants coexpressed with β 1 b in tsA-201 cells before (P1) or after (P2) DPP to 100 mV. Voltage protocol is represented in dotted box above. (D) Mean fitted plots of Ca V 2.1-WT, Ca V 2.1-T698F, and Ca V 1.2-F737T mutants in response to DPP ranging from 0 to 180 mV. Cell numbers are represented in brackets. (E) Bar plots display the maximum VDF at 120 mV DPP for Ca V 2.1-WT, Ca V 2.1-T698F, and Ca V 1.2-F737T mutants. Unpaired Student’s t-test used statistical significance. All plots represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **p < 0.0001, ns, p > 0.05 (non-significant).

Article Snippet: In experiments involving P/Q-type currents, the cDNA of Ca V 2.1 (α 1A , human, Addgene ID: 140575 ) was co-transfected with β 1 b at a 1:0.75 ratio.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Aligned amino acid sequences of DIIS6, DIIP2, and DIP1 PD helices from rat (r) and rabbit (rb) L-type (Ca V 1.1–1.4) and Ca V 2 (2.1–2.3) channels. Conserved and unique residues participating in the π-H and π-π quadrangle interactions are highlighted. (B and C) Representative whole-cell current traces (top) with Current density (pA/pF) and normalized conductance (G/G max ) vs. voltage plots (below) of WT or mutants of Ca V 1.2/Ca V 2.1 channels. Voltage protocol is represented in dotted box above. Insets display half-maximal activation voltages. (D and E) Normalized current traces (left) of WT and mutant Ca V 1.2/Ca V 2.1 channels at 0 mV showing channel inactivation. Time constant of inactivation (τ) plots (right) from exponential fits of 0 mV traces. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. *p ≤ 0.05, **p ≤ 0.01, ns, non-significant.

Article Snippet: In experiments involving P/Q-type currents, the cDNA of Ca V 2.1 (α 1A , human, Addgene ID: 140575 ) was co-transfected with β 1 b at a 1:0.75 ratio.

Techniques: Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b subunit in tsA-201 cells recorded in external solutions stored in glass bottles (black) or PP tubes (magenta). Voltage protocol is represented in dotted box above. (C and D) Current density (pA/pF) and normalized conductance (G/G max ) versus voltage plots of Ca V 1.2 (C) and Ca V 1.3 (D) show no significant differences when recorded in solutions stored in glass bottles (black) and PP tubes (magenta). Insets show half-maximal activation voltages. (E and F) Normalized current traces of Ca V 1.2 (E) and Ca V 1.3 (F) at 0 mV show channel inactivation in glass (black) or PP tubes (magenta). Insets display time constants of inactivation (τ) from exponential fits of 0 mV traces. R 400 plots represent the ratio of residual current at 400 msec to the peak current amplitude at voltage steps ranging from -50 to +40 mV. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b and α 2 δ 1 subunit in tsA-201 cells recorded in external solutions stored in glass bottles (black) or PP tubes (magenta). Voltage protocol is represented in dotted box above. (C and D) Current density (pA/pF) and normalized conductance (G/G max ) versus voltage plots of Ca V 1.2 (C) and Ca V 1.3 (D) show no significant differences when recorded in solutions stored in glass bottles (black) and PP tubes (magenta). Insets show half-maximal activation voltages. (E and F) Normalized current traces of Ca V 1.2 (E) and Ca V 1.3 (F) at 0 mV show channel inactivation in glass (black) or PP tubes (magenta). Insets display time constant of inactivation (τ) from exponential fits of 0 mV traces. R 400 plots represent the ratio of residual current at 400 msec to the peak current amplitude at voltage steps ranging from -50 to +40 mV. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of L-type Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b subunit in tsA-201 cells recorded in external solutions stored in glass bottles (black) or polypropylene (PP) tubes (magenta). Voltage protocol is represented in dotted box above. (C and D) Bar plot quantifications depict percentage facilitation of Ca V 1.2 (C) and Ca V 1.3 (D) whole-cell currents, measured by the difference between peak current amplitudes of P2 and P1 (P2-P1) voltage steps, in buffer solutions stored either in glass bottles (black) or PP tubes (magenta). Cell numbers represented in brackets. Two-way ANOVA used for statistical significance. (E and F) Exemplary cell-attached single-channel current traces of Ca V 1.2 coexpressed with the accessory subunit β 1 b in tsA-201 cells recorded in external solutions stored in glass bottles (E) or PP tubes (F) . Voltage protocol is represented in dotted box above. (G and H) Bar plots show open probability and dwell time analysis, while histogram plots display the single-channel current amplitudes of Ca V 1.2 cell-attached recordings obtained at 0 mV step of 2 sec in buffer solutions stored in glass bottles (G) or PP tubes (H) before (P1) or after (P2) the DPP to 100 mV for 100 msec. n = number of cells, N = number of single-channel traces. Paired Student’s t-test used for statistical significance. All plots show mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ns, p > 0.05 (non-significant).

