Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Used mouse model for labelling the endogenous population of Cav2.1 channels and the strategy how to use the N-terminal citrine-tag as defined epitope. The binding site of antibodies on the C-terminus is indicated as well, which allowed us to control the specificity of intrabody labelling. B) Hippocampal neurons 16 DIV from the Cacna1a Citrine KI mouse labelled with anti-GFP nanobody (ATTO647N), anti-bassoon as presynaptic scaffold, and anti-Homer as postsynaptic scaffold protein. Within the enlarged view the arrows point to spots where all three label co- localize. The scale bars correspond to 20 µm (overview) and 2 µm (enlarged picture). C) Four constructs that were used to develop intracellular labelling approach to visualize endogenous Ca V 2.1 channels or manipulate their subcellular distribution. D-F) Left panels correspond to the expression of the intrabody-Halo-Tag-CCR5 , CRY2- intrabody-Halo-Tag-CCR5 or CAG-Halo-Tag- intrabody-ΔCCR5 in Ca V 2.1::neurons. Middel panels represent the expression of intrabody-Halo- Tag-CCR5, CRY2- intrabody-Halo-Tag-CCR5 or CAG-Halo-Tag-intrabody-ΔCCR5 in Cre-induced Ca V 2.1-KO neurons by transfection of neurons with Cre-Td-Tomato. Right panel shows quantification of fluorescent intensities of CAG-Halo-Tag-intrabody-CCR5 (control: n ROI = 6, N cultures = 2, Cre; n ROI = 11, N cultures = 2. Unpaired t-test, p-value<0.0001), CRY2- intrabody-Halo-Tag- CCR5 (control: n ROI = 10, N cultures = 2, Cre; n ROI = 10, N cultures = 2. Unpaired t-test, p-value<0.0001), and CAG-Halo-Tag-intrabody-ΔCCR5 (control; n ROI = 10, N cultures = 2, Cre; n ROI = 8, N cultures = 2. Unpaired t-test, p-value = 0.412). Scale bar = 20 µm. G) Quantification of the colocalization of Ca V 2.1 channels in bassoon-positive spots (0.60 ± 0.01, n ROI = 23, N cultures = 3), Ca V 2.1 channels colocalize with Bassoon and Gephyrin (0.85 ± 0.02, n ROI = 20, N cultures = 3), and Ca V 2.1 channels colocalize with Bassoon and Homer (0.94 ± 0.006, n ROI = 16, N cultures = 3). H) Normalized intensity of Ca V 2.1 channels in inhibitory synapses (1.00 ± 0.08, n ROI = 27, N cultures = 3), and excitatory (1.08 ± 0.14, n ROI = 30, N cultures = 3) synapses, unpaired t-test, p-value = 0.612. Data are presented as mean ± SEM. I) Quantification of the occupancy rate of Ca V 2.1-channels labelled with the halotag- intrabody against GFP and C-terminal anti-Ca V 2.1 antibody (construct I: 0.47 ± 0.04, n ROI = 11, N cultures = 2, construct II: 0.75 ± 0.01, n ROI = 30, N cultures = 3, construct III: 0.82 ± 0.01, n ROI = 29, N cultures = 3). Data are presented as mean ± SEM. Number of replicates are summarized in suppl. table Fig .

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Binding Assay, Control, Construct, Expressing, Transfection

