ca v 1 3  (Addgene inc)

Journal: Frontiers in Synaptic Neuroscience

Article Title: Patterned pre-sensory spontaneous activity drives the structural refinement of developing cochlear ribbon synapses

doi: 10.3389/fnsyn.2026.1730181

Figure Lengend Snippet: 2D-STED nanoscopy reveals a positive correlation between the sizes of ribbons and Ca v 1.3 clusters. (A) Representative examples of single optical sections of IHC synapses stained with antibodies against Ca v 1.3 (green; KO-verified) and CtBP2 (magenta) visualized using 2-color 2D-STED nanoscopy, while Homer1 (blue) – indicating the PSD context – was acquired in confocal mode. The individual image dimensions are 1 × 1 μm. (B) Presynaptic ribbon and Ca v 1.3 cluster sizes obtained from 2D-STED images display a moderate positive correlation as assessed by CtBP2 and Ca v 1.3 areas. The red line indicates a linear regression with the associated 95% confidence interval. N animals = 3, n ribbons = 99; r s = 0.35, *** p = 0.0004.

Article Snippet: For all conditions except Ca v 1.3 -KO, datasets were analyzed in Imaris x64 9.6.1, with 3D reconstructions of ribbon puncta generated using the surface algorithm with local background subtraction enabled.

Techniques: Staining

Journal: Frontiers in Synaptic Neuroscience

Article Title: Patterned pre-sensory spontaneous activity drives the structural refinement of developing cochlear ribbon synapses

doi: 10.3389/fnsyn.2026.1730181

Figure Lengend Snippet: Effects of acute pharmacological inhibition and genetics-based functional disruption of murine auditory ribbon synapses. (A) Representative temporal maximum intensity projections of GCaMP6 fluorescence in afferent SGN terminals over a 10 min timelapse interval in an organotypically-cultured organ of Corti of early postnatal Snap25-T2A-GCaMP6s-D mice (P5DIV1). Pharmacological block of presynaptic Ca v 1.x-mediated Ca 2+ influx by application of isradipine (Isra., 10 μM) abolished the spontaneous synaptic activity detected in the postsynaptic SGNs; panels display a representative time interval prior (A) and post (A’) isradipine application. Here, across the analysis time window, early Ca 2+ waves are represented in dark blue, late activity waves in yellow – the latter are completely absent upon isradipine treatment, thus illustrating complete block of synaptic transmission in this condition upon wash-in of the drug. Scale bar: 10 μm. (B) Normalized GCaMP6 fluorescence intensity plots of four different organ of Corti cultures treated with isradipine, prior and post application. (B’) Quantification of the peak frequency in SGN terminal GCaMP6 fluorescence, showing a dramatic reduction of activity bursts upon isradipine application. ( N animals = 4, n Corti = 4, imaged prior and post application). Paired Student’s t -test: ** p < 0.01. (C,C’) Representative maximum projections of confocal z -stacks of WT organ of Corti cultures treated with either (C) vehicle (DMSO) or (C’) 10 μM isradipine for 1 h at P5DIV1. IHCs were stained against Parvalbumin (blue), RibeyeA (green) and PSD95 (magenta). Scale bars: 5 μm. (D) Average ribbon counts in vehicle- and isradipine-treated IHCs at P5DIV1 ( N animals = 4, n Corti = 4 for both conditions) reveal no statistically significant differences between treatments. Two-way ANOVA with Holm-Šídák’s multiple comparisons correction: n.s. = no significant. (E,E’) Violin plots showing (E) unchanged integrated fluorescence intensity of presynaptic ribbons, but (E’) an increase in PSD integrated fluorescence intensity in isradipine-treated IHCs at P5DIV1 after 1 h of treatment. N animals = 4, n Corti = 4, n ribbons = 2,524, n PSD = 1,038 for vehicle- and N animals = 4, n Corti = 4, n ribbons = 2,713, n PSD = 1,076 for isradipine-treated conditions for both conditions. Median is indicated by solid lines inside the violin plots, while upper and lower quartiles are indicated by dashed lines. Mann–Whitney U -test: *** p < 0.001. (F,F’) Representative maximum projections of confocal z -stacks of (F) WT and (F’) Ca v 1.3 -KO organ of Corti cultures at P5DIV1. IHCs were stained against Parvalbumin (blue), RibeyeA (green) and PSD95 (magenta). Scale bars: 5 μm. (G) Quantification of average ribbon counts reveals a decrease in cytosolic ribbons in Ca v 1.3 -KO IHCs at P5DIV1 ( N animals = 3, n Corti = 6 for both genotypes). Two-way ANOVA with Holm-Šídák’s multiple comparisons correction: *** p < 0.001. (H,H’) Violin plots reveal (H) a decrease in ribbon integrated fluorescence intensity and (H’) an increase in PSD integrated fluorescence intensity in Ca v 1.3 -KO IHCs at P5DIV1. N animals = 3, n Corti = 6, n ribbons = 2,555, n PSD = 1,575 for each WT and N animals = 3, n Corti = 6, n ribbons = 2,555, n PSD = 1,466 for Ca v 1.3 -KOs. Median is indicated by solid lines inside the violin plots, while upper and lower quartiles are indicated by dashed lines. Mann–Whitney U -test: *** p < 0.001. (I,I’) Representative maximum projections of confocal z -stacks of (I) WT and (I’) Otof -KO organ of Corti cultures at P5DIV1. IHCs were stained against Parvalbumin (blue), RibeyeA (green) and PSD95 (magenta). Scale bars: 5 μm. (J) Quantification of synaptic as well as cytoplasmic ribbon counts reveals WT-like ribbon numbers in Otof -KO IHCs at P5DIV1 ( N animals = 3, n Corti = 6 for both genotypes). Two-way ANOVA with Holm-Šídák’s multiple comparisons correction. (K,K’) Violin plots showing (K) unchanged integrated fluorescence intensity in ribbons but (K’) an increase in PSD integrated fluorescence intensity in Otof -KO IHCs at P5DIV1. N animals = 3, n Corti = 6, n ribbons = 3,912, n PSD = 1,423 for each WT and N animals = 3, n Corti = 6, n ribbons = 3,105, n PSD = 1,064 Otof -KO. Median is indicated by solid lines inside the violin plots, while upper and lower quartiles are indicated by dashed lines. Mann–Whitney U -test: *** p < 0.001. Refer to for ribbon/PSD volume data.