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques:

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of L-type Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b and α 2 δ 1 subunits in tsA-201 cells recorded in external solutions stored in glass bottles (black) or PP tubes (magenta). Voltage protocol is represented in dotted box above. (C and D) Bar plot quantifications depict percentage facilitation of Ca V 1.2 (C) and Ca V 1.3 (D) whole-cell currents, measured as peak current difference between P2 and P1 voltage steps (P2-P1), in solutions stored in glass bottles (black) or PP tubes (magenta). Cell numbers represented in brackets. Two-way ANOVA used for statistical significance. (E) Bar plots depict percentage VDF (P2 over P1 at 0 mV after 120 mV DPP) for indicated L-type channel combinations. Unpaired Student’s t-test used for statistical significance. All plots represent mean ± SEM (cell numbers in brackets). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001; ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques:

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of L-type Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b subunit in tsA-201 cells recorded in external solutions containing 500 nM 2,4-DTBP (magenta) or DMSO (black) before (P1) or after (P2) the DPP to 100 mV. Mean fitted plots (right) show maximum facilitation of Ca V 1.2 (A) and Ca V 1.3 (B) channel currents with 100, 250, or 500 nM 2,4-DTBP or DMSO control in response to DPP ranging from 0 to 180 mV. Voltage protocol is represented in dotted box above. Cell numbers are denoted in brackets. (C and D) Exemplary cell-attached single-channel current traces of Ca V 1.2 coexpressed with β 1 b subunit in tsA-201 cells recorded with DMSO (C) or 500 nM 2,4-DTBP (D) . Voltage protocol is represented in dotted box above. Bar plots below display open probability and dwell time analyses; histograms show single-channel current amplitudes before and after DPP (100 mV, 100 ms). n = number of cells, N = number of single-channel traces. Paired Student’s t-test used for statistical significance. All plots represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ns, p > 0.05 (non-significant).

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Control

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of L-type Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b subunit in tsA-201 cells recorded in external solutions containing 1 μM 1,3-DTBB (red), DEHP (green), or 2,4-DTBP (magenta), or DMSO (black). Voltage protocol is represented in dotted box above. (C and D) Bar plot quantifications depict VDF percentage of Ca V 1.2 (C) and Ca V 1.3 (D) whole-cell currents, measured by the difference between peak current amplitudes of P2 and P1 (P2-P1) voltage steps, in external solutions containing 1,3-DTBB (red), DEHP (green), or 2,4-DTBP (magenta), or DMSO (black). Two-way ANOVA used for statistical significance. All plots represent mean ± SEM (cell numbers in brackets). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques:

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of L-type Ca V 1.2 (A) and Ca V 1.3 (B) channels coexpressed with β 1 b and α 2 δ 1 subunits in tsA-201 cells recorded in external solutions containing DMSO or 500 nM 2,4-DTBP before (P1) or after (P2) the DPP to 100 mV. Mean fitted plots (below) show maximum facilitation of Ca V 1.2 (A) and Ca V 1.3 (B) channel currents with DMSO, or 2,4-DTBP at 100, 250, or 500 nM in response to DPP ranging from 0 to 180 mV. Voltage protocol is represented in dotted box above (C and D) Representative whole-cell current traces of Ca V 1.2 ( C ) and Ca V 1.3 ( D ) channels coexpressed with β 1 b and α 2 δ 1 subunits in tsA-201 cells recorded in external solutions containing DMSO (black) or 500 nM 2,4-DTBP (magenta). Voltage protocol is represented in dotted box above. Current density (pA/pF) and normalized conductance (G/G max ) voltage plots (below) of Ca V 1.2 ( C ) and Ca V 1.3 ( D ) show no significant differences between DMSO control (black) vs 500 nM 2,4-DTBP (magenta). Insets display half-maximal activation voltages. (E and F) Normalized current traces of Ca V 1.2 ( E ) and Ca V 1.3 ( F ) at 0 mV show channel inactivation with DMSO (black) or 500 nM 2,4-DTBP (magenta). Insets display time constants of inactivation (τ) from exponential fits of 0 mV traces. R 400 plots represent the ratio of residual current at 400 msec to the peak current amplitude at voltage steps ranging from -50 to +40 mV. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Control, Activation Assay