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Hippocampal neurons transfected with the calcium sensor jRGECO1a::synaptophysin, which also serves as synaptic marker. Calcium transients are shown for two regions along the axon. B) Same ROI as A) with expression of Halo-tag::intrabody, subregions are enlarged in C) and D) to illustrate individual trajectories of CaV2.1 channels inside a synapse and along the axon. E) Individual trajectories of synaptic and axonal localized Ca V 2.1 channels, the colour code corresponds to immobile (pink), confined (orange) or free mobile (blue). F) Quantification of calcium transients from control neurons expressing jRGECO1a::synaptophysin (2.24 ± 0.12, n synapse = 189, N = 3) and intrabody expressing neurons (2.30 ± 0.07, n synapse = 135, N = 3) unpaired t-test, p-value = 0.707. G) Quantification of the mean square displacement (MSD) of Ca V 2.1- channels in synaptic (black line) and extra-synaptic (gray line) compartments. H) Frequency distribution of the diffusion coefficient of Ca V 2.1 channels in synaptic (0.027 ± 0.001, n synapses = 205, N cultures = 3) and extra-synaptic compartments (0.059 ± 0.004, n axon segment = 184, N cultures = 3), unpaired t test p-value<0.0001. I) Frequency distribution of weighted mean jumping distance (MJD) of Ca V 2.1-channels in synaptic (black line) and extra-synaptic (grey line) compartments. J) Quantification of the proportions of different motions of Ca V 2.1-channels based on MJD in synaptic and extra-synaptic compartments, Chi-square test, p-value = 0.026. Data are presented as mean ± SEM. K-N) Quantification of Ca V 2.1-channel mobility in synaptic and extra-synaptic compartments based on MJD under different conditions. K, L) Preincubation of neurons with Nocodazol (1µM) or Latrunculin A (5µM) for 30 min. (Nocodazol: synaptic Chi-square test, p-value = 0.386; extra-synaptic Chi-square test, p-value = 0.001, Latrunculin: synaptic Chi-square test, p- value = 0.653; extra-synaptic Chi-square test, p-value = 0.001) M) Preincubation of neurons with TTX (1µM) for 30 min (synaptic Chi-square test, p-value = 0.0257; extra-synaptic Chi-square test, p-value = 0.911). N) Preincubation of neurons with BAPTA-AM (20 µM) for 30 min inside synapses (synaptic Chi-square test, p-value = 0.0063; extra-synaptic Chi-square test, p-value = 0.379). O) Example image of axons expressing iGluSnFR3 and intrabody::Halo-tag. Example traces of glutamate responses and bleaching curve ( P ) of synaptic concentrated Ca V 2.1 channels are shown. Q) Correlation of failure rate with channel number of synapses, Pearson correlation coefficient r = -0.4637. Sample sizes for trajectories/synapses/cultures are given in Suppl. Table Fig .

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Transfection, Marker, Expressing, Control, Diffusion-based Assay

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Maximum projection of axonal segments expressing iGluSnFR3 stimulated 30x1Hz with a field electrode indicating several presynaptic locations. B) Voronoi segmentation of Ca V 2.1 localisations recorded from SPT experiments. Tresholding based on the localisation density within neuronal structures allow to identify distinct regions of higher localisation density, often associated with synapses. Occasionally channels more clustered in nanodomains as exemplified in synapse 2 (C) . The example synapses are represented with their corresponding glutamate responses and density borders for synaptic objects and nanoclusters (black region within synapse 2). D) Surface distribution of objects and nanoclusters E) density of localisations in object and nanoclusters. Sample sizes for synapses/cultures are given in Suppl. Table Fig . F) [Ca 2+ ] spikes at time t 0 of peak Ca 2+ inflow (upper) and at a time of high vesicle fusion rate (t 0 + 0.2 ms) (lower). Blue bar denotes the simulated release site location. Simulation area size is denoted by x = y = 0.5 µm. G) pVrs for each topology calculated as the ratio of each scenario compared to the pVr of the channel at 30 nm alone (second column). The grey line indicates the simulated pVr when the indicated channels were simulated together, whereas the black crosses show the pVr predicted assuming independent channel contributions, calculated from the individually simulated pVr of each channel (see Methods). ( H, K) Schematic representation of the simulated channel arrangements. Dispersed (orange): channels placed independently at random positions. Clustered (green): all channels placed at the same randomly selected position. Mixed (yellow): half of the channels clustered and half dispersed. I) Distribution of simulated pVr values observed with N = 5, 10 and 15 open channels for dispersed and clustered conditions. J) Comparison of median pVr for N = 5, 10 and 15. (L + M) Distributions of pVrs for mixed and clustered conditions for N = 10 shown as scatter plot (G) and histogram (M). N) Distributions of simulated failure rates for mixed and clustered conditions for N = 10. Asterisks denote significant difference between mean values (****: p < 0.0001; two-sample t-test).

