Figure 6. Sin3B reduces native sodium currents in N1E-115 neuroblastoma cells.

(A) Typical sodium currents recorded under voltage-clamp in transfected N1E-115 cells. Currents were elicited by depolarizing steps from a holding potential of −80 mV to +40 mV. Upper trace: control cell, expressing DsRed alone. Lower trace: cell expressing Sin3B 293 and DsRed. (B) Current-Voltage relationship. Sodium currents were recorded at different test voltages from a holding potential of −80 mV. Control cells (black dots) showed a larger current at all voltages tested than Sin3B-expressing cells (gray dots). (C) Conductance-voltage curves were calculated for each cell, and normalized to the average G_max of control cells (black dots). A strong reduction of G_max was observed in Sin3B-expressing cells (gray dots). Analysis of data showed no changes in V_G1/2 or k_G. (D) Inactivation curves. I_Na was recorded at −10 mV after inactivating pre-pulses of 200 ms. Peak currents were normalized to the average of I_Na recorded in control cells (black dots) and plotted as function of the pre-pulse voltage. The data from each cell were fitted with Boltzmann equations to calculate V_h1/2 and k_h. On average, a small (5 mV) but statistically significant shift to the right was observed in Sin3B-expressing cells (gray dots). (E) Immunoreactivity for Na_v channels from whole cell lysates, from three independent experiments, was measured by densitometry and normalized to the immunoreactivity of actin in control (black) and Sin3B-expressing cells (white). Inset: representative inmmunoblots with Pan Na_v (upper panel) and anti-actin (lower panel) antibodies in control N1E-115 cells (lane 1) and cells expressing Sin3B 293 (lane 2). (F) In contrast, potassium currents were unaffected by overexpression of Sin3B at any voltage tested.