Neuron, Vol. 18, 665–673, April, 1997, Copyright 1997 by Cell Press Hanatoxin Modifies the Gating of a Voltage-Dependent K؉ Channel through Multiple Binding Sites

Kenton J. Swartz*† and Roderick MacKinnon*‡§ acid sequence, HaTx seems to have little in common *Department of Neurobiology with the previously described pore-blocking Kϩ channel Harvard Medical School inhibitors (Swartz and MacKinnon, 1995). By what mech- Boston, Massachusetts 02115 anism does HaTx inhibit voltage-gated Kϩ channels? §Laboratory of Molecular Neurobiology Voltage-gated Kϩ channels contain a Kϩ selective pore and Biophysics that opens and closes in response to changes in mem- The Rockefeller University brane voltage. So HaTx might inhibit the channel either New York, New York 10021 by physically plugging the pore or by interfering with the way that changes in membrane voltage control opening and closing of the pore. In the present paper, we de- Summary scribe experiments that address the mechanism of inhi- bition and the stoichiometry of the interaction between We studied the mechanism by which Hanatoxin (HaTx) HaTx and the drk1 Kϩ channel. Using a tail current proto- inhibits the drk1 voltage-gated K؉ channel. HaTx inhib- col, we discovered that the drk1 Kϩ channel can open its the K؉ channel by shifting channel opening to more with HaTx bound to it. bound channels require depolarized voltages. Channels opened by strong de- strong depolarization to open and deactivate much polarization in the presence of HaTx deactivate much faster upon repolarization, suggesting that HaTx inhibits faster upon repolarization, indicating that toxin bound by modifying the gating of the drk1 voltage-gated Kϩ channels can open. Thus, HaTx inhibits the drk1 K؉ channel. We then developed a way of measuring the channel, not by physically occluding the ion conduc- fractional occupancy of the channel by HaTx using tail tion pore, but by modifying channel gating. Occupancy current kinetics as an indicator of open channel occu- of the channel by HaTx was studied using various pancy by HaTx. The concentration dependence for equi- strength depolarizations. The concentration depen- librium occupancy as well as the kinetics of onset and dence for equilibrium occupancy as well as the kinet- recovery from inhibition indicate that multiple HaTx mol- ϩ ics of onset and recovery from inhibition indicate that ecules can simultaneously bind to a single K channel. multiple HaTx molecules can simultaneously bind to a single K؉ channel. These results are consistent with Results a simple model in which HaTx binds to the surface of ϩ the drk1 K؉ channel at four equivalent sites and alters Inhibition of the drk1 voltage-gated K channel by HaTx ϩ the energetics of channel gating. was studied by expressing the K channel in Xenopus oocytes and controlling membrane voltage with a con- Introduction ventional two-electrode voltage clamp. Under physio- logical ionic conditions, and at voltages where the chan- A number of from scorpion and snake nel closes to near completion, the deactivation kinetics ϩ inhibit Kϩ channels by binding with 1:1 stoichi- of the drk1 K channel are faster than the settling time ϩ ometry to the outer pore region and physically blocking of the voltage clamp. We therefore used 50 mM Rb in Kϩ conduction (Miller, 1988; MacKinnon and Miller, the extracellular solution to slow channel deactivation 1988, 1989; Anderson et al., 1988; Hurst et al., 1991; (Swenson and Armstrong, 1981; Matteson and Swen- Stocker et al., 1991; Park and Miller, 1992). The Kϩ chan- son, 1986) and facilitate measurement of inward tail cur- nel inhibitors from scorpion venom were initially used rents. We wanted to be able to completely block ion ϩ to identify the pore region of a voltage-gated Kϩ channel flux through the drk1 K channels so as to identify and and have been subsequently used to probe the molecu- subtract leak and background conductances. The wild- ϩ lar structure of the outer pore vestibule (MacKinnon and type drk1 K channel is rather insensitive to the pre- Miller, 1989; MacKinnon et al., 1990; Stocker and Miller, viously described pore-blocking peptide inhibitors (Gar- 1994; Stampe et al., 1994; Hildago and MacKinnon, cia et al., 1994; unpublished data). We therefore used a ϩ 1995; Naranjo and Miller, 1996; Aiyar et al., 1996; Ranga- mutant drk1 K channel (drk1⌬7; Aggarwal, 1996) that is blocked completely, and with high affinity, by the nathan et al., 1996). pore-blocker Agitoxin (AgTx ; Garcia et al., 1994; Aggar- Hanatoxin (HaTx) is a 4.1 kDa toxin that we 2 2 wal, 1996). Figure 1A shows a number of current records previously isolated from the venom of the spider Gram- for the drk1⌬7 Kϩ channel obtained using Rbϩ to slow mostola spatulata (Swartz and MacKinnon, 1995) by deactivation and AgTx to subtract the leak and back- screening for activity against the drk1 voltage-gated Kϩ 2 ground conductances. The Rbϩ tail currents decayed channel (Frech et al., 1989). At the level of the amino with time constants ranging from 50–60 ms, far slower 1msinف) than the settling time of the voltage clamp †Present address: Molecular Physiology and Biophysics Unit, Na- the example in Figure 1). The amplitude of the Rbϩ tail tional Institute of Neurological Disorders and Stroke, National Insti- current measured at Ϫ50 mV is plotted as a function of tutes of Health, Building 36, Room 2C19, 36 Convent Drive, MSC 4066, Bethesda, Maryland 20892. voltage of the preceding test depolarization (Figure 1B). ‡ Present address: Laboratory of Molecular Neurobiology and Bio- The activation curve in Figure 1B shows that the tail physics, The Rockefeller University, 1230 York Avenue, New York, current amplitude is a saturable function of the test New York 10021. voltage. Neuron 666