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Examples of axons transfected with CRY2-intrabody-Halo-tag before and after blue light stimulation, arrows point to regions of induced clusters. B) Synaptic calcium transients of jRGECO1a::synaptophysin before and after blue light with average transient in gray (con) or red (CRY2). C) Quantification of synaptic calcium transients before light: ΔF/F 0 = 2.13 ± 0.07 and after light, ΔF/F 0 = 2.11 ± 0.03, unpaired t-test, p-value = 0.8. D) Frequency distribution of MJD of Ca V 2.1 channels before (black line) and after blue light (red line). E) Quantification of synaptic Ca V 2.1 channels number within synapses with and without x-link (Unpaired t-test, p-value = 0.735). F, G) Example images of Ca V 2.1 localisations with and after light induced clustering. Accumulated Ca V 2.1 localisations labelled with the intrabody::Halo-tag (F) or the intrabody-Cry2::Halo-tag. Selected regions (1, 2) correspond to presynaptic CaV2.1 channels distributed evenly or clustered within the synapse. The red line corresponds to the border of the synaptic localisation, black indicate the size of Ca V 2.1 nano-clusters) H) Quantification of nanocluster area and I) localisation density before and after x-link in Ca V 2.1 channels. J) Frequency distribution of glutamate responses to 1 Hz stimulation for 1 min from neurons co-transfected with intrabody and glutamate sensor. Fraction of monovesicular release events is not different between intrabody and x-link. K) Comparison between control and Cre induced KD reveal no difference of glutamate responses or fraction of monovesicular release. L) Failure rate of glutamate responses in the different conditions as indicated. M) Distribution of Ca V 2.1 channels (gray dots) within a synaptic region overlayed by the localisations of glutamate release after 1 Hz stimulation for 1 min. N) Membrane area of glutamate release based on localisation of individual glutamate responses over time for the conditions as indicated. O) Average traces of synaptic glutamate responses to 51 stimuli of 10, 20 and 50 Hz. The immobilisation of Ca V 2.1 channels (red) lead to a depression of transmitter release during repetitive stimulation in comparison to control conditions (grey). P) Area under the curve plotted for x-linked (red) and control (grey) conditions. Sample sizes for trajectories/synapses/cultures are given in Suppl. Table Fig .

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Transfection, Comparison, Control, Membrane

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Examples of presynaptic calcium transients in response to 1AP stimuli measured before and after application of baclofen (5 µM) using GCamp:synaptophysin. B) Quantification of calcium transients before and after baclofen application. C) Fraction of silenced (grey) and active (dark grey) synapses after activation of GABAB receptors for the indicated conditions. D) Distribution of mean jumping distance (MJD) for silenced (grey) or active (black) synapses after application of baclofen (5µM) E) Fractions of immobile (red), confined (orange) and fast moving (blue) Ca V 2.1 channels in silenced and responding synapses (Chi-square test, p-value <0.0001). F) Example traces of glutamate responses with (green) and without (grey) activation of GABAB receptors or I) after x-link before (red) and after (green) activation of GABAB receptors. G) Frequency distribution of glutamate responses before (grey) and after activation (green) of GABAB receptors. The insert show fraction of monovesicular release under the different conditions. J) Similar data as in G) but after x-link of Ca V 2.1. H) Quantification of failure rate for the indicated conditions. K) Release area calculated from the localisation of glutamate responses over time under the different conditions. Sample sizes for trajectories/synapses/cultures are given in Suppl. Table Fig. 5.

