García E , Scanlon M , Naranjo D .

	Scheme S1.
	Figure 1. Effect of the κ-PVIIA on Shaker IR. Two-electrode voltage clamp recordings of an oocyte expressing Shaker H4 Δ(6-46) in a control solution (A) and in the presence of 100 nM κ-PVIIA (B). Currents were elicited from a holding voltage of −90 mV by activating pulses of −60 to +50 mV with intervals of 10 mV. Dotted lines indicate the zero current level. (C) Potassium conductance measured from the current at the end of the activating pulse. • and ○ indicate conductance with and without toxin, respectively. Values are taken from the records shown in A and B. After leak subtraction, each value of conductance was calculated assuming a reversal potential of −100 mV. The normalizing value, gmax, is the conductance measured at the end of the control record taken at +40 mV.
	Figure 2. Voltage dependence of κ-PVIIA inhibition. (A) After leak subtraction, records taken in the presence of 100 nM κ-PVIIA at each pulse voltage (Fig. 1 B) were divided point-by-point by leak-subtracted control record taken at corresponding voltages. Shown are the results of such an operation for the interval 2–100 ms after the beginning of the 0 to +50 activating pulses. For the pulses to −10 and −20 mV, the points intervals 15–100 and 18–100, respectively, are shown. The thin lines correspond to fits of an exponential equation of the from: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}f(t)\;=\;a\;-\;b\;{\cdot}\;{\mathrm{e}}^{-}t/{\mathrm{{\tau}}}\end{equation*}\end{document}, where a, b, and τ correspond to the steady state fractional current, the amplitude extrapolated to the beginning of the pulse, and the time constant, respectively. Note that the IPVIIA/Icontrol curves converge to similar values when extrapolated to the beginning of the activating pulse. The mean ± SEM value is: 0.35 ± 0.004 for the −10- to +50-mV pulses. These values correspond to the resting inhibition at holding potential. (B) Plot of the results from the kinetic analysis shown in A. Plotted, as mean ± SEM for four separate experiments, are the time constant and steady state fractional current, (IPVIIA/Icontrol)∞, as a function of the activating pulse voltage (a and τ in A). The continuous line over the time constant data (•) was drawn according to with \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}_{(0)}\;=\;39\;{\mathrm{ms\;and\;V}}_{{\mathrm{s}}}\;=\;49\;{\mathrm{mV}}\end{equation*}\end{document}.
	Figure 3. Dose dependence of κ-PVIIA inhibition. (A) Four superimposed TEVC records taken with activating pulse to +40 mV in the presence of the indicated nanomolar concentrations of the toxin. Record taken with 300 nM toxin corresponds to a different experiment, but for the sake of comparison was scaled to represent the actual blockade. The dotted line indicates the zero current level. (B) Records divided point-by-point by their respective controls with no toxin added. Thin lines are single exponential fits extrapolated to the beginning of the voltage pulse as described in Fig. 2 A. (Inset) Dose dependence of the inhibition at resting obtained from the values extrapolated to the beginning of the pulse. Measurements made for 30, 100, and 300 nM represent mean ± SEM for three to four oocytes. The value for 1 μM corresponds to a single measurement. The solid line corresponds to a simple inhibition hyperbola with a half-saturation at 65 nM. (C) Rate of relaxation measured as the reciprocal of the time constants obtained from the single exponential fits to data as shown in B. Straight lines are linear regression to the data. For a 1:1 stoichiometry, this rate is represented by . At each voltage, the slope is the second order rate constant, kon, and the intercept at zero toxin corresponds to the dissociation rate, koff. (D) Plot of kon and koff as a function of the applied voltage pulse for each measurement made in C. Solid lines were drawn according to . The values are shown in Table .
