Key role of segment IS4 in Cav1.2 inactivation: link between activation and inactivation

Fig. 1. Neutralization of segment IS4 modulates Cav1.2 inactivation. a Alignment of Cav1.2 segments IS4-IVS4. Charged residues are highlighted in blue. b Superimposed typical normalized IBa through WT and mutant IS4(K1Q/R2Q/R3Q/R4Q). IBa through WT and quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) during 3 s depolarizations from −80 mV to the voltages of the maximum of the current-voltage relationship (WT: 10 mV; IS4(K1Q/R2Q/R3Q/R4Q): −20 mV). Note the faster development of inactivation in IS4(K1Q/R2Q/R3Q/R4Q). Current decay was fitted to a monoexponential function yielding time constants of τ inact(WT) = 393 ± 24 ms and τinact(IS4(K1Q/R2Q/R3Q/R4Q)) = 235 ± 29 ms, respectively (see “Methods”). Solid lines represent the fitted function. c Steady-state inactivation curves of WT and the indicated IS4 mutants. Voltages of half-maximal inactivation (V 0.5,inact) where −18.3 ± 1.1 mV (WT), −13.2 ± 3.5 mV (IS4(K1Q)), −20.3 ± 1.2 mV (IS4(K1Q/R2Q)), −38.1 ± 0.8 mV ((IS4(K1Q/R2Q/R3Q)), and −45.0 ± 0.7 mV ((IS4(K1Q/R2Q/R3Q/R4Q)). d Inactivation time constants (τ inact) at different voltages were obtained by fitting the IBa decay over an interval of 3000 ms by a mono-exponential function. Time constants for…

Fig. 2. IS4 charge neutralizations cause correlating changes in activation and inactivation. a Steady-state activation of Cav1.2 mutants with gradually neutralized IS4 charges. b Activation currents of IBa through WT and mutant channel constructs with partially neutralized S4 segments (IS4(K1Q) and IS4(K1Q/R2Q/R3Q/R4Q)). Currents were recorded by applying a 100-ms pulse from holding potential (−80 mV) with 10 mV steps (WT −30 mV − 10 mV, K1Q −20 mV − 20 mV, K1Q/R2Q/R3Q/R4Q −50 mV − −20 mV). IS4(K1Q/R2Q/R3Q/R4Q) activates at more negative voltages than IS4(K1Q/R2Q/R3Q) and WT while IS4(K1Q) activates at more positive voltages (Table 1). Charge neutralization in IS4 (K1Q/R2Q/R3Q/R4Q) notably accelerates inactivation. Compare typical current recordings of WT (upper panel) with mutants K1Q (middle panel) and quadruple mutant IS4(K1Q/R2Q/R3Q/R4Q) (lower panel). c, Correlation between potentials of half-maximal activation (V 0.5,act) and half-maximal inactivation (V 0.5,inact) of IS4 mutants (Table 1). Linear regression analysis (solid line) yielded a statistically significant linear correlation (p < 0.01) with a correlation coefficient of 0.95

Fig. 3. Effect of charge neutralization in IIS4 and IIIS4 on steady-state inactivation and activation. Steady-state inactivation (left panels) and activation (right panels) curves of WT, IIS4, IIIS4, and IVS4 mutants. Representative barium currents through WT and mutant channels during a 3000-ms depolarization from −80 mV to the peak potential (PP) of the I–V curves are shown in corresponding insets. WT (PP 10 mV); IIS4(R2Q/R3Q/R4Q/K5Q/R6Q) (PP 10 mV); IIIS4(K1Q/R2Q/R3Q/R4Q) (PP 10 mV); IVS4(R2Q) (PP 20 mV)

Fig. 4. Charge neutralizations in Cav1.2 segments IS4–IVS4 differently affect the position and slope of the inactivation curve. a–c Shifts of the midpoint voltages (V 0.5,inact closed bars,V 0.5,act open bars) of a IS4 , b IIS4 and IIIS4, and c IVS4-mutants compared to WT. d Slope factors of inactivation curves were significantly (*p < 0.05) decreased by charge neutralization in IS4 and IVS4. Strongest changes were observed for mutants IS4((K0Q/R1Q/R2Q) with k inact. = 8.2 ± 0.6 mV; IS4(K1Q/R2Q/R3Q/R4Q) with k inact. = 6.2 ± 0.7 mV; IVS4(R2Q) with k inact. = 10.2 ± 1.2 mV; IVS4(R4Q) with k inact. = 9.7 ± 0.7 mV and IVS4(K5Q) with k inact. = 8.1 ± 0.7 mV. Neutralization of either single or combined charge neutralizations in IIS4 and IIIS4 had no significant effect on the slope factors of steady-state inactivation curves (Table 1)