Hanatoxin Modifies the Gating of a Voltage-Dependent K+ Channel through Multiple Binding Sites  Kenton J. Swartz, Roderick MacKinnon  Neuron  Volume 18, Issue 4, Pages 665-673 (April 1997) DOI: 10.1016/S0896-6273(00)80306-2

Figure 1 Voltage-Dependent Gating of the drk1Δ7 K+ Channel (A) Records for currents elicited by various strength depolarizations for 200 ms from a holding potential of −80 mV. Tail potential was −50 mV. RbCl (50 mM) was present in the extracellular solution. Current records were generated by subtracting records obtained in the absence and presence of 100 nM AgTx2 (see Experimental Procedures). Fifteen records are shown beginning at −30 mV and ending at +40 mV. (B) Tail current amplitude measured at −50 mV plotted as a function of the preceding test depolarization. Tail current amplitude was averaged for 1 ms beginning 2 ms after repolarization to −50 mV. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 2 HaTx Shifts the Opening of the drk1Δ7 K+ Channel to More Depolarized Voltages (A) Current records in the absence or presence of 5 μM HaTx for depolarization to either −5 mV or +50 mV for 200 ms from a holding potential of −80 mV. (B) Tail current amplitude measured at −50 mV plotted as a function of the preceding test depolarization in either the absence or presence of 5 μM HaTx. Tail current amplitude was averaged for 1 ms beginning 2 ms after repolarization to −50 mV. (C) Scaled tail currents following repolarization from +50 mV. The tail current in the presence of HaTx was scaled so that outward steady-state current at +50 mV equaled that in control. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 3 Tail Currents Elicited Following Various Strength Depolarizations in Different Concentrations of HaTx (A) Tail currents elicited following depolarization to various voltages in the absence or presence of 200 nM, 1 μM, and 5 μM HaTx. The bottom row shows scaled records for all three concentrations of toxin for depolarizations to +20, +50, and +100 mV. For the scaled records at 0 mV, only the 200 nM record is shown along with the control because at this voltage 1 and 5 μM toxin completely inhibit the opening of channels. In all cases, the test voltage was 200 ms in duration from a holding potential of −80 mV. (B) Plot of 1/τ deactivation against HaTx concentration for various strength depolarizations. (C) Plot of 1/τ deactivation against the test voltage for various concentrations of HaTx. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 4 Multiple HaTx Molecules Bind to the drk1Δ7 K+ Channel (A) Tail current amplitude measured at −50 mV plotted as a function of the preceding test depolarization in either the absence or presence of 200 nM or 5 μM HaTx. Tail current amplitude was averaged for 1 ms beginning 2 ms after repolarization to −50 mV. (B) Tail current records elicited at −50 mV following depolarization to −30 mV in the absence or presence of 200 nM or 5 μM HaTx. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 5 Model Activation Curves for Channels with Different HaTx Occupancy Levels Activation curves illustrating five populations of channels, each with different numbers of HaTx molecules bound and different gating energetics. Activation curves for the unliganded and fully liganded (four HaTxs bound) channels are from the data in Figure 2. Activation curves for the intermediate occupancy levels (one HaTx to three HaTxs bound) are drawn with intermediate placement on the voltage axis and intermediate maximal open probabilities. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 6 Measurement of the Fractional Occupancy of the drk1Δ7 K+ Channel by HaTx (A) Fraction of uninhibited tail currents elicited by various strength depolarizations. I is tail current amplitude in the presence of HaTx. Io is tail current amplitude in the absence of HaTx. Tails were elicited by repolarization to −50 mV. Test depolarizations were 200 ms in duration from a holding potential of −80 mV. (B) Tail currents elicited in the absence and presence of 100 nM HaTx following depolarization to either −10 mV or +40 mV. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 7 Concentration Dependence for Frac- tional Occupancy of the drk1Δ7 K+ Channel by HaTx Plot of probability unbound against HaTx concentration. Data points are mean ± SEM for the fraction of uninhibited tail current at different HaTx concentrations. Fraction of uninhibited tail current was measured in the plateau phase of the relations shown in Figure 6A, typically following depolarization from between −30 and −20 mV. Tail potential was −50 mV and holding potential was −80 mV; n = 3 for all data points. The solid line is a fit of the data to ρ0 = (1−p)4 where ρ0 is the probability of the channel having zero HaTxs bound, p = [HaTx] [HaTx] + Kd with Kd = 102 nM. This equation assumes four equivalent and independent binding sites. The dashed line is a fit of the data to ρ0 = (1−p), a single site binding equation with p given above and Kd = 19 nM. The dotted line is drawn according to ρ4 = (p)4 where ρ4 is the probability of all four sites being occupied and p is the same as given above with Kd=102 nM, assuming four equivalent and independent binding sites. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 8 Onset of Inhibition by HaTx Monitored at Different Voltages (A) Current records obtained using a double-pulse protocol. Currents were elicited in the absence and presence of 200 nM HaTx. A 200 ms delay was present between the first and second pulse. (B) Plot of tail current amplitude against time for both weak (−10 mV) and strong (+100 mV) depolarizations. Tail current amplitude was averaged over 1 ms beginning 2 ms after repolarization to −50 mV. The pulse protocol illustrated in (A) was delivered every 20 s. In the lower graph, the tail current amplitudes for the two pulses have been normalized to better compare the inhibition kinetics monitored using two different strength depolarizations. Single exponential fits to the data gave time constants of 41 and 93 s for −10 mV and +100 mV, respectively. See Figure 9 legend for an estimate of kon. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)

Figure 9 Recovery from Inhibition by HaTx Monitored at Different Voltages (A) Current records obtained using a triple-pulse protocol. Records were elicited before washing in HaTx, in the presence of 4 μM HaTx, or 400 s after washing HaTx from the bath. (B) Plot of tail current amplitude against time following depolarization to either −20 mV, +50 mV, or +100 mV. Tail current amplitude was averaged over 1 ms beginning 2 ms after repolarization to −50 mV. The pulse protocol illustrated in (A) was delivered every 20 s. Recovery at +50 mV and +100 mV could be fit by single exponential functions with τ = 261 s and τ = 213 s, respectively. In the lower graph, the tail current amplitude following weak depolarization to −20 mV is shown on an expanded scale to better illustrate the lag in recovery following washing out HaTx from the recording chamber. The solid line is a fit of the data to I = A(1−e−t koff)4 with koff = 4.1 × 10−3 s−1 and A = 0.53. A kon value of 4 × 104 M−1 s−1 is predicted from the ratio of koff/Kd (4.1 × 10−3 s−1/102 × 10−9 M), in reasonable agreement with the onset of inhibition kinetics shown in Figure 8. (C) Plot of log I versus log (1−e−t koff) where I is tail current following depolarization to −20 mV, t is time, and koff = 4.1 x 10−3 s−1. The solid line is a fit of the data to log (I) = mlog(1−e−t koff) + b where m is the slope (3.7), and b is the y intercept. Neuron 1997 18, 665-673DOI: (10.1016/S0896-6273(00)80306-2)