TI loop mutations differently affect the voltage sensor dynamics.

On-sensing currents of Ci-VSP were recorded from a holding potential of −60 mV in response to variable test pulses between −40 to+160 mV at maximum (increment: 20 mV, duration: 500 ms). Off-sensing currents were monitored by stepping back to −50 mV after the test potential phase (off-pulse duration: 500 ms). (A–D) Representative full current traces are shown for the WT and the denoted mutants. From these signals, selected off-currents are zoomed out (40 mV increments). As described in Materials and Methods, Q_off,all-V-distributions were calculated by integrating the off-sensing currents. These distributions are plotted against the potential of the preceding test pulse phase. From the whole amount of translocated sensing charges (Q_off,all), the fast (Q_off,fast) and the slow (Q_off,slow) component was determined as described in Materials and Methods. All calculated Q_off-values were normalized to the Q_off,all-value corresponding to the test potential of+120 mV. Q_off,all-V-distributions were approximated with a Boltzmann-type function (see Materials and Methods). Parameters of voltage-dependence (V_0.5, z_q) are given in Table S3.