Figure 4.

Expression of exogenous PRRT2 increases P/Q-type Ca²⁺ currents in excitatory neurons and HEK293 cells

(A) Somatic voltage-gated Ca²⁺ currents were recorded in excitatory hippocampal neurons transduced with PRRT2. Left: representative voltage-gated Ca²⁺ currents evoked by a voltage step at −10 mV lasting 200 ms from a holding potential of −70 mV. Right: individual data and means ± SEMs of current density (I_density) recorded at −10 mV.

(B) Ca²⁺ current density versus voltage relationships in PRRT2-overexpressing neurons. PRRT2 overexpression did not affect membrane capacitance (58.31 and 55.45 pF for mCherry- and PRRT2-mCherry-infected neurons, respectively).

(C) Conductance-voltage (G/V) curves obtained in PRRT2-overexpressing neurons by measuring the amplitude of the tail currents on repolarization to −120 mV following step depolarizations from −70 to +70 mV (V_h = −80 mV). For further details, see Figure 3 legend.

(D) Representative fluorescence images of non-permeabilized HEK293 cells transiently co-transfected with Cav2.1-HA/α2δ-1/β4 and either mCherry alone or PRRT2-mCherry (red) and retrospectively stained with anti-HA antibodies (green). The white line, corresponding to the major axis of the cell, was used to measure the fluorescence intensity of Cav2.1-HA immunostaining. Scale bar, 10 μm.

(E) Intensity profiles of Cav2.1-HA fluorescence in non-permeabilized HEK293 cells expressing either mCherry (black lines) or PRRT2-mCherry (light blue lines).

(F) Normalized mean fluorescence intensity (n = 57 and 52 cells for mCherry and PRRT2-mCherry, respectively) of Cav2.1-HA immunoreactivity in HEK293 cells expressing either mCherry (black bars) or PRRT2-mCherry (light blue bars). PRRT2 significantly increases membrane expression of Cav2.1. Data are means ± SEMs.

(G) Left: representative traces of voltage-gated Ca²⁺ currents evoked by a 200 ms voltage step at 0 mV (V_h = −70 mV) 2 days after transfection of HEK293 cells with Cav2.1/α2δ-1/β4 and either mCherry alone (black) or PRRT2-mCherry (light blue). Right: individual data and means ± SEMs of current density (I_density) values recorded in HEK293 cells expressing Cav2.1/α2δ-1/β4 in the absence (black) or presence (light blue) of PRRT2.

(H) I_density versus voltage (V) relationships for HEK293 cells expressing Cav2.1/α2δ-1/β4 in the absence (black) or presence (light blue) of PRRT2.

(I) Representative Ca²⁺ currents recorded in HEK293 cells expressing Cav2.1/α2δ-1/β4 in the absence (black) or presence (light blue) of PRRT2. Holding potential −80 mV, steps between −70 and +60 mV for 20 ms in 10-mV increments, repolarization to −120 mV.

(J) Normalized conductance-voltage curves of HEK293 cells expressing Cav2.1/α2δ-1/β4 in the absence (black) or presence (light blue) of PRRT2. Curves were fitted to the Boltzmann equation.

^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001 unpaired Student’s t test/Mann-Whitney U test (n = 19 for both mCherry- and PRRT2-mCherry-infected neurons; n = 51 and 53 for Cav2.1/α2δ-1/β4 expressing HEK293 cells in the absence or presence of PRRT2, respectively.