A novel region in the CaV2.1 α1 subunit C-terminus regulates fast synaptic vesicle fusion and vesicle docking at the mammalian presynaptic active zone

Abstract

In central nervous system (CNS) synapses, action potential-evoked neurotransmitter release is principally mediated by Ca_V2.1 calcium channels (Ca_V2.1) and is highly dependent on the physical distance between Ca_V2.1 and synaptic vesicles (coupling). Although various active zone proteins are proposed to control coupling and abundance of Ca_V2.1 through direct interactions with the Ca_V2.1 α1 subunit C-terminus at the active zone, the role of these interaction partners is controversial. To define the intrinsic motifs that regulate coupling, we expressed mutant Ca_V2.1 α₁ subunits on a Ca_V2.1 null background at the calyx of Held presynaptic terminal. Our results identified a region that directly controlled fast synaptic vesicle release and vesicle docking at the active zone independent of Ca_V2.1 abundance. In addition, proposed individual direct interactions with active zone proteins are insufficient for Ca_V2.1 abundance and coupling. Therefore, our work advances our molecular understanding of Ca_V2.1 regulation of neurotransmitter release in mammalian CNS synapses.

Keywords: biophysics; calcium channels; exocytosis; mouse; neuroscience; presynapse; structural biology; synaptic plasticity; synaptic transmission.

Figure 1.. Ca_V2.1 can be selectively ablated and functionally rescued at the calyx of Held.

(A) Cartoon depicting Ca_V2.1 α₁ subunit distal C-terminal interaction partners. (B) Amino acid sequence of the distal Cav2.1 C-terminus indicating interaction sites and truncation mutants. (C) left: Schematic view of stereotactic surgery to inject/coinject HdAd vectors expressing Cre + eGFP and Ca_V2.1 constructs + mCherry into the aVCN at age P1. Right: top: Experimental timeline from virus injection at P1 to electrophysiological recordings at P9-P11 prior to the onset of hearing (P12). Middle and bottom: schematic view of the viral constructs used, expressing either Cre + eGFP or Ca_V2.1 constructs + mCherry, respectively, driven by individual promotors. (D) Calyx of Held terminals transduced with Cre + eGFP (top) and Ca_V2.1 + mCherry (middle). eGFP and mCherry signals overlap with those of a calyx of Held loaded with Lucifer Yellow via a patch pipette (bottom). (E) Pharmacological isolation of presynaptic Ca_V2 isoforms in wildtype, CKO and Ca_V2.1 full transcript rescue calyxes. Traces in absence of any blockers (black), after blocking Ca_V2.1 fraction with 200 nM ω-AgaIVA (brown), after blocking Ca_V2.2 fraction with 2 µM ω-GVIA (blue) and after blocking all Ca_V2 channels with 50 µM Cd²⁺ (gray). (F) Relative Ca_V2 current fractions in wildtype, CKO and Ca_V2.1 full transcript rescue calyxes (n = 3 for each condition). DOI: http://dx.doi.org/10.7554/eLife.28412.003

Figure 2.. C-terminal deletions in Ca_V2.1 do not affect Ca_V2 abundance at the presynaptic terminal.

(A) Cartoons depicting Ca_V2.1 full transcript or mutants (top) with corresponding exemplary Ca²⁺ currents (bottom) triggered by 10 ms voltage steps from d -50 mV to 50 mV in 5mV steps. (B–C) Current-voltage relationship of absolute Ca²⁺ currents (B) and normalized current-voltage relationships (I/I_max; C). (D) Mean absolute Ca²⁺ currents. (E–F) Absolute tail currents (E) and normalized (I/I_max; F) tail currents as a function of voltage. (G) Mean tail Ca²⁺ currents at +40 mV. For Ca_V2.1 α₁ CKO (n = 11), wildtype (n = 12), Ca_V2.1 α₁full transcript (n = 10), Δ2365–2368 (n = 10), Δ2213–2368 (n = 10) and Δ2016–2368 (n = 10). All data are depicted as mean ± SEM. Detailed values can be derived from Table 1. DOI: http://dx.doi.org/10.7554/eLife.28412.004

Figure 2—figure supplement 1.. Ca_v2.1 rescue after CKO does not affect biophysical properties of the Ca²⁺ current at the calyx of Held.

(A) top: Raw peak Ca²⁺ current amplitudes and, bottom: normalized peak currents (I/I_masx) as a function of voltage. Continuous curves represent fits according to Equation 1. (B) top: Averaged raw and, bottom: normalized tail currents (I/I_max) (bottom) as a function of voltage. Continuous curves represent Boltzmann fits according to Equation 2. (C) top: Quantification of maximal Ca²⁺ peak currents and, bottom: cell capacitance. All data are depicted as mean ± SEM with wildtype (n = 12), full transcript (n = 10) and CKO (n = 11). DOI: http://dx.doi.org/10.7554/eLife.28412.005

Figure 3.. A novel role for a C-terminal region between amino acids 2042 and 2061 that regulates fast release independent of Ca_V2.1 abundance.

