Effects of mutation within the hydrophobic spine of Dr-VSP as estimated from KCNQ2/3 currents. (A) Schematic view of the protocol for examining VSP enzyme activity in the whole-cell patch configuration. The pulse protocol used to estimate VSP activity (bottom panel) consisted of a 300-ms step pulse to 0 mV (test pulse) and a 300-ms depolarization pulse to 75 mV. This protocol was repeated 21 times without an interval. The holding potential was −60 mV. (B) Representative traces of outward KCNQ2/3 currents elicited by depolarization to 0 mV in HEK293T cells coexpressing Dr-VSP WT, L223F, or L223Q. The traces of KCNQ2/3 currents elicited by the 21-times repeated stimuli are superimposed. (C) Plots of the time-dependent changes in the amplitudes of the outward KCNQ2/3 currents. Values were measured at the end of the depolarizing test pulses (arrows in B) and normalized by the current amplitude during the first stimulation. Gray unfilled circles (WT), black squares (L223F), and black unfilled triangles (L223Q) were drawn based on the data shown in B. Average values were connected by lines. Symbols depict the mean ± SD of data from nine WT, nine L223F, or four L223Q cells. (D) Rate constants for the declines in the normalized KCNQ2/3 currents with single exponential fitting are shown as phosphatase activities of WT, L223F, and L223Q. Single exponential fitting was performed with the dataset from each cell. Data are the mean ± SD from four to nine cells, and each symbol shows the data from each cell. Values in parentheses indicate the numbers of cells. Upper bars show P values from a two-tailed Student’s t test.