Figure 7. Syn II modulates GABA asynchronous release by interacting with presynaptic Ca²⁺ channels.

(a) Representative traces of eIPSCs from WT and KO dentate gyrus granule neurons before (black and grey for WT and KO, respectively) and after blockade of P/Q-type Ca²⁺ current with 0.5 μM ω-Agatoxin-IVA (blue) or of N-type Ca²⁺ current with 1 μM ω-Conotoxin-GVIA (green). (b) Aligned dot plot of the per cent reduction in peak amplitude of eIPSCs in WT (closed circles) and KO (open circles) neurons after the addition of either ω-Agatoxin-IVA (AGA; 0.5 μM) or ω-Conotoxin-GVIA (CONO; 1 μM). (c) Representative traces of the delayed asynchronous inhibitory response in WT and KO neurons (black and grey, respectively) and its modulation by Ca²⁺-channel blockers. Inset traces represent the first second after the end of the stimulation under control conditions and after the addition of ω-Agatoxin-IVA (0.5 μM; blue) or ω-Conotoxin-GVIA (1 μM; green). (d) Plots of the mean (±s.e.m.) charge of the delayed asynchronous release as a function of time after the end of the stimulation train in WT and KO neurons. Data recorded under control conditions (black/grey circles) or in the presence of ω-Agatoxin-IVA (0.5 μM; closed/open blue circles) were normalized to the respective pre-train spontaneous release. *P<0.05, two-tailed paired Student’s t-test. n=5 neurons from both WT (4 mice) and KO (4 mice), respectively. (e) Same as for d, but in the presence of ω-Conotoxin-GVIA (1 μM; closed/open green circles) **P<0.01, two-tailed paired Student’s t-test. n=5 and n=6 neurons from WT (4 mice) and KO (3 mice), respectively. (f,g) Cortical neuron lysates were immunoprecipitated with anti-SynII antibodies or anti-HA antibodies, as indicated (immunoprecipitation (IP)). After electrophoretic separation of the immunocomplexes, membranes were probed with antibodies specific for anti-Cav2.1 (P/Q-type), anti-Cav2.2 (N-type) and anti-Cav2.3 (R-type) Ca²⁺ channel subunits, as indicated (western blotting (WB)). A representative immunoblot is shown (f), together with the quantification of the immunoreactive signal in the immunoprecipitated samples (g), normalized to the binding to the HA control (means±s.e.m.; n=3 independent experiments). T, 100 μg of total lysate; FT, 100 μg of flow-through after immunoprecipitation.