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A and B) Representative whole-cell current traces of Ca V 1.2 ( A ) and Ca V 1.3 ( B ) channels coexpressed with β 1 b subunit in tsA-201 cells recorded in external solutions containing 500 nM 2,4-DTBP (magenta) or DMSO control (black). Voltage protocol is represented in dotted box above. (C and D) Current density (pA/pF) and normalized conductance (G/G max ) versus voltage plots of Ca V 1.2 ( C ) and Ca V 1.3 ( D ) show no significant differences between 500 nM 2,4-DTBP (magenta) and DMSO control (black). Insets display half-maximal activation voltages. (E and F) Normalized current traces of Ca V 1.2 ( E ) and Ca V 1.3 ( F ) at 0 mV show channel inactivation with DMSO (black) or 500 nM 2,4-DTBP (magenta). Insets display time constants of inactivation (τ) from exponential fits of 0 mV traces. R 400 plots represent the ratio of residual current at 400 msec to the peak current amplitude at voltage steps ranging from -50 to +40 mV. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Control, Activation Assay

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: ( A ). Side view (left) and top view (right) of human Ca V 1.2 α 1C protein backbone showing 2,4-DTBP binding at the DI–DII PD interface. (B) Zoomed view of 2,4-DTBP binding pocket at DI–DII fenestration. Key residues forming hydrophobic interactions and H-bonds with 2,4-DTBP in DIS6, DIS5, and DIIS6 are marked. (C) Root mean square deviation (RMSD) of protein backbone and 2,4-DTBP during MD simulation. (D) Time evolution of H-bond formation between 2,4-DTBP and polar N741 residue at the DIIS6 helix. (E) Zoomed view of DI–DII PD interface showing π-H and π-π quadrangle interactions between F357, F737, W708, and N741. (F) PCA of F737 and N741 side chains reveal distinct conformational clusters, with representative orientations shown below. (G) Centroid-to-centroid distances between F737-F357 and F737-N741 over simulation trajectories (above) with distance distributions represented as histograms (below). (H) Comparison of S6 helix movements with and without bound 2,4-DTBP.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Binding Assay, Residue, Comparison

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Schematic of DI–DII PD fenestration in Ca V 1.2 WT, F737A, F737Y, N741A, and N741Q mutants. Possible π-H and π-π quadrangle interactions shown as dotted lines. (B) Representative whole-cell current traces of Ca V 1.2 point mutants at DI–DII PD fenestration coexpressed with β 1 b in tsA-201 cells before (P1) or after (P2) DPP to 100 mV. (C) Mean fitted plots of Ca V 1.2 WT and DI–DII PD mutants coexpressed with β 1 b in response to DPP ranging from 0 to 180 mV. Cell numbers are denoted in brackets. (D) Bar plots depict maximum VDF at 120 mV DPP for Ca V 1.2 WT and DI–DII PD mutants. Unpaired Student’s t-test used for statistical significance. (E) Cell-attached single-channel traces of Ca V 1.2-F737A and Ca V 1.2-F737Y mutants coexpressed with β 1 b before and after DPP to 100 mV. Bar plots below display open probability and dwell time analyses; histograms show single-channel current amplitudes before and after DPP. Voltage protocol is represented in dotted box. n = number of cells, N = number of single-channel traces. Paired Student’s t-test used for statistical significance. All plots represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p < 0.0001, ns, p > 0.05 (non-significant).

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques:

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Current density (pA/pF) vs. voltage plots of Ca V 1.2 WT and specified mutants. (B) Normalized conductance (G/G max ) vs. voltage plots of Ca V 1.2 WT and specified mutants. (C) Bar plots showing half-maximal activation voltages (V a ) for Ca V 1.2 WT and mutants. (D) Representative normalized whole-cell current traces of Ca V 1.2 WT and mutants at 0 mV, displaying inactivation differences. (E) Bar plot quantifications of the time constant of inactivation (τ) for Ca V 1.2 WT and mutants. All plots show mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns (non-significant).