Figure 1. Voltage-Dependent Gating of the drk1⌬7 Kϩ Channel (A) Records for currents elicited by various strength depolarizations for 200 ms from a holding potential of Ϫ80 mV. Tail potential was Ϫ50 mV. RbCl (50 mM) was present in the extracellular solution. Current records were generated by subtracting records obtained ϩ in the absence and presence of 100 nM AgTx2 (see Experimental Figure 2. HaTx Shifts the Opening of the drk1⌬7 K Channel to Procedures). Fifteen records are shown beginning at Ϫ30 mV and More Depolarized Voltages ending at ϩ40 mV. (A) Current records in the absence or presence of 5 ␮M HaTx for (B) Tail current amplitude measured at Ϫ50 mV plotted as a function depolarization to either Ϫ5mVorϩ50 mV for 200 ms from a holding of the preceding test depolarization. Tail current amplitude was potential of Ϫ80 mV. averaged for 1 ms beginning 2 ms after repolarization to Ϫ50 mV. (B) Tail current amplitude measured at Ϫ50 mV plotted as a function of the preceding test depolarization in either the absence or pres- ence of 5 ␮M HaTx. Tail current amplitude was averaged for 1 ms Voltage Dependence of Inhibition by HaTx beginning 2 ms after repolarization to Ϫ50 mV. (C) Scaled tail currents following repolarization from ϩ50 mV. The We initially examined the inhibitory effects of HaTx at tail current in the presence of HaTx was scaled so that outward different membrane voltages. Figure 2 shows activation steady-state current at ϩ50 mV equaled that in control. curves obtained in the absence and presence of a large concentration of extracellular HaTx. HaTx shifted the opening of the drk1⌬7 Kϩ channel to more depolarized HaTx. Unscaled records can be seen in Figure 2A. The voltages. At negative voltages (Ϫ30 to Ϫ5 mV), 5 ␮M channels opened by strong depolarization in the pres- HaTx completely inhibited current through the drk1⌬7 ence of HaTx deactivate much faster than the control Kϩ channel (Figures 2A and 2B). In contrast, the drk1⌬7 channels. This result suggests that either the toxin re- Kϩ channel opened and conducted at test voltages posi- mains bound to the drk1⌬7 Kϩ channel when it opens tive to 0 mV while in the continual presence of 5 ␮M or that the toxin unbinds rapidly upon depolarization HaTx (Figures 2A and 2B). In addition, HaTx decreased and rebinds rapidly upon repolarization. The binding the maximal tail current amplitude elicited following kinetics of HaTx (5 ␮M) at Ϫ80 mV are three orders of (s at 5 ␮M; data not shown 4 ف even strong depolarizations (e.g., ϩ100 mV). magnitude slower (␶ ms), indicating 5 ف In principle, a number of distinct mechanisms might than the kinetics of this tail current (␶ explain why HaTx shifts the opening of the drk1⌬7 Kϩ that the speeding of deactivation is not caused by re- channel to more depolarized voltages. An important clue binding of the toxin. This is reinforced by the finding to the actual mechanism can be seen by looking closely that tail current decay kinetics reach a saturating value at the kinetics of deactivation. Scaled tail currents elic- as HaTx concentrations are increased (see Figure 3). ited following depolarization to ϩ50 mV are shown in These results suggest that the channels opened by Figure 2C, both in the presence and absence of 5 ␮M strong depolarization in the presence of HaTx have the Hanatoxin Modifies the Gating of a Kϩ Channel 667