Article Snippet: For all conditions except Ca v 1.3 -KO, datasets were analyzed in Imaris x64 9.6.1, with 3D reconstructions of ribbon puncta generated using the surface algorithm with local background subtraction enabled.

Techniques: Inhibition, Functional Assay, Disruption, Fluorescence, Cell Culture, Blocking Assay, Activity Assay, Transmission Assay, Staining, MANN-WHITNEY

Journal: Frontiers in Synaptic Neuroscience

Article Title: Patterned pre-sensory spontaneous activity drives the structural refinement of developing cochlear ribbon synapses

doi: 10.3389/fnsyn.2026.1730181

Figure Lengend Snippet: Summary of the observed structural changes of IHCs ribbon synapses following acute pharmacological, genetic, or optogenetic manipulation. Acute pharmacological inhibition of presynaptic Ca 2+ influx using the L-type calcium channel inhibitor isradipine, or functional disruption of exocytosis due to absence of Otoferlin, resulted in unchanged ribbon size but an increase in PSD size. Genetic ablation of Ca v 1.3 channels caused structural collapse of IHC ribbon size but also led to an increase in PSD size. Temporally-patterned optogenetic stimulation ( oSparse ) caused structural upscaling of both, ribbon and PSD size. In contrast, prolonged opening time of L-type calcium channels, induced by 1 h application of BayK led to a reduction in ribbon and PSD size. Notably, temporally-patterned optical stimulation in form of bursts ( oBurst ) similarly led to a reduction in ribbon size but failed to dramatically attenuate PSD size. Please note that we omitted possible postsynaptic Ca v 1. x channels from this schematic drawing due to current lack of experimental evidence for their presence in the PSD. PD, presynaptic density; SV, synaptic vesicle.

Article Snippet: For all conditions except Ca v 1.3 -KO, datasets were analyzed in Imaris x64 9.6.1, with 3D reconstructions of ribbon puncta generated using the surface algorithm with local background subtraction enabled.

Techniques: Inhibition, Functional Assay, Disruption

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 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 1.3 α 1D 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 N740 residue at the DIIS6 helix. (E) Zoomed view of DI–DII PD interface showing π-H and π-π quadrangle interactions between F358, F736, W707, and N740. (F) PCA of F736 and N740 side chains reveal distinct conformational clusters, with representative orientations shown below. (G) Centroid-to-centroid distances between F736-F358 and F736-N740 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