	Figure 4. Comparison between pulse and chronic applications of κ-PVIIA to outside-out patches. A and D show current records from two different outside-out patches elicited by 200-ms long voltage pulses from −60 to +50 mV, with intervals of 10 mV. Bath and pipette K+ concentration were 2 and 15 mM, respectively (see methods). (B) Rapid applications of 1 mM TEA+/500 nM κ-PVIIA to an outside-out patch ∼40 ms after the beginning of the activating voltage pulse. The shaded area (Pulse) indicates the duration of the toxin application. Records shown are the average of four individual records at each voltage. (Inset) A single record taken at 0 mV showing the inflection in the K currents produced by the presence of 1 mM TEA+ in the toxin-containing solution. The TEA+-induced inflection is lost in the average because of the variable latency of the solenoid valve. (E) Chronic application of 1 mM TEA+/500 nM κ-PVIIA to the outside-out patch. The solenoid valve was open while the acquisition lasted. C and F show point-by-point divisions of the leak-subtracted TEA+/toxin records by their respective leak-subtracted controls. Single-exponential fits to each resulting relaxation while the toxin was present were performed (thin lines shown alternately for clarity). The sections including current at holding potential and capacitative transient were eliminated. (F) Exponential functions were extrapolated to the beginning of the voltage pulse and converge to the same value. In A, B, D, and E, the dotted line indicates the zero current level.
	Figure 5. Results from the kinetic analysis to the relaxation resulting in the point-by-point division of the patch clamp experiments shown in Fig. 4. (A) Time constants of the exponential fits to the relaxations are as those shown in Fig. 4C and Fig. F. Data are plotted as mean ± SEM. ○ and • are for pulse and chronic relaxations in the presence of the toxin, respectively. □ are the time constant for recovery resulting from removal of the toxin at the end of the pulse application. Solid lines are best fits to , with: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}_{{\mathrm{on}}}\;=\;27\;{\pm}\;2\;{\mathrm{ms\;and\;V}}_{{\mathrm{s\;on}}}\;=\;80\;{\pm}\;17\;{\mathrm{mV}}\end{equation*}\end{document} for pulse applications, and \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{{\tau}}}_{{\mathrm{off}}}\;=\;73\;{\pm}\;2\;{\mathrm{ms\;and\;V}}_{{\mathrm{s\;off}}}\;=\;56\;{\pm}\;4\;{\mathrm{mV}}\end{equation*}\end{document} for recovery after toxin removal (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}n\;=\;13\end{equation*}\end{document}; pooled data from seven experiments in 100-Kin//1-Kex, and six in 15-Kin//1-Kex). For chronic applications: τ = 29 ± 3 ms and Vs = 141 ± 58 mV (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}n\;=\;11\end{equation*}\end{document}; pooled data from four experiments with 100-kin and seven with 15-kin in the pipette). No significantly different values for the time constant of onset relaxation are detected (○ and •; P > 0.05; paired t test for the data between −20 and +40 mV). (B) Steady state inhibition determined from the asymptotic values of the single exponential fits. The levels of steady state fractional current reached with both protocols are significantly different (P < 0.00001; paired t test for the data between −20 and +40 mV).
	Figure 6. Kinetic values of the κ-PVIIA inhibition. Small symbols represent different solutions to the system of and , while the big symbols (○ and •) represent arithmetic averages of the kon and koff values obtained by the different methods (see text). Small open symbols represent kon; ○ and □ result from the pulse application protocol, while the (▵) plot results from the chronic application protocol. Small filled symbols represent koff; • and ▪ from the pulse application protocol, and ▴ from the chronic application protocol. Solid lines are single exponential fits of . The rate constant values that describe the data are shown in Table , under pooled patch clamp data.
	Figure 7. Effect of TEA+ on the kinetic parameters of κ-PVIIA inhibition. Average values of kon (filled symbols) and koff (open symbols) at different TEA+ concentrations. All values shown were normalized by the rates measured at zero applied potential in the absence of TEA+. Symbols represent different concentrations of TEA+: 0 mM (▪ and □), 1 mM (• and ○), 5 mM (♦ and ⋄), and 10 mM (▴ and ▵). The continuous lines correspond to the normalized values of kon and koff measured in 10 mM NaCl added on top of the normal recording solution. Each value represents the average ± SEM from two to five different oocytes.