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Used mouse model for labelling the endogenous population of Cav2.1 channels and the strategy how to use the N-terminal citrine-tag as defined epitope. The binding site of antibodies on the C-terminus is indicated as well, which allowed us to control the specificity of intrabody labelling. B) Hippocampal neurons 16 DIV from the Cacna1a Citrine KI mouse labelled with anti-GFP nanobody (ATTO647N), anti-bassoon as presynaptic scaffold, and anti-Homer as postsynaptic scaffold protein. Within the enlarged view the arrows point to spots where all three label co- localize. The scale bars correspond to 20 µm (overview) and 2 µm (enlarged picture). C) Four constructs that were used to develop intracellular labelling approach to visualize endogenous Ca V 2.1 channels or manipulate their subcellular distribution. D-F) Left panels correspond to the expression of the intrabody-Halo-Tag-CCR5 , CRY2- intrabody-Halo-Tag-CCR5 or CAG-Halo-Tag- intrabody-ΔCCR5 in Ca V 2.1::neurons. Middel panels represent the expression of intrabody-Halo- Tag-CCR5, CRY2- intrabody-Halo-Tag-CCR5 or CAG-Halo-Tag-intrabody-ΔCCR5 in Cre-induced Ca V 2.1-KO neurons by transfection of neurons with Cre-Td-Tomato. Right panel shows quantification of fluorescent intensities of CAG-Halo-Tag-intrabody-CCR5 (control: n ROI = 6, N cultures = 2, Cre; n ROI = 11, N cultures = 2. Unpaired t-test, p-value<0.0001), CRY2- intrabody-Halo-Tag- CCR5 (control: n ROI = 10, N cultures = 2, Cre; n ROI = 10, N cultures = 2. Unpaired t-test, p-value<0.0001), and CAG-Halo-Tag-intrabody-ΔCCR5 (control; n ROI = 10, N cultures = 2, Cre; n ROI = 8, N cultures = 2. Unpaired t-test, p-value = 0.412). Scale bar = 20 µm. G) Quantification of the colocalization of Ca V 2.1 channels in bassoon-positive spots (0.60 ± 0.01, n ROI = 23, N cultures = 3), Ca V 2.1 channels colocalize with Bassoon and Gephyrin (0.85 ± 0.02, n ROI = 20, N cultures = 3), and Ca V 2.1 channels colocalize with Bassoon and Homer (0.94 ± 0.006, n ROI = 16, N cultures = 3). H) Normalized intensity of Ca V 2.1 channels in inhibitory synapses (1.00 ± 0.08, n ROI = 27, N cultures = 3), and excitatory (1.08 ± 0.14, n ROI = 30, N cultures = 3) synapses, unpaired t-test, p-value = 0.612. Data are presented as mean ± SEM. I) Quantification of the occupancy rate of Ca V 2.1-channels labelled with the halotag- intrabody against GFP and C-terminal anti-Ca V 2.1 antibody (construct I: 0.47 ± 0.04, n ROI = 11, N cultures = 2, construct II: 0.75 ± 0.01, n ROI = 30, N cultures = 3, construct III: 0.82 ± 0.01, n ROI = 29, N cultures = 3). Data are presented as mean ± SEM. Number of replicates are summarized in suppl. table Fig .

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Binding Assay, Control, Construct, Expressing, Transfection

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Hippocampal neurons transfected with the calcium sensor jRGECO1a::synaptophysin, which also serves as synaptic marker. Calcium transients are shown for two regions along the axon. B) Same ROI as A) with expression of Halo-tag::intrabody, subregions are enlarged in C) and D) to illustrate individual trajectories of CaV2.1 channels inside a synapse and along the axon. E) Individual trajectories of synaptic and axonal localized Ca V 2.1 channels, the colour code corresponds to immobile (pink), confined (orange) or free mobile (blue). F) Quantification of calcium transients from control neurons expressing jRGECO1a::synaptophysin (2.24 ± 0.12, n synapse = 189, N = 3) and intrabody expressing neurons (2.30 ± 0.07, n synapse = 135, N = 3) unpaired t-test, p-value = 0.707. G) Quantification of the mean square displacement (MSD) of Ca V 2.1- channels in synaptic (black line) and extra-synaptic (gray line) compartments. H) Frequency distribution of the diffusion coefficient of Ca V 2.1 channels in synaptic (0.027 ± 0.001, n synapses = 205, N cultures = 3) and extra-synaptic compartments (0.059 ± 0.004, n axon segment = 184, N cultures = 3), unpaired t test p-value<0.0001. I) Frequency distribution of weighted mean jumping distance (MJD) of Ca V 2.1-channels in synaptic (black line) and extra-synaptic (grey line) compartments. J) Quantification of the proportions of different motions of Ca V 2.1-channels based on MJD in synaptic and extra-synaptic compartments, Chi-square test, p-value = 0.026. Data are presented as mean ± SEM. K-N) Quantification of Ca V 2.1-channel mobility in synaptic and extra-synaptic compartments based on MJD under different conditions. K, L) Preincubation of neurons with Nocodazol (1µM) or Latrunculin A (5µM) for 30 min. (Nocodazol: synaptic Chi-square test, p-value = 0.386; extra-synaptic Chi-square test, p-value = 0.001, Latrunculin: synaptic Chi-square test, p- value = 0.653; extra-synaptic Chi-square test, p-value = 0.001) M) Preincubation of neurons with TTX (1µM) for 30 min (synaptic Chi-square test, p-value = 0.0257; extra-synaptic Chi-square test, p-value = 0.911). N) Preincubation of neurons with BAPTA-AM (20 µM) for 30 min inside synapses (synaptic Chi-square test, p-value = 0.0063; extra-synaptic Chi-square test, p-value = 0.379). O) Example image of axons expressing iGluSnFR3 and intrabody::Halo-tag. Example traces of glutamate responses and bleaching curve ( P ) of synaptic concentrated Ca V 2.1 channels are shown. Q) Correlation of failure rate with channel number of synapses, Pearson correlation coefficient r = -0.4637. Sample sizes for trajectories/synapses/cultures are given in Suppl. Table Fig .

