Figure 5. Countercharges E87 and E90 differentially regulate kinetics of voltage sensing domain (VSD) I transitions and current activation.

(A,K) Schematic of VSD I in activated and resting states, showing the loss of ionic interactions upon mutation of E87A or E90A. (B–G) In Ca_V1.1e E87A right-shifted voltage dependence of activation without affecting kinetics (wildtype [red], E87A [lime]). (L–Q) The E90A mutation accelerated kinetics >4-fold and right-shifted voltage dependence of activation (wildtype [red], E87A [purple]). (B,L) Normalized representative currents show acceleration of activation in E90A (L) but not in E87A (B). (C,M) Time to peak (p=0.47 in C, p=0.00017 in M); (D,N) current-voltage relationship; (E,O) maximum current density (p=0.08 in E, p=0.04 in O); (F,P) voltage dependence of activation; (G,Q) voltage at half-maximal activation (V½) (p=0.000014 in G, p=0.008 in Q). Mean ± SEM; p-values calculated with Student’s t-test. (H–J) The time-lagged independent component analysis (tICA) free energy surface and schematic 1D representation of E87A show four macrostates corresponding to resting states 1, 2, 3 and the activated state with energy barriers similar to wildtype (gray) and transition kinetics in the higher μs timescale. (R–T) E90A shows three macrostates corresponding to the resting states 1 and 3 and the activated state, reduced energy barriers, and transition kinetics in the low μs timescale.

Figure 5—figure supplement 1. Deactivation kinetics are fast in Ca_V1.1e wildtype (WT) and in slowly (E87A) and fast-activating (E90A) voltage sensing domain (VSD) I mutants.

(A) Voltage clamp protocol used to examine deactivation kinetics (top) and representative current traces (bottom). Upon repolarization from +60 mV to varying negative potentials (at 10 mV increments), activated channels conduct a transient inward calcium current (downward spike) before they deactivate and close the channel pore. The decay of this so-called tail current was fitted with a mono-exponential function to determine the time constants of deactivation. (B) The time constants of deactivation are equally fast in Ca_V1.1e WT, E90A, and E87A (in contrast to their distinct activation kinetics; cf. Figure 5—figure supplement 2). At −20 mV significant kinetic differences start to occur, because at such weak repolarization part of the channels fail to deactivate and thus contaminate the tail current with a slowly inactivating current component (p=0.019; two-way repeated measures ANOVA and Holm-Sidak post hoc test). (C) Simplified model explaining slow activation and fast deactivation of the channel. We assume that at least two VSDs need to be in the up-state for the channel gate to open (at +60 mV). In response to the depolarizing voltage step, the slow VSD (orange; VSD I in Ca_V1.1) will be rate-limiting and thus endow the channel with slow activation kinetics. On repolarization, the rapid downward movement of another VSD (green) will close the channel gate with fast deactivation kinetics, thus masking the continuing downward movement of the slow VSD.