Carbamazepine action on the decay phase of macroscopic Na⁺ currents in different Y1618 mutant channels. (A) 300 μM carbamazepine (CBZ) slightly accelerates the decay of the macroscopic current in the wild-type (WT) channel but evidently slows current decay in the Y1618K mutant channel. The two superimposed traces show the representative currents elicited by a step depolarization to +10 mV from a holding potential of −120 mV in the absence (black line) and presence (red line) of 300 μM carbamazepine. The dotted line indicates zero current level. Note that in the Y1618K channel, the slowing of current decay is not discernible from the peak but only becomes evident later on (the arrow indicates an arbitrary “starting point” for the slowing of decay). (B; Top) The same experiments as those in A were repeated in different Y1618 mutant channels. 300 μM carbamazepine (red lines) slightly slows the macroscopic current decay in the Y1618D and Y1618R, yet accelerates the decay in the Y1618W channels. The dotted line indicates zero current level. (Bottom) The relative decay rate (the decay rate in 300 μM carbamazepine normalized to the decay rate in control) is compared among different Y1618 mutant (Y1618K, D, R, W, P, and A) channels (n = 3–9). The decay rate is obtained from the inverse of the time constant of the monoexponential fit to the current decay. (C) Estimation of carbamazepine binding rate to the open Y1618W mutant channels and binding affinity to the resting Y1618K mutant channels. (Left) The differences between the inverses of the current decay time constants in drug and in control are plotted against the concentration of carbamazepine in the Y1618W mutant channel (n = 3). The line is the linear regression fit to the data points of the form: blocking rate (s⁻¹) = 0.177 × D + 8, where D is the concentration of carbamazepine in μM. The slope of the line (∼1.8 × 10⁵ M⁻¹s⁻¹) gives a rough estimate of the binding rate of carbamazepine to the open Y1618W channel. (Right) The relative decay rates of the Y1618K mutant channel are plotted against different concentrations of carbamazepine. The relative decay rates in 30, 100, and 300 μM carbamazepine are 0.78 ± 0.028, 0.55 ± 0.046, and 0.48 ± 0.023, respectively (n = 4–9). The error bars are omitted in the plot. The curve is the fit of the data points with a one-to-one binding function: relative decay rate = (1 + 0.38 × D/46.4)/(1 + D/46.4), where D is the concentration of carbamazepine in μM.