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Transfection, Marker, Expressing, Control, Diffusion-based Assay

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Maximum projection of axonal segments expressing iGluSnFR3 stimulated 30x1Hz with a field electrode indicating several presynaptic locations. B) Voronoi segmentation of Ca V 2.1 localisations recorded from SPT experiments. Tresholding based on the localisation density within neuronal structures allow to identify distinct regions of higher localisation density, often associated with synapses. Occasionally channels more clustered in nanodomains as exemplified in synapse 2 (C) . The example synapses are represented with their corresponding glutamate responses and density borders for synaptic objects and nanoclusters (black region within synapse 2). D) Surface distribution of objects and nanoclusters E) density of localisations in object and nanoclusters. Sample sizes for synapses/cultures are given in Suppl. Table Fig . F) [Ca 2+ ] spikes at time t 0 of peak Ca 2+ inflow (upper) and at a time of high vesicle fusion rate (t 0 + 0.2 ms) (lower). Blue bar denotes the simulated release site location. Simulation area size is denoted by x = y = 0.5 µm. G) pVrs for each topology calculated as the ratio of each scenario compared to the pVr of the channel at 30 nm alone (second column). The grey line indicates the simulated pVr when the indicated channels were simulated together, whereas the black crosses show the pVr predicted assuming independent channel contributions, calculated from the individually simulated pVr of each channel (see Methods). ( H, K) Schematic representation of the simulated channel arrangements. Dispersed (orange): channels placed independently at random positions. Clustered (green): all channels placed at the same randomly selected position. Mixed (yellow): half of the channels clustered and half dispersed. I) Distribution of simulated pVr values observed with N = 5, 10 and 15 open channels for dispersed and clustered conditions. J) Comparison of median pVr for N = 5, 10 and 15. (L + M) Distributions of pVrs for mixed and clustered conditions for N = 10 shown as scatter plot (G) and histogram (M). N) Distributions of simulated failure rates for mixed and clustered conditions for N = 10. Asterisks denote significant difference between mean values (****: p < 0.0001; two-sample t-test).

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Expressing, Comparison

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Examples of axons transfected with CRY2-intrabody-Halo-tag before and after blue light stimulation, arrows point to regions of induced clusters. B) Synaptic calcium transients of jRGECO1a::synaptophysin before and after blue light with average transient in gray (con) or red (CRY2). C) Quantification of synaptic calcium transients before light: ΔF/F 0 = 2.13 ± 0.07 and after light, ΔF/F 0 = 2.11 ± 0.03, unpaired t-test, p-value = 0.8. D) Frequency distribution of MJD of Ca V 2.1 channels before (black line) and after blue light (red line). E) Quantification of synaptic Ca V 2.1 channels number within synapses with and without x-link (Unpaired t-test, p-value = 0.735). F, G) Example images of Ca V 2.1 localisations with and after light induced clustering. Accumulated Ca V 2.1 localisations labelled with the intrabody::Halo-tag (F) or the intrabody-Cry2::Halo-tag. Selected regions (1, 2) correspond to presynaptic CaV2.1 channels distributed evenly or clustered within the synapse. The red line corresponds to the border of the synaptic localisation, black indicate the size of Ca V 2.1 nano-clusters) H) Quantification of nanocluster area and I) localisation density before and after x-link in Ca V 2.1 channels. J) Frequency distribution of glutamate responses to 1 Hz stimulation for 1 min from neurons co-transfected with intrabody and glutamate sensor. Fraction of monovesicular release events is not different between intrabody and x-link. K) Comparison between control and Cre induced KD reveal no difference of glutamate responses or fraction of monovesicular release. L) Failure rate of glutamate responses in the different conditions as indicated. M) Distribution of Ca V 2.1 channels (gray dots) within a synaptic region overlayed by the localisations of glutamate release after 1 Hz stimulation for 1 min. N) Membrane area of glutamate release based on localisation of individual glutamate responses over time for the conditions as indicated. O) Average traces of synaptic glutamate responses to 51 stimuli of 10, 20 and 50 Hz. The immobilisation of Ca V 2.1 channels (red) lead to a depression of transmitter release during repetitive stimulation in comparison to control conditions (grey). P) Area under the curve plotted for x-linked (red) and control (grey) conditions. Sample sizes for trajectories/synapses/cultures are given in Suppl. Table Fig .