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Activation Assay

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Structural representation of DII–DIII, DIII–DIV, and DI–DIV PD interfaces of Ca V 1.2 showing key aromatic and polar residues. (B) Mean fitted plots showing the maximum VDF of specified Ca V 1.2 mutant channels to DPP, ranging from 0-180 mV. (C) Bar plots show VDF quantifications of specified Ca V 1.2 mutants in comparison with Ca V 1.2-WT to DPP of 120 mV. (D) Bar plots comparing current density of specified Ca V 1.2 mutant channels vs WT. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001, ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Mutagenesis, Comparison

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Structural representation of DI–DII PD interface in Ca V 2.1-WT and Ca V 2.1-T698F mutant. Possible π-H and π-π quadrangle interaction in Ca V 2.1-T698F mutant shown as dotted lines. (B) Aligned amino acid sequences of DIIS6, DIIP2, and DIP1 PD helices from human (h) and mouse (m) L-type (Ca V 1.1–1.4) and Ca V 2 (2.1–2.3) channels. Conserved and unique residues participating in π-H and π-π quadrangle interactions are highlighted. (C) Representative whole-cell current traces of Ca V 2.1-WT, Ca V 2.1-T698F, and Ca V 1.2-F737T mutants coexpressed with β 1 b in tsA-201 cells before (P1) or after (P2) DPP to 100 mV. Voltage protocol is represented in dotted box above. (D) Mean fitted plots of Ca V 2.1-WT, Ca V 2.1-T698F, and Ca V 1.2-F737T mutants in response to DPP ranging from 0 to 180 mV. Cell numbers are represented in brackets. (E) Bar plots display the maximum VDF at 120 mV DPP for Ca V 2.1-WT, Ca V 2.1-T698F, and Ca V 1.2-F737T mutants. Unpaired Student’s t-test used statistical significance. All plots represent mean ± SEM. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, **p < 0.0001, ns, p > 0.05 (non-significant).

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Aligned amino acid sequences of DIIS6, DIIP2, and DIP1 PD helices from rat (r) and rabbit (rb) L-type (Ca V 1.1–1.4) and Ca V 2 (2.1–2.3) channels. Conserved and unique residues participating in the π-H and π-π quadrangle interactions are highlighted. (B and C) Representative whole-cell current traces (top) with Current density (pA/pF) and normalized conductance (G/G max ) vs. voltage plots (below) of WT or mutants of Ca V 1.2/Ca V 2.1 channels. Voltage protocol is represented in dotted box above. Insets display half-maximal activation voltages. (D and E) Normalized current traces (left) of WT and mutant Ca V 1.2/Ca V 2.1 channels at 0 mV showing channel inactivation. Time constant of inactivation (τ) plots (right) from exponential fits of 0 mV traces. All plots represent mean ± SEM (cell numbers in brackets). Unpaired Student’s t-test used for statistical significance. *p ≤ 0.05, **p ≤ 0.01, ns, non-significant.

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Activation Assay, Mutagenesis

Journal: bioRxiv

Article Title: L-type channel voltage-dependent facilitation results from asymmetric π-H and π-π quadrangle interactions at DI–DII domains

doi: 10.64898/2026.01.23.701029

Figure Lengend Snippet: (A) Cell-attached single-channel traces of Ca V 1.2-F737T mutant coexpressed with β 1 b before and after DPP to 100 mV. Voltage protocol is represented in dotted box. (B) Bar plots showing open probability and dwell time analyses; histograms display single-channel current amplitudes before and after DPP. n = number of cells, N = number of single-channel traces. Paired Student’s t-test was used for statistical significance. All plots represent mean ± SEM. ns, p > 0.05 (non-significant).

Article Snippet: 20-24 hr after seeding, 2-3 μg of cDNAs of wild type (WD) or point mutated α1 subunit of L-type channel isoform Ca V 1.2 (α 1C , mouse, Addgene ID: 26572, ) or Ca V 1.3 (α 1D , rat, Addgene ID: 49332, ) in pcDNA6 backbone, was co-transfected with accessory subunits β 1 b (rat), β 2 a (rat), or α 2 δ 1 (rat) in pcDNA3.1 , into tsA201 cells in varied combinations, specifically mentioned in the results section.

Techniques: Mutagenesis

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