Figure 9.

An enhanced version of Dr-VSP by mutation of K to M in the background of eVSP (Dr-VSP L223F). Enhanced voltage-dependent PI(4,5)P₂ phosphatase activity shown by measuring activities of KCNQ2/3 channels in HEK293T cells. (A) Representative KCNQ2/3 current traces in HEK293T cells expressing L223F (eVSP) and L223F/K303M mutant when the membrane was depolarized to 0 and 25 mV. The pulse protocol is shown in the top panel. All 11 traces recorded during the test pulse are superimposed. Dotted lines indicate zero current level. (B) Time-dependent change of the magnitude of KCNQ2/3 current at the end of the test pulse (arrows in A) with conditioning depolarization to 0, 25, 50, and 75 mV. The current amplitudes were normalized to that in the first trace. (C) Decay rate constant of normalized KCNQ2/3 current was plotted against membrane potential. KCNQ2/3 were coexpressed with L223F (eVSP) or L223F/K303M. Decay rate constant was acquired as rate constant of decay time course of KCNQ2/3 fitted with single exponential function. ^∗p < 0.05; ^∗∗p < 0.01; n.s., statistically not significant, two-tailed unpaired t-test. (D) Normalized charge was plotted against membrane potential. Q_OFF-V curves were fitted with the Boltzmann equation: Q_OFF(V)/Q_MAX = 1/[1 + exp(-ZF(V-V_Half)/RT)], where F is Faraday constant, R is the gas constant, T is the absolute temperature, Z is the effective valency, V is voltage, and V_Half is the voltage that gives half of the maximum Q_OFF (Q_MAX). Values of (V_Half, Z) are (122.9 ± 1.3 mV, 1.0) and (122.0 ± 0.8 mV, 1.1) for L223F/K303M and L223F (eVSP), respectively. Black curve, L223F/K303M; black dotted curve, L223F (eVSP). In (B) to (D), data are presented as mean ± SE.