Fig 2: Interaction of Prrt2 with the GluA1 subunit in vivo and in vitro. (A) Protein levels of the truncated Prrt2-FLAG plasmid (p.R223X, the corresponding mutant of p.R217X in human PRRT2) in HEK 293T cells. Antibodies against the N-terminus of Prrt2 and FLAG were used to detect Prrt2-FLAG. Western blotting results demonstrated that the mutant Prrt2 protein is undetectable. (B, C, D) In vivo co-immunoprecipitation assays using the adult mouse hippocampus. After pull-down with GluA1, GluA2, and GluA3 antibodies, Western blotting results demonstrated interactions between Prrt2 and both GluA1 and GluA2, but not GluA3. (E) In vitro co-immunoprecipitation using cell extracts from HEK 293T cells co-transfected with HA-tagged GluA1, GluA2, and GluA3 and FLAG-tagged Prrt2. After pull-down with the FLAG antibody, Western blotting results demonstrated direct interactions between Prrt2 and GluA1. (F) Total and membrane proteins were extracted from HEK 293T cells co-transfected with HA-tagged GluA1, FLAG-tagged Prrt2, and FLAG-tagged mutant Prrt2 (p.R223X). (G) Western blotting results demonstrated decreased total amounts of GluA1 after co-transfection with Prrt2 (*p = 0.024 Prrt2 + GluA1 vs. Prrt2mut + GluA1; *p = 0.010 Prrt2 + GluA1 vs. GluA1; ns. p = 0.729 Prrt2mut + GluA1 vs. GluA1; n = 3). (H) Western blotting results demonstrated decreased surface expression levels of GluA1 after co-transfection with Prrt2 (**p = 0.003 Prrt2 + GluA1 vs. Prrt2mut + GluA1; *p = 0.025 Prrt2 + GluA1 vs. GluA1; ns. p = 0.195 Prrt2mut + GluA1 vs. GluA1; n = 3)

Fig 3: PRRT2 deficiency specifically affects P/Q-type Ca2+ currentsSomatic voltage-gated Ca2+ currents were recorded in excitatory hippocampal neurons in which PRRT2 was acutely silenced (top panels) or constitutively deleted (bottom panels).(A) Representative voltage-gated Ca2+ currents evoked by a voltage step at -10 mV lasting 200 ms from a holding potential of -70 mV and means ± SEMs current density (Idensity) recorded at -10 mV with superimposed individual data obtained in PRRT2-silenced (upper panel) and PRRT2 KO (lower panel) neurons.(B) Ca2+ current density versus voltage relationships in PRRT2 KD (upper panel) and PRRT2 KO (lower panel) neurons. PRRT2 depletion did not affect membrane capacitance.(C) Representative Ca2+ currents recorded in Scr- and Sh4-infected excitatory (GAD67-GFP-) hippocampal neurons (13 DIV). Holding potential -80 mV, steps between -70 and +60 mV for 20 ms in 10-mV increments, repolarization at -120 mV.(D) Conductance-voltage (G/V) curves obtained in PRRT2 KD (upper panel) and PRRT2 KO (lower panel) neurons by measuring the amplitude of the tail currents on repolarization to -120 mV following step depolarizations from -70 to 60 mV (Vh = -80 mV). The tail Idensity, obtained by dividing the tail current by the cell capacitance, was further normalized to the peak tail Idensity. The G/V curves were fitted to the Boltzmann equation. The inset in the upper panel shows an enlargement of the Ca2+ current recordings shown in (C), highlighting the fast deactivation of the tail currents.(E and H) Left: representative voltage-gated Ca2+ currents, evoked by a 200 ms voltage step at -10 mV (Vh = -70 mV) in Scr/Sh4-infected excitatory hippocampal neurons in the absence (Ctrl) or presence of either Cono-GIVA (E) or Aga-IVA (H) to identify N- and P/Q-type Ca2+ currents. Pure N- and P/Q-type peak Idensity, Idensity/V and G/V relationships were measured by subtracting the toxin-insensitive current recorded in the presence of either Cono-GIVA or Aga-IVA, from the current recorded under control conditions. Right: individual data and means ± SEMs and of N-type (E) and P/Q-type (H) Idensity in PRRT2-silenced (solid bars) and PRRT2 KO (open bars) neurons.(F and I) Current density (Idensity) versus voltage (V) relationships for N-type (F) and P/Q-type (I) VGCC currents obtained in PRRT2-silenced (left) and PRRT2 KO (right) neurons.(G and J) Normalized G/V curves of N-type (G) and P/Q-type (J) channels, obtained in PRRT2-silenced (left) and PRRT2 KO (right) neurons. The G/V curves were fitted to the Boltzmann equation.*p < 0.05, **p < 0.01, unpaired Student’s t test/Mann-Whitney U test. (A)–(D) n = 18 and 22 for Scr- and Sh4-infected neurons, respectively; n = 16 and 19 for WT and PRRT2 KO neurons, respectively. (E)–(G) n = 10, 10, 10, and 11 for Scr-infected, Sh4-infected, WT, and PRRT2 KO neurons, respectively. (H)–(J) n = 9, 10, 11, 10 for Scr-infected, Sh4-infected, WT, and PRRT2 KO neurons, respectively.