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 ␮M, and 5 ␮M HaTx. The bottom row shows scaled records for all three concentrations of toxin for depolarizations to ϩ20, ϩ50, and ϩ100 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 ␮M toxin completely inhibit the opening of channels. In all cases, the test voltage was 200 ms in duration from a holding potential of Ϫ80 mV. (B) Plot of 1/␶ deactivation against HaTx concentration for various strength depolarizations. (C) Plot of 1/␶ deactivation against the test voltage for various concentrations of HaTx. toxin continually bound to them. We conclude that HaTx deactivation in the presence of HaTx became faster with is a gating modifier and that it shifts channel opening stronger depolarization and with higher concentrations to more depolarized voltages by altering the energetics of HaTx. For all HaTx concentrations, the tail current of channel gating. decay rate reached a saturating value at test voltages Figure 3A shows tail currents elicited following depo- of about ϩ50 mV. Likewise, at all test voltages, the tail larization to various voltages in the presence of different current decay kinetics saturated between 1 and 5 ␮M concentrations of HaTx. In the absence of HaTx, the HaTx. We interpret these results to suggest that multiple decay of the tail could be relatively well fit by a single populations of channels exist in the presence of HaTx ms and was rather and that the dependence of this distribution on toxin 50 ف exponential function with ␶ unaffected by the voltage of the preceding depolariza- concentration arises from multiple HaTx occupancy lev- tion. In contrast, in the presence of HaTx, at voltages els. Moreover, the relative distribution of channels positive to 0 mV, the decay of tail current was multiexpo- opened by any given depolarization varies as a function nential and depended on both the test voltage and toxin of the strength of that depolarization because channels concentration. The fastest component had a time con- with different numbers of HaTx molecules bound have .ms. At least two additional components distinct gating energetics 5ف stant of were present that had time constants between the 5 ms component and the control 50 ms component. In Figure 3B, the decay of the tail, although a multiexponential Multiple HaTx Molecules Bind to the Channel process, was fit by a single exponential function, and The concentration dependence of the effect of HaTx on the reciprocal time constant was plotted against HaTx channels opened by various strength depolarizations concentration for test voltages between ϩ20 and ϩ100 indicates that a single drk1⌬7 Kϩ channel can be occu- mV. In Figure 3C, the time constant for tail current decay pied by multiple toxin molecules. Activation curves are is plotted against the voltage of the preceding depolar- shown in Figure 4A in the absence or presence of either ization for various HaTx concentrations. The kinetics of 200 nM or 5 ␮M HaTx. In the example in Figure 4, 200 Neuron 668

Figure 4. Multiple HaTx Molecules Bind to the drk1⌬7 Kϩ Channel (A) Tail current amplitude measured at Ϫ50 mV plotted as a function of the preceding test depolarization in either the absence or presence of 200 nM or 5 ␮M HaTx. Tail current amplitude was averaged for 1 ms beginning 2 ms after repolarization to Ϫ50 mV. (B) Tail current records elicited at Ϫ50 mV following depolarization to Ϫ30 mV in the ab- sence or presence of 200 nM or 5 ␮M HaTx.

