FIGURE 6.

Slow inactivation in the presence of 150 nM CTX. (A) Two-electrode voltage clamp current traces elicited by a 400 ms pulse to 0 mV from a holding voltage of −90 mV in the absence (thin trace) and in the presence (thick trace) of 150 nM CTX. In addition to the inhibitory effect of CTX on the potassium channels, it introduced a downward component to the ionic current due to the lower affinity that the toxin has for the closed channels (shaded area). This effect is reminiscent of the κ−PVIIA blockade of Shaker K-channels in symmetrical potassium concentrations (25). Traces were leak-subtracted. (B) Point-by-point ratio of the toxin and control traces. The solid line is a fit of a single exponential function with time constant of 60 ms plus a steady-state component of 0.55, an inhibition level equivalent to a K_Do = 183 nM. The extrapolation of the trace to the beginning of the voltage pulse yields to 0.64 as the extent of the unblocked closed channels, equivalent to a K_Dc = 267 nM. (C) Responses to a 15 s pulse to 0 mV from a holding voltage of −90 mV in the absence (thin trace) and in the presence (thick trace) of 150 nM CTX. Note the hump in the toxin trace corresponding to the relaxation to the higher affinity toxin-channel equilibrium in the open state. Single exponential fits to the decays of the traces shown gave time constants of 6.5 s and 7.1 s for the control and the toxin traces, respectively. Traces were leak subtracted. (D) Recovery from inactivation. (Inset) A two-pulse protocol was applied to the oocyte in the presence of 150 nM CTX as in Fig. 4. The recovered currents were measured 400 ms into the test pulse and normalized by the peak amplitude of the prepulse. The solid line is a two exponential function with time constants of 0.1 and 2.0 s as in Fig. 4 superimposed to the experimental points with an offset of 0.09. Error bars are standard error of five oocytes at CTX concentrations ranging from 150 to 700 nM.