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: PLOS Biology

Article Title: Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons

doi: 10.1371/journal.pbio.3002879

Figure Lengend Snippet: (A) Left panel: schematic illustration of the paired recording. Single MFBs were stimulated in the tight-seal, cell-attached configuration, while postsynaptic CA3-PNs were simultaneously recorded in the whole-cell voltage-clamp configuration. Right top panel: 50-Hz train of 10 stimuli. Right bottom panel: overlay of average EPSCs before (“Ctrl,” gray) and in the presence of 50 μM forskolin (“FSK,” red). (B) Normalized EPSC 1 peak amplitude plotted against experimental time. Red horizontal line indicates the application of forskolin (“FSK”). Dashed line indicates 100% baseline. Note that the onset of forskolin application is the time point of switching solutions from forskolin-free ACSF to forskolin-containing ACSF. Forskolin-containing ACSF reached the recording chamber about 60–90 s after the onset of forskolin application. Data from 8 pairs (8 rats). (C) Summary bar graph of EPSC 1 peak amplitudes before (“Ctrl,” gray) and in the presence of 50 μM forskolin (“FSK,” red). Bars and whiskers show mean + SEM; P = 0.0078, Wilcoxon signed-rank test. (D) Summary bar graph of PPR (EPSC 2 /EPSC 1 ) before (“Ctrl,” gray) and in the presence of 50 μM forskolin (“FSK,” red). Bars and whiskers show mean + SEM; P = 0.0156, Wilcoxon signed-rank test. (E) Cumulative plot of EPSC peak amplitudes during a 50-Hz train with 10 stimuli before (“Ctrl,” gray) and in the presence of 50 μM forskolin (“FSK,” red). Data points during the last 4 stimuli (at time points ≥120 ms) were fit by linear regression and back-extrapolated to time point 0. (F–H) Summary bar graphs of RRP (F; P = 0.0078), P r (G; P = 0.0391), and refilling rate (H; P = 0.7422, Wilcoxon signed-rank tests), estimated from the cumulative EPSC plot (E), before (“Ctrl,” gray) and in the presence of 50 μM forskolin (“FSK,” red). Bars and whiskers show mean + SEM. See and Figs. Numerical values for this figure are detailed at https://doi.org/10.15479/AT:ISTA:18296 . ACSF, artificial cerebrospinal fluid; EPSC, excitatory postsynaptic current; MFB, mossy fiber bouton; PN, pyramidal neuron; PPR, paired-pulse ratio; RRP, readily releasable pool; SEM, standard error of the mean.

Article Snippet: Then, replicas were incubated in the first primary antibody, guinea pig anti-Ca V 2.1 (P/Q-type; Synaptic Systems, Cat # 152 205, RRID:AB_2619842, 1.3 μg ml −1 ) [ ] in 2% BSA in TBS overnight, shaking at 15°C.

Techniques:

Journal: PLOS Biology

Article Title: Presynaptic cAMP-PKA-mediated potentiation induces reconfiguration of synaptic vesicle pools and channel-vesicle coupling at hippocampal mossy fiber boutons

doi: 10.1371/journal.pbio.3002879

Figure Lengend Snippet: (A) Example TEM micrograph from acute hippocampal slices, showing putative MFBs (white asterisks) with apparent postsynaptic spines (black asterisks) in stratum lucidum of CA3 region. MFBs were recognized based on characteristic morphological features: large bouton size, high density of clear SVs, presence of large dense-core vesicles, high-density of mitochondria, and multiple synaptic contacts with large spines. Black arrowhead points to an AZ shown in (C). (B, C) Higher magnification TEM micrographs showing AZs (white line) and docked SVs (pink circles and white arrowheads) in putative MFBs in DMSO control (B, “Ctrl”) and after 50 μM forskolin (C, “FSK”). (D) Schematic representation of the time course of the experiment with 5-min forskolin (“FSK”) treatment. (E) Summary bar graph of the number of docked vesicles per 100 nm of AZ profile length in DMSO control (“Ctrl,” gray) and after forskolin application (“FSK,” red). Note zero values indicate AZs without any observed docked vesicles. Bars and whiskers show mean + SD. Horizontal black lines indicate median values. P < 0.0001, Mann–Whitney test. (F) Cumulative plots of the data displayed in (E), color scheme is identical to (E). P < 0.0001, Mann–Whitney test. (G) Summary bar graph of the diameter of docked vesicles measured in DMSO control (“Ctrl,” gray) and after forskolin treatment (“FSK,” red). Bars and whiskers show mean + SD. Horizontal black lines indicate median values. P = 0.1689, Mann–Whitney test. (H) Cumulative plots of the data displayed in (G), color scheme is identical to (G). P = 0.2317, Mann–Whitney test. Scale bar sizes are indicated on the figure panels. Numerical values for this figure are detailed at https://doi.org/10.15479/AT:ISTA:18296 . AZ, active zone; DMSO, dimethyl sulfoxide; MFB, mossy fiber bouton; SD, standard deviation; SV, synaptic vesicle; TEM, transmission electron microscopy.

Article Snippet: Then, replicas were incubated in the first primary antibody, guinea pig anti-Ca V 2.1 (P/Q-type; Synaptic Systems, Cat # 152 205, RRID:AB_2619842, 1.3 μg ml −1 ) [ ] in 2% BSA in TBS overnight, shaking at 15°C.

Techniques: Control, MANN-WHITNEY, Standard Deviation, Transmission Assay, Electron Microscopy