	Figure 8. Effect of internal K+ removal on the dissociation rate of κ-PVIIA. (A) Pulse application of 1 mM TEA+/500 nM κ-PVIIA (shaded area) to an outside-out patch. External solution contained 100 mM K+ and the pipette was filled with 100 mM NMG+ (0-Kin//100-Kex; see methods). Pipette potential was maintained at −90 mV and 400-ms pulses were applied from −70 to +50 in 10-mV increments. These records represent the average of four identical traces. The dotted line is the zero current level. (B) Voltage dependence of the dissociation rate in the absence of internal K+. A single exponential function was fitted to each time course of current recovery after toxin removal. Dissociation rate constants were calculated from the reciprocal of the resulting time constants. The solid line was traced with the following parameters: \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}k_{{\mathrm{off}}(0)}\;=\;11\;{\pm}\;1\;{\mathrm{s}}^{-}1\;{\mathrm{and}}\;z{\mathrm{{\delta}}}\;=\;0.28\;{\pm}\;0.05\end{equation*}\end{document}. Each data point represents mean ± SEM for four different patches. For comparison, the dashed line represents the voltage dependence of the dissociation rate measured in whole oocyte bathed in 100 mM external K+ \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \usepackage[Euler]{upgreek} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}(k_{{\mathrm{off}}(0)}\;=\;22\;{\mathrm{s}}^{-}1\;{\mathrm{and}}\;z{\mathrm{{\delta}}}\;=\;0.66)\end{equation*}\end{document}.
	Figure 9. κ-PVIIA protects the Shaker pore from collapsing. Traces show 10 current records from outside-out patches with 100 mM external K+ and 100 NMG+ in the pipette (0-Kin//100-Kex, see methods). Except for the initial control in A, traces shown correspond to one in every four acquired. The dotted line indicates the zero current level. The lower cartoon under each set of traces depicts the kinetic states that the Shaker K channels are putatively visiting during the experimental protocol: closed, open-conducting, open-empty, and pore-collapsed. (A) Exposing channels to zero potassium produces a cumulative reduction of the current. Every 5 s, the pipette voltage was stepped from −90 to −40 mV for 200 ms to open channels. After an initial control trace, distinguished by the inward tail current, ∼15 ms near the end of the depolarization, the pulse of a zero K+ solution is given (0-Kin//0-Kex). We estimated that the channels are exposed for 5–8 ms to this 0-Kin//0-Kex experimental condition. During this time, K+ residing in the pore are allowed to escape, reducing the channel pore occupancy. The fraction of channels not entering nonconductive state(s) promoted by this experimental manipulation is plotted (C, ○). (B) Exposing channels to toxin does not produce reduction in the current. Similar to A, every 5 s, the pipette voltage was stepped from −90 to −40 mV for 200 ms to open channels. At ∼150 ms after the beginning of the voltage pulse, a 150-ms long TEA+/toxin pulse was applied to block the channels (shaded area). During this period, ions residing in the pore of the open/blocked channel are expected to escape away into the intracellular side of the channel. The activating voltage pulse ends when ∼60% of the channels are blocked and near equilibrium. The toxin residence time at this voltage is three orders of magnitude larger than that of possibly the last occupying ion (150 μs vs. 150 ms, see Fig. 8 B; Baukrowitz and Yellen, 1996). Also, this residence time is 20–30-fold larger than the 5–8 ms of exposure to zero K+ needed to observe reduction in the current as shown in A. If the toxin does not protect, and if the 60% toxin binding at the end of the pulse is in equilibrium for more than 5–8 ms, at least a 40% reduction in the current is expected to be produced at the 40th pulse. This is a very conservative low limit that was obtained from the exponential fitting to the 0-Kex data in C. (C) Fractional current averages of non–leak-subtracted records. Averages were measured in the 50–100-ms interval after the beginning of the voltage pulse for both experimental conditions and the error bars represent SD in each interval. A solid line was drawn according to a single exponential with a decay constant of 44 pulses.

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