Figure 4.

Effect of deletions from the II/III loop of α_1B influence N-type channel inactivation properties and affect responsiveness of N-type channels to syntaxin 1A modulation. (A) Voltage-dependent availability of wild-type N-type channels (filled circles, V_1/2 = −76 mV), Nd2 (open squares, V_1/2 = −68 mV), Nd5 (open circles, V_1/2 = −36 mV), Nd6 (downward triangles, V_1/2 = −35 mV), Nd7 (upward triangles, V_1/2 = −41 mV). N-type channel mutant constructs are described in Materials and Methods. Data points are averages derived from at least three independent experiments for each channel mutant (error bars indicate ± SEM). (B) Analysis of midpoint shifts (ΔV_1/2) produced by coexpression of syntaxin. (Left) Wild-type N-type channels (with synprint site shown as thick line), shown schematically above a series of II/III loop deletion constructs Nd2, Nd5, Nd6, Nd7 (extent of deletion indicated by thin dotted line). (Right) Coexpression with syntaxin (filled symbols) induced a hyperpolarizing shift in the inactivation properties of wild-type channels and each of the II/III loop mutants relative to their behavior in control (open symbols). The shift was estimated as the displacement along the voltage axis that would be needed to align a data point in the presence of syntaxin (left vertical arrow) with the voltage-dependent curve describing inactivation in the absence of syntaxin (right vertical arrow). Estimates of the shift were as follows: WT, ΔV_1/2 > 28 mV; Nd2, ΔV_1/2 =16 mV; Nd5, ΔV_1/2 =12 mV; Nd6, ΔV_1/2 =10 mV; Nd7, ΔV_1/2 =12 mV. The shift was statistically significant (P < 0.01) in all cases. Where not shown, error bars (SEM) were smaller than the symbols.