nM HaTx inhibits tail currents elicited following weak reaches a plateau. This suggests that when even a single depolarization to Ϫ30 mV by 94%. This indicates that HaTx molecule binds to a channel, the channel-open at least 94% of the channels in this oocyte have at least probability is shifted sufficiently to the right so that a a single HaTx molecule bound to them. Subsequent bound channel will not open at these voltages. Thus, at application of a higher concentration of HaTx (5 ␮M) negative voltages, the fraction of uninhibited tail current produces an additional shift in the activation curve to reflects the fraction of unbound channels. Analysis of the right. The additional shift in the activation curve the kinetics of tail currents elicited following various obtained by changing from 200 nM to 5 ␮M HaTx cannot strength depolarizations supports this conclusion. Fol- be accounted for by going from 94% to near full occu- lowing weak depolarizations, the tail currents that remain pancy, and strongly suggests that there is more than one in various concentrations of HaTx have similar kinetics site on the drk1⌬7 Kϩ channel to which HaTx can bind. when compared to the control tail currents (Figure 6B). This is not the case for stronger depolarizations. Follow- Equilibrium Occupancy of the Channel by HaTx ing strong depolarizations, the tail currents that remain We wanted to study the concentration dependence for in the presence of HaTx have much faster kinetics than occupancy of the channel by HaTx at equilibrium. This the control tail currents (Figure 6B). We conclude that is not completely straightforward since toxin bound at negative voltages, the fractional tail current is a good channels can open and since there are multiple toxin measure of the fraction of channels unbound by HaTx. binding sites on each channel. Figure 5 illustrates a way Figure 7 shows how the fraction of unbound channels to think about this problem that motivated all of the (␳0) varies as a function of HaTx concentration. The data experiments that follow. We do not know exactly how points are mean fractions of uninhibited tail current mea- many toxins can bind to a channel, but since the channel sured at negative voltages for various concentrations that we are studying is a tetramer with four identical of HaTx (Figure 7). The data conformed well to equation subunits, maybe as many as four HaTxs can bind to a 1, which assumes four equivalent and independent bind- channel, one per subunit. If this is true, then at least ing sites, each with an equilibrium dissociation constant five different channel populations should exist, unbound (Kd) of 102 nM (Figure 7; solid line). channels and channels with between one and four toxins [HaTx] 4 bound to them. Given the analysis of tail current kinetics, ␳0 ϭ 1 Ϫ (1) ΂ [HaTx] ϩ Kd΃ it is likely that the different channel populations have distinct gating energetics. We know what the activation The data for the fraction of uninhibited current deviated curve looks like for the unliganded channel since this considerably from an equation for a single site (Figure can be obtained in the absence of toxin. We think that the activation curve for the fully liganded channel is that which we observe in 5 ␮M HaTx since concentrations in this range seem to be saturating (e.g., Figure 3). The intermediate channel populations (one to three toxins bound) will have intermediate activation curves some- thing like that illustrated in Figure 5. If the actual situation is anything like that illustrated in Figure 5, then the most direct way to measure occu- pancy of the channel by HaTx is at negative voltages where even a single bound toxin molecule is sufficient to completely inhibit opening of the channel. Since HaTx Figure 5. Model Activation Curves for Channels with Different HaTx bound channels deactivate much faster than the unli- Occupancy Levels ganded channel, the tail current kinetics should confirm Activation curves illustrating five populations of channels, each with that the fraction of uninhibited current at negative volt- different numbers of HaTx molecules bound and different gating ages arises from unbound channels. Figure 6 shows the energetics. Activation curves for the unliganded and fully liganded (four HaTxs bound) channels are from the data in Figure 2. Activation concentration dependence for fractional inhibition of tail curves for the intermediate occupancy levels (one HaTx to three currents for various strength depolarizations. At nega- HaTxs bound) are drawn with intermediate placement on the voltage tive voltages, the fraction of uninhibited tail current axis and intermediate maximal open probabilities. Hanatoxin Modifies the Gating of a Kϩ Channel 669

Figure 6. Measurement of the Fractional Oc- cupancy of the drk1⌬7 Kϩ Channel by HaTx (A) Fraction of uninhibited tail currents elic- ited by various strength depolarizations. I is tail current amplitude in the presence of

HaTx. Io is tail current amplitude in the ab- sence of HaTx. Tails were elicited by repolar- ization to Ϫ50 mV. Test depolarizations were 200 ms in duration from a holding potential of Ϫ80 mV. (B) Tail currents elicited in the absence and presence of 100 nM HaTx following depolar- ization to either Ϫ10 mV or ϩ40 mV.

7; dashed line). Figure 7 also shows the predicted con- achieving a maximal effect is consistent with a simple centration dependence for occupancy of all four sites binding model with four equivalent and independent

(␳4) given by equation 2, again assuming four equivalent sites. and independent sites (Figure 7; dotted line). [HaTx] 4 Kinetics of Inhibition by HaTx ␳4 ϭ (2) The kinetics of the onset and recovery from inhibition ΂[HaTx] ϩ Kd΃ by HaTx were examined using various strength depolar- Given a Kd for each site of 102 nM, the channel is pre- izations to monitor occupancy of the channel by HaTx. dicted to approach full occupancy by HaTx at concen- Consider the following sequential binding scheme that trations just Ͼ10 ␮M(␳4 ϭ0.96 at 10 ␮M toxin). To a incorporates four equivalent and independent HaTx first approximation, this is consistent with the finding binding sites: that the inhibitory effect of HaTx for moderate to strong ␮M (e.g., Figure 3). 4 kon 3 kon 2 kon kon 5ف depolarizations saturates at * * * * These results confirm that multiple HaTx molecules bind C ) CH1 ) CH2 ) CH3 ) CH4 koff 2 koff 3 koff 4 koff to the drk1⌬7 Kϩ channel. The concentration depen- dence for the fraction of unbound channels (␳0) and for C is the unbound channel, and Cx are channels with