(A) Cartoon depicting truncated regions in our Ca_V2.1 α₁ deletion mutants including the binding sites for Ca_Vβ4, CASK, RBP, PXXP, RIM1/2 and Mint-1 along with the effects of C-terminal truncations on I_Ca. (B) Averaged traces of RRP and total releasable pool measurements from mice expressing Cre + full transcript Ca_V2.1 rescue (grey), Δ2365–2368 (cyan), Δ2213–2368 (yellow), Δ2061–2368 (purple), Δ2042–2368 (green) or Δ2016–2368 (blue). I_Ca (top) and EPSCs (bottom) triggered by 3 ms and 30 ms pulses, plotted on top of each other (n = 10 for each group, except for Δ2212–2368: n = 8). (C–H) Quantification of I_Ca charge (3 ms: C; and 30 ms: F), max. EPSC amplitudes (3 ms: D; 30 ms: G) and the 10–90% rise of the EPSCs (3 ms: E; 30 ms: H). All data are depicted as mean ± SEM. Detailed values can be derived from Table 2. DOI: http://dx.doi.org/10.7554/eLife.28412.006

Figure 3—figure supplement 1.. Ca_v2.1 Full transcript rescue does not affect synaptic transmission at the calyx of Held/MNTB synapse.

(A–B) Averaged traces of SV pool measurements in noninjected control mice (black; a) and mice expressing Cre + Full length rescue construct (grey; B). Top: stimulation protocol driving SV release by depolarization from −80 mV to 70 mV for 2 ms, followed by 0 mV for 3 ms or 30 ms. Below: Traces depicting the resulting presynaptic I_Ca (middle) with corresponding EPSCs (bottom). (C–D) Summary graphs of SV release rates (c) and the cumulative synaptic vesicle release rates (D) after 3 ms and 30 ms stimulation. (E–F) Summary graphs depicting normalized SV release after 3 ms and 30 ms stimulation (E) and normalized cumulative SV release after 30 ms stimulation (F). (G–L) Quantification of I_Ca amplitude (G), charge of Ca²⁺ currents (H I), EPSC amplitude (J), 10–90% rise time of the EPSC (K) after 3 ms and 30 ms stimulation, respectively, as well as the number of SVs released by 30 ms stimulation (L). Data are represented as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.28412.007

Figure 4.. The novel C-terminal region between amino acids 2042 and 2061 regulates size of the fast and total releasable pool and synaptic vesicle release kinetics.

(A–B) Average release rate trace after 3 ms (A) or 30 ms stimulation (B) from calyces expressing either Cre + full transcript rescue (grey), Δ2042–2368 (green) or Δ2016–2368 (blue); n = 10 for each group; (C) Averaged cumulative release after 3 ms and 30 ms stimulation. (D) Normalized cumulative release of the total releasable pool triggered by 30 ms stimulation. Inset presents a magnified view of the area encircled by the dashed box. (E–G) Quantification of SV numbers released by 3 ms (E) and 30 ms (F) as well as the ratio of SVs released by 3 ms and 30 ms stimulation (G). All data are depicted as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.28412.011

Figure 5.. The novel C-terminal region between amino acids 2042 and 2061 regulates the number of docked synaptic vesicles at the active zones.

(A–C) Representative EM images showing AZs from nontransduced calyces (A), and calyces transduced with Δ2042–2368 (B) or Δ2016–2368 (C). Transduced cells were identified by pre-embedded nanogold immunolabelling for eGFP (black dots in B and C). (D–G) Quantification of mean AZ length and docked SVs (within 5 nm of the membrane) of calyces transduced with Δ2042–2368 (n = 120; D E) or Δ2016–2368 (n = 100; F G), compared to AZs from the nontransduced contralateral MNTB, respectively. (H–I) Quantification of the mean distribution of SVs up to 200 nm distant from AZs for calyces expressing Δ2042–2368 (n = 120; H) or Δ2016–2368 (n = 100; I). Insets show SVs in closest proximity to the membrane (up to 20 nm). All data are depicted as mean ± SEM. DOI: http://dx.doi.org/10.7554/eLife.28412.012

Figure 6.. A novel C-terminal region in Ca_V2.1 α₁ subunit regulates SV docking at the active zone and RRP size independent of Ca²⁺ signaling.

Cartoon depicting truncated regions in our Ca_V2.1 α₁ deletion mutants including the binding sites for Ca_Vβ, CASK, RBP, RBP, RIM1/2, Mint-1 as well as PXXP motifs and the effects of truncations on I_Ca, SV docking, SV coupling as well as size of the fast and the total releasable synaptic vesicle pools. The critical region 2042–2061 is highlighted in grey. DOI: http://dx.doi.org/10.7554/eLife.28412.013