Fig 4: PRRT2 deficiency specifically decreases the contribution of P/Q channels to evoked EPSCs(A, D, and G) Representative eEPSCs recorded before (1, control) and during (2, toxins) application of Nife; (5 µM), Cono-GVIA (3 µM), or Aga-IVA (0.2 µM) in WT autaptic neurons. eEPSCs were elicited by clamping the cell at –70 mV and stimulating it with 0.5 ms voltage steps to +40 mV applied at 0.1 Hz. Toxins were applied after 100 s of baseline recording, only when the Ca2+ current rundown was lower than 10%. In all of the traces, the stimulation artifacts were blanked for clarity.(B and C) Upper panels: eEPSC amplitude (means ± SEMs) recorded under control conditions and in the presence of Nife in Scr- (n = 6; red bars) and Sh4- (n = 7; blue bars) infected neurons (B) and in WT (n = 8; red open bars) and PRRT2 KO (n = 7; blue open bars) neurons (C). The line-connected symbols represent the individual responses before and after Nife application. Lower panels: individual data and means ± SEMs of the percent blockade of eEPSC amplitude (left) and of Nife-sensitive EPSC amplitude (right) in the corresponding experimental groups. p = 0.58 (Scr versus Sh4; % blockade), p = 0.68 (Scr versus Sh4; Nife sensitivity), p = 0.61 (WT versus KO; % blockade), p = 0.62 (WT versus KO; Nife sensitivity), paired Student’s t test.(E and F) Recordings performed as in (B) and (C) under control conditions and in the presence of Cono-GVIA (3 µM) in the following experimental groups: Scr- (n = 11; red bars) and Sh4- (n = 13; blue bars) infected neurons (E) and in WT (n = 15; red open bars) and PRRT2 KO (n = 14; blue open bars) neurons (F). p = 0.32 (Scr versus Sh4; % blockade), p = 0.06 (Scr versus Sh4; Cono sensitivity), p = 0.36 (WT versus PRRT2 KO; % blockade), p = 0.12 (WT versus PRRT2 KO; Cono sensitivity), paired Student’s t test.(H and I) Recordings performed as in (B) and (C) under control conditions and in the presence of Aga-IVA (5 µM) in the following experimental groups: Scr- (n = 15; red bars) and Sh4- (n = 10; blue bars) infected neurons (H) and in WT (n = 14; red open bars) and PRRT2 KO (n = 10; blue open bars) neurons (I). **p < 0.01 (Scr versus Sh4; % blockade), ***p < 0.001 (Scr versus Sh4; Aga sensitivity), p = 0.2 (WT versus PRRT2 KO; % blockade), **p < 0.01 (WT versus PRRT2 KO; Aga sensitivity), paired Student’s t test.

Fig 5: The loss of PRRT2 in hippocampal developing neurons plated on laminin disrupted growth cone size and complexity. (A) Representative images of 1 DIV WT and PRRT2 KO hippocampal neurons plated on poly-l-lysine and laminin (PLL + LN). Neurons were labelled with phalloidin to stain F-actin and βIII-tubulin for neuronal microtubules. Scale bars = 20 µm. (B) Quantitative evaluation of actin protrusion areas. Actin protrusion area was calculated by subtracting the area of βIII-tubulin signal from F-actin area. Data are expressed as median values ± interquartile range of n = total number of neurons from four independent cultures (WT = 130, PRRT2 KO = 134). Median F-actin protruding area (µm2): WT = 126.2, PRRT2 KO = 110.8. Statistical significance was determined by Mann–Whitney test. (C) The biggest growth cone was selected from the mask of the phalloidin channel on the neurons described in (A), and its area, perimeter, and circularity were measured. Scale bars = 20 µm. (D) Quantitative analysis of growth cone parameters. Data are expressed as median ± interquartile range of n = total number of growth cones from three independent cultures (WT = 119, PRRT2 KO = 142). Median growth cone area (µm2): WT = 70.35, PRRT2 KO = 42.47; median growth cone perimeter (µm): WT = 72.28, PRRT2 KO = 50.04; median growth cone circularity (A.U.): WT = 0.160, PRRT2 KO = 0.206. Statistical significance was determined by Mann–Whitney test; *** p < 0.001. (E) The NeuronJ plugin of the ImageJ software was used to trace the neurites on the βIII-tubulin channel of neurons in (A). Scale bar = 20 μm. (F) Quantitative analysis of neuritic elongation expressed as median neurite length, total neurite length, and number of neurites/neurons. Data are expressed as median ± interquartile range of n = total number of neurons from three independent cultures (WT = 78; PRRT2 KO = 84). Median neurite length (µm): WT = 25.33, PRRT2 KO = 24.67; median total neurite length (µm): WT = 119.9, PRRT2 KO = 114.4; median neurites number/neuron: WT = 4, PRRT2 KO = 4. Statistical significance was determined by Mann–Whitney test.