Figure 7. Concentration Dependence for Frac- tional Occupancy of the drk1⌬7 Kϩ Channel by HaTx Plot of probability unbound against HaTx concentration. Data points are mean Ϯ SEM for the fraction of uninhibited tail current at different HaTx concentrations. Fraction of un- inhibited tail current was measured in the pla- teau phase of the relations shown in Figure 6A, typically following depolarization from between Ϫ30 and Ϫ20 mV. Tail potential was Ϫ50 mV and holding potential was Ϫ80 mV; n ϭ 3 for all data points. The solid line is a fit 4 of the data to ␳0 ϭ (1Ϫp) where ␳0 is the probability of the channel having zero HaTxs [HaTx] bound, p ϭ with Kd ϭ 102 nM. [HaTx] ϩ Kd This equation assumes four equivalent and independent binding sites. The dashed line

is a fit of the data to ␳0 ϭ (1Ϫp), a single site

binding equation with p given above and Kd ϭ 19 nM. The dotted line is drawn according to 4 ␳4 ϭ (p) where ␳4 is the probability of all four sites being occupied and p is the same as

given above with Kdϭ102 nM, assuming four equivalent and independent binding sites. Neuron 670

between one and four HaTx molecules bound. kon is the second order association rate constant (MϪ1sϪ1) for

HaTx binding to a single site, and koff is the first order dissociation rate constant (sϪ1) for HaTx unbinding from a single site. When monitoring channel occupancy at negative voltages, the onset of inhibition should be times faster at high toxin concentrations) than 4ف) faster predicted by the microscopic rate constant kon because there are four possible sites, and any one occupied will completely inhibit opening (see Figure 5). At negative voltages, if we start with a fully liganded channel (CH4), the recovery from inhibition is expected to be sigmoidal because the channel will not open until all toxins have dissociated. For example, if there are four equivalent and independent sites, then the appearance of unliganded channels should obey equation 3, which predicts a delay before unliganded channels are seen. 4 Ϫtkoff ␳0 ϭ ΂1 Ϫ e ΃ (3) The sigmoidal shape predicted by equation 3 would become less pronounced with stronger depolarizations where HaTx bound channels can open. For example, at voltages where channels with three or fewer HaTxs can open, an exponential recovery would be expected. Figure 8 shows an experiment where the kinetics of onset of inhibition were studied using two different strength depolarizations to monitor channel occupancy by HaTx. Two pulses are given in rapid succession where the first is a weak depolarization to Ϫ10 mV, and the second is a strong depolarization to ϩ100 mV. The whole pulse protocol is given every 20 s. The inhibition kinetics observed when monitoring channel occupancy at negative voltages (␶ϭ41 s) is considerably faster than observed when monitoring channel occupancy at positive voltages (␶ϭ93 s), even though the two mea- surements are being made almost instantaneously. Figure 9 shows an experiment where the kinetics of recovery from inhibition were studied using various strength depolarizations to monitor channel occupancy by HaTx. Three pulses are given in rapid succession Figure 8. Onset of Inhibition by HaTx Monitored at Different where the first is a weak depolarization to Ϫ20 mV, the Voltages second is a moderately strong depolarization to ϩ50 (A) Current records obtained using a double-pulse protocol. Cur- mV, and the third is a strong depolarization to ϩ100 mV. rents were elicited in the absence and presence of 200 nM HaTx. A 200 ms delay was present between the first and second pulse. The whole pulse protocol is given every 20 s. HaTx (4 (B) Plot of tail current amplitude against time for both weak (Ϫ10 ␮M) was used so that the channel would be nearly fully mV) and strong (ϩ100 mV) depolarizations. Tail current amplitude liganded (␳4 ϭ 0.9). Upon removal of toxin from the re- was averaged over 1 ms beginning 2 ms after repolarization to Ϫ50 cording chamber, the current elicited by the strong de- mV. The pulse protocol illustrated in (A) was delivered every 20 s. polarizations to ϩ50 and ϩ100 mV began to recover In the lower graph, thetail current amplitudes for the two pulses have been normalized to better compare the inhibition kinetics monitored immediately and proceed along an approximately expo- using two different strength depolarizations. Single exponential fits nential time course. In contrast, the current elicited by to the data gave time constants of 41 and 93 s for Ϫ10 mV and weak depolarization recovered along a sigmoidal time ϩ100 mV, respectively. See Figure 9 legend for an estimate of kon. s. The graph in Figure 80ف course with a clear lag of 9C shows a double log plot of tail current measured following weak depolarization to Ϫ20 mV as a function Discussion Ϫt koff Ϫ3 of (1Ϫe ). The line corresponds to koff ϭ 4.1 ϫ 10 sϪ1 with a slope of 3.7, in reasonable agreement with In this study, we attempt to explain the mechanism by the fourth power relationship proposed in equation 3. which HaTx inhibits the drk1 voltage-gated Kϩ channel. This result perhaps most clearly demonstrates that there We observed that HaTx shifts the opening of channels are multiple, probably four, sites on the drk1⌬7 Kϩ chan- to more depolarized voltages and that the kinetics of nel to which HaTx binds. deactivation were hastened quite dramatically (Figures Hanatoxin Modifies the Gating of a Kϩ Channel 671

