Fig. 3.

A454T prevents modulation of P/Q channel activity by syntaxin 1A and SNAP-25. (A) Representative current traces from cells expressing WT (Left) or A454T (Right) P/Q channels in the presence of syntaxin 1A (to compare in the absence of syntaxin 1A see Fig. 2B). Western blot of plasma membrane proteins obtained from HEK 293 cells transfected with CFP-α_1A, Ca_Vβ_2a, and α₂δ P/Q channel subunits in the absence or presence of syntaxin 1A and probed with anti-GFP, syntaxin 1A, and β-actin antibodies. Heterologous expression of P/Q channel subunits does not induce the expression of endogenous syntaxin 1A. Methodological details can be found in SI Methods. (B and C) Steady-state inactivation curves. V_1/2,inact and k_inact values were (in mV): WT (○, n = 11) –3 ± 0.6 and –4.5 ± 0.5; WT + syntaxin 1A (, n = 22) –12.8 ± 0.5 and –5.1 ± 0.4; A454T (•, n = 14) –9.2 ± 0.5 and –4.6 ± 0.5; A454T + syntaxin 1A (, n = 15) –7.8 ± 0.5 and –4.8 ± 0.5. (D) Inhibition of WT and A454T P/Q channels alone (Left) or coexpressed with SNAP-25 (Right) evoked by a 20-ms test pulse to +20 mV following a 30-s prepulse to −80 mV (black trace) or −20 mV (green trace). (E) Average percentage I_Ca inhibition of WT and A454T P/Q channels in the absence or presence of SNAP-25. Ca²⁺ currents obtained as indicated in D were normalized to the current following the −80 mV prepulse. *P < 0.05 vs. WT. (F) Representative current traces from HEK 293 cells expressing WT P/Q channels coexpressed with WT (I-II_WT loop) or A454T (I-II_A545T loop) intracellular loops connecting domains I and II in the absence or presence of syntaxin 1A (stx 1A). Ca²⁺ currents were evoked by a 20-ms test pulse to +20 mV following a 30-s prepulse to −80 mV (black trace) or −20 mV (green trace). (G) Average percentage of WT P/Q I_Ca inhibition obtained under the experimental conditions shown in F (*P < 0.01).