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Transfection, Comparison, Control, Membrane

Journal: bioRxiv

Article Title: Functional relevance of mobile and clustered Ca V 2.1 channels in central synapses

doi: 10.64898/2026.07.13.737722

Figure Lengend Snippet: A) Examples of presynaptic calcium transients in response to 1AP stimuli measured before and after application of baclofen (5 µM) using GCamp:synaptophysin. B) Quantification of calcium transients before and after baclofen application. C) Fraction of silenced (grey) and active (dark grey) synapses after activation of GABAB receptors for the indicated conditions. D) Distribution of mean jumping distance (MJD) for silenced (grey) or active (black) synapses after application of baclofen (5µM) E) Fractions of immobile (red), confined (orange) and fast moving (blue) Ca V 2.1 channels in silenced and responding synapses (Chi-square test, p-value <0.0001). F) Example traces of glutamate responses with (green) and without (grey) activation of GABAB receptors or I) after x-link before (red) and after (green) activation of GABAB receptors. G) Frequency distribution of glutamate responses before (grey) and after activation (green) of GABAB receptors. The insert show fraction of monovesicular release under the different conditions. J) Similar data as in G) but after x-link of Ca V 2.1. H) Quantification of failure rate for the indicated conditions. K) Release area calculated from the localisation of glutamate responses over time under the different conditions. Sample sizes for trajectories/synapses/cultures are given in Suppl. Table Fig. 5.

Article Snippet: We tested a monoclonal mouse anti-Ca V 2.1 antibody (synaptic system, cat#152211) as well as a polyclonal rabbit anti-Ca V 2.1 antibody (synaptic system, cat#152203) targeting the C-terminus of Ca V 2.1 channels (Synaptic systems, Göttingen).

Techniques: Activation Assay

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Schematic illustrating imaging at the plasma membrane (PM). B Left: representative single-plane Airyscan confocal images of the PM showing Ca V 1.2 immunolabeling in control (CTL, black) and PFF-treated (red) neurons. Inset: MAP2 (pink) neuronal marker. Right: quantification of Ca V 1.2 cluster size, cluster density, and mean gray value (MGV) in the soma (a.) and dendrites (b.) of CTL (black) and PFF-treated (red) neurons. Dendritic measurements are shown separately for excitatory (dark blue) and inhibitory (light blue) populations. n = 20 somata per condition; n = 20 dendrites per group (CTL excitatory, CTL inhibitory, PFF excitatory, PFF inhibitory); two independent isolations with each isolation containing 8-10 pups. C Left: representative super-resolution TIRF localization maps showing Ca V 1.2 immunolabeling in CTL (black) and PFF-treated (red) neurons. Right: quantification of PM Ca V 1.2 cluster size, cluster density, and nearest-neighbor distance in the somatic region. n = 16 neurons per condition; two independent isolations. D Same experimental design as in ( B ), with neurons immunolabeled for Ca V 2.1. n = 19 (CTL) and n = 20 (PFF) somata; n = 20 dendrites per group (CTL excitatory, CTL inhibitory, PFF excitatory, PFF inhibitory); two independent isolations. E Same experimental design as in ( C ), with neurons immunolabeled for Ca V 2.1. n = 16 neurons per condition; two independent isolations. Error bars represent SEM. Statistical significance was determined using two-tailed Mann-Whitney or unpaired two-tailed t-tests. ns, not significant; *P ≤ 0.05; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Imaging, Clinical Proteomics, Membrane, Immunolabeling, Control, Marker, Isolation, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Schematic representation of the CCAD peptide mechanism of action. B Left: representative single-plane Airyscan confocal images of the PM in CTL and PFF-treated neurons co-incubated with SCRBL or CCAD peptides and co-immunolabeled for Ca V 1.2 and K V 2.1. Conditions are shown as CTL;SCRBL (black), PFF;SCRBL (red), CTL;CCAD (gray), and PFF;CCAD (yellow). Right: quantification of Ca V 1.2 cluster size, K V 2.1 cluster size, and Ca V 1.2-K V 2.1 overlap area in the somatic region. n = 19 (CTL;SCRBL), n = 19 (PFF;SCRBL), n = 20 (CTL;CCAD), and n = 20 (PFF;CCAD) neurons; two independent isolations. C Schematic representation of the proximity ligation assay (PLA). D Top: representative Airyscan confocal PLA images showing Ca V 1.2-K V 2.1 proximity. Images are maximum intensity projections from Z-stacks spanning whole cells. Color coding as in ( B ). Bottom: quantification of PLA puncta density. n = 18 neurons per condition; two independent isolations. E Same experimental design as in ( D ), but assessing Ca V 2.1-K V 2.1 proximity. n = 18 neurons per condition; two independent isolations. Error bars represent SEM. Statistical significance in panels ( B, D-E ) was determined using two-way ANOVA with appropriate post hoc tests. ns, not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment; CCAD, calcium channel association domain peptide; SCRBL, scrambled control peptide.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Incubation, Immunolabeling, Proximity Ligation Assay, Control

Journal: bioRxiv

Article Title: Nanoscale CaV channel reorganization links α-synuclein pathology to calcium-dependent transcriptional dysregulation

doi: 10.64898/2026.05.01.719272

Figure Lengend Snippet: A Left: representative FV4000 confocal images showing co-immunolabeling of CDK5 and K V 2.1 at the PM in control (CTL, black) and PFF-treated (red) neurons. Images are maximum intensity projections from three optical sections acquired at the PM. Right: quantification of CDK5-K V 2.1 overlap area and CDK5 puncta density in the somatic region. n = 18 (CTL) and n = 19 (PFF) neurons; two independent isolations. B Schematic representation of the roscovitine mechanism of action. C Top: representative FV4000 confocal images of the PM in CTL and PFF-treated neurons incubated with or without roscovitine and immunolabeled for pS603-K V 2.1. Conditions are shown as CTL (black), PFF (red), CTL;Rosco (blue), and PFF;Rosco (purple). Images are maximum intensity projections from Z-stacks spanning whole cells. Bottom: quantification of somatic pS603-Kv2.1 occupancy (% of soma area), cluster MGV, and cluster density. n = 19 (CTL), n = 20 (PFF), n = 20 (CTL;Rosco), and n = 20 (PFF;Rosco) neurons; two independent isolations. D Left: representative FV4000 confocal PLA images showing Ca V 1.2-K V 2.1 proximity. Images are maximum intensity projections from Z-stacks spanning whole cells. Color coding as in ( C ). Right: quantification of PLA puncta density. n = 20 neurons per condition; two independent isolations. E Same experimental design as in ( D ), but assessing Ca V 2.1-K V 2.1 proximity. n = 20 (CTL), n = 20 (PFF), n = 21 (CTL;Rosco), and n = 20 (PFF;Rosco) neurons; two independent isolations. Error bars represent SEM. Statistical significance in panel ( A ) was determined using two-tailed Mann-Whitney test; panels ( C – E ) were analyzed using two-way ANOVA with appropriate post hoc tests. ns, not significant; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. CTL, control; PFF, α-synuclein pre-formed fibril treatment; Rosco, roscovitine; pS603-K V 2.1, K V 2.1 phosphorylated at serine 603.

Article Snippet: The following combinations were used: K V 2.1 (NeuroMab, K89/34) with Ca V 1.2 (Alomone, ACC-003); K V 2.1 (NeuroMab, K89/34) with Ca V 2.1 (Alomone, ACC-001); and K V 2.1 (NeuroMab, K89/34) with K V 2.1 (NeuroMab, Drk1).

Techniques: Immunolabeling, Control, Incubation, Two Tailed Test, MANN-WHITNEY

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