2 and 3). The effect of HaTx on deactivation kinetics turns out to be of seminal importance. First, it allows us to conclude that HaTx bound channels can in fact open and that they do so with altered energetics. This strongly argues that HaTx inhibits the drk1 voltage- gated Kϩ channel by modifying its gating. Second, the effect of HaTx on deactivation kinetics serves as a marker for a HaTx bound channel. Even though the HaTx bound channels can open, we can distinguish them from the unbound channels by virtue of the fact that they deactivate faster (Figure 6). A number of observations in the present study argue that there are multiple sites on the drk1 Kϩ channel to which HaTx binds. The concentration dependence of the equilibrium occupancy of the channel by HaTx was considerably steeper than predicted by a simple 1:1 stoichiometry between HaTx and the channel (Figures 6 and 7). In fact, the equilibrium occupancy data were well fit by a binding model with four equivalent and independent sites (Figure 7). The concentration depen- dence for complete occupancy (four toxins bound) pre- dicted from this model (Figure 7) is consistent with the concentration dependence that we observed for the maximal effects of HaTx assayed using both moderate and strong depolarizations (Figure 3). The kinetics of the onset and recovery from inhibition by HaTx were monitored using various strength depolarizations. The observed kinetics would have been independent of the strength of the test voltage used to assay occupancy if there were only a single site on the drk1 Kϩ channel to which HaTx bound. We observed that the kinetics dramatically depended on the strength of the test depo- larization used to assay occupancy. For example, there was a pronounced lag in the recovery upon washout of HaTx when monitoring with weak depolarizations where channels would not be expectedto recover until all HaTx molecules had dissociated. Finally, the kinetics of deac- tivation were multiexponential in the presence of HaTx, and these kinetics depended on both HaTx concentra- Figure 9. Recovery from Inhibition by HaTx Monitored at Different tion and the strength of the depolarization. This seems Voltages indicative of multiple populations of channels arising (A) Current records obtained using a triple-pulse protocol. Records from different HaTx stoichiometries. In a previous study, were elicited before washing in HaTx, in the presence of 4 ␮M HaTx, we observed that the concentration dependence for or 400 s after washing HaTx from the bath. both equilibrium occupancy and the kinetics of binding (B) Plot of tail current amplitude against time following depolariza- tion to either Ϫ20 mV, ϩ50 mV, or ϩ100 mV. Tail current amplitude and unbinding were consistent with 1:1 stoichiometry ϩ was averaged over 1 ms beginning 2 ms after repolarization to Ϫ50 between HaTx and the drk1 K channel (Swartz and mV. The pulse protocol illustrated in (A) was delivered every 20 s. MacKinnon, 1995). The apparent 1:1 stoichiometry re- Recovery at ϩ50 mV and ϩ100 mV could be fit by single exponential sulted because multiple occupancy is evident only at functions with ␶ϭ261 s and ␶ϭ213 s, respectively. In the lower high toxin concentrations and at the extremes of mem- graph, the tail current amplitude following weak depolarization to Ϫ20 mV is shown on an expanded scale to better illustrate the lag brane voltage. These conditions were not investigated in recovery following washing out HaTx from the recording chamber. in the previous study. Ϫt koff 4 The solid line is a fit of the data to I ϭ A(1Ϫe ) with koff ϭ 4.1 ϫ In summary, we conclude that HaTx binds to multiple Ϫ3 Ϫ1 4 Ϫ1 Ϫ1 ϩ 10 s and A ϭ 0.53. A kon value of 4 ϫ 10 M s is predicted sites on the K channel and the data are compatible Ϫ3 Ϫ1 Ϫ9 from the ratio of koff/Kd (4.1 ϫ 10 s /102 ϫ 10 M), in reasonable with the idea that each of the four subunits contain agreement with the onset of inhibition kinetics shown in Figure 8. one HaTx binding site. In contrast to the pore-blocking (C) Plot of log I versus log (1ϪeϪt koff) where I is tail current following Ϫ3 Ϫ1 toxins, the HaTx binding site must be located away from depolarization to Ϫ20 mV, t is time, and koff ϭ 4.1 x 10 s . The solid line is a fit of the data to log (I) ϭ mlog(1ϪeϪt koff) ϩ b where the channel’s central axis. That is, in order to allow for m is the slope (3.7), and b is the y intercept. multiple occupancy by HaTx, the sites must be nonover- lapping. Finally, when HaTx binds to its receptor on a channel subunit, it causes the voltage-activation relation to shift to more depolarized membrane voltages. Such Neuron 672

coupling of toxin binding to channel gating requires that work was supported by a grant from the National Institutes of Health HaTx bind more tightly to the closed state of the channel. (GM43949). In other words, the toxin binding affinity must change when the channel gates. Thus, HaTx senses the confor- Received February 7, 1997; revised March 18, 1997. mational changes associated with channel gating. References Where are these conformational changes taking place when the channel gates, and which residues Aggarwal, S.K. (1996). Ph.D. dissertation. Analysis of the voltage- form the HaTx binding sites? In the accompanying paper sensor in a voltage-activated . Harvard Univer- (Swartz and MacKinnon, 1997 [this issue of Neuron]), sity, Cambridge, Massachusetts. we examine which residues on the channel contribute to Aiyar, J., Withka, J.M., Rizzi, J.P., Singleton, D.H., Andrews, G.C., form the HaTx binding sites and determine the minimal Lin, W., Boyd, J., Hanson, D.C., Simon, M., Dethlefs, B., Lee, C.-I., distance of these amino acid residues from the central Hall, J.E., Gutman, G.A., and Chandy, K.G. (1996). Topology of the ϩ pore axis. pore-region of a K channel revealed by the NMR-derived structures of scorpion toxins. Neuron 15, 1169–1181. Anderson, C.S., MacKinnon, R., Smith, C., and Miller, C. (1988). Experimental Procedures Charybodotoxin block of single Ca2ϩ-activated Kϩ channels. J. Gen. Physiol. 91, 317–333. A Mutant drk1 K؉ Channel Sensitive to Scorpion Toxins Frech, G.C., VanDongen, A.M.J., Schuster, G., Brown, A.M., and The wild-type drk1 Kϩ channel is insensitive to the pore-blocking Joho, R.H. (1989). A novel potassium channel with delayed rectifier peptide Agitoxin (AgTx ; Garcia et al., 1994) because it contains a 2 2 properties isolated from rat brain by expression cloning. Nature 340, number of residues in critical positions in the S5–S6 linker that are 642–645. very unfavorable for AgTx2 binding (Gross et al., 1994). A mutant drk1 Kϩ channel was previously constructed containing seven point Garcia, M.L., Garcia-Calvo, M., Hildago, P., Lee, A.W., and MacKin- mutations in the S5–S6 linker that rendered it highly sensitive to non, R. (1994). Purification and characterization of three inhibitors ϩ of voltage-dependent Kϩ channels from Leiurus quinquestriatus var. -pM; Aggarwal, 1996). This drk1⌬7 K channel con 70 ف AgTx2 (Kd tained the following mutations: Thr355Ser, Lys356Gly, Ala362Asp, hebraeus venom. Biochemistry 33, 6834–6839. Ser363Ala, Ile379Met, Tyr380Thr, and Lys382Val (Aggarwal, 1996). Gross, A., Abramson, T., and MacKinnon, R. (1994). Transfer of ϩ AgTx2 (100 nM) completely blocked drk1⌬7 K channel currents at the scorpion toxin receptor to an insensitive channel. Neuron 13, voltages ranging from Ϫ50 to ϩ100 mV while having no effect on 961–966. background conductances observed in uninjected oocytes. For all Hildago, P., and MacKinnon, R. (1995). Revealing the architecture of the experiments described in this paper, 100 nM AgTx2 was used of a Kϩ channel pore through mutant cycles with a peptide inhibitor. ϩ to selectively block drk1⌬7 K channel currents so that leak and Science 268, 307–310. background conductances could be identified and subtracted from Hurst, R.S., Busch, A.E., Kavanaugh, M.P., Osborne, P.B., North, drk1⌬7 Kϩ channel currents. HaTx inhibits the drk1 and drk1⌬7 Kϩ R.A., and Adelman, J.P. (1991). Identification of amino acid residues channels with similar affinities. The drk1⌬7 Kϩ channel gene, in a involved in dendrotoxin block of rat voltage-dependent potassium bluescript vector, was linearized with Not1 and transcribed using channels. Mol. Pharmacol. 40, 572–576. T7 RNA polymerase. MacKinnon, R., and Miller, C. (1988). Mechanism of charybdotoxin 2ϩ ϩ Electrophysiological Recordings block of the high-conductance, Ca -activated K channel. J. Gen. Physiol. 91, 335–349. Oocytes from Xenopus laevis frogs were removed surgically and incubated with agitation for 1.5 hr in a solution containing: NaCl (82.5 MacKinnon, R., and Miller, C. (1989). Mutant potassium channels mM), KCl (2.5 mM), MgCl2 (1 mM), HEPES (5 mM), and collagenase (2 with altered binding of charybdotoxin, a pore-blocking peptide in- mg/ml; Worthington Biochemical Corp.), pH 7.6 with NaOH. Defolli- hibitor. Science 245, 1382–1385. culated oocytes were injected with cRNA encoding the drk1⌬7 Kϩ MacKinnon, R., Heginbotham, L., and Abramson, T. (1990). Mapping channel and incubated at 17ЊC in a solution containing: NaCl (96 the receptor site for charybdotoxin, a pore-blocking potassium mM), KCl (2 mM), MgCl2 (1 mM), CaCl2 (1.8 mM), HEPES (5 mM), channel inhibitor. Neuron 5, 767–771. and gentamicin (50 g/ml; GIBCO BRL), pH 7.6 with NaOH for 16–24 ␮ Matteson, D.R., and Swenson, R.P. (1986). External monovalent cat- hr prior to electrophysiological recording. Stage 4–6 oocytes were ions that impede the closing of Kϩ channels. J. Gen. Physiol. 87, used for the experiments described in this paper. The recordings 795–816. shown in Figures 2–4, 8, and 9 were obtained from stage 4 oocytes Miller, C. (1988). Competition for block of a Ca2ϩ-activated Kϩ chan- because they have much faster settling times following a voltage nel by charybodotoxin and tetraethylammonium. Neuron 1, 1003– step. Oocyte membrane voltage was controlled using an Axoclamp- 2A two-electrode voltage clamp (Axon Instruments). Data were fil- 1006. tered at 2 kHz (8-pole Bessel) and digitized at 5 kHz. Microelectrode Naranjo, D., and Miller, C. (1996). A strongly interacting pair of resi- resistances were 0.1–0.8 M⍀ when filled with 3 M KCl. Oocytes were dues on the contact surface of charybdotoxin and a shaker Kϩ studied in a 100 ␮l recording chamber that was perfused with an channel. Neuron 16, 123–130. extracellular solution containing: RbCl (50 mM), NaCl (50 mM), MgCl2 Park, C.-S., and Miller, C. (1992). Interaction of charybdotoxin with (1 mM), CaCl2 (0.3 mM), and HEPES (5 mM), pH 7.6 with NaOH. permeant ions inside the pore of a Kϩ channel. Neuron 9, 307–313. s to reach 10ف Solution exchange in the recording chamber required Ranganathan, R., Lewis, J.H., and MacKinnon, R. (1996). Spatial completion as determined by changing between two solutions con- localization of the Kϩ channel selectivity filter by mutant cycle-based taining different Kϩ concentrations. All experiments were carried out structure analysis. Neuron 16, 131–139. 22ЊC). HaTx was purified from Grammostolaف) at room temperature spatulata venom (Spider Pharm, Feasterville, PA) as previously de- Stampe, P., Kolmakova-Partensky, L., and Miller, C. (1994). Intima- tions of Kϩ channel structure from a complete functional map of scribed (Swartz and MacKinnon, 1995). Recombinant AgTx2 was produced as previously described (Garcia et al., 1994). Concentra- the molecular surface of charybdotoxin. Biochemistry 33, 443–450. tions of both toxins were determined by amino acid analysis. Stocker, M., and Miller, C. (1994). Electrostatic distance geometry in a Kϩ channel vestibule. Proc. Natl. Acad. Sci. USA 91, 9509–9513. Acknowledgments Stocker, M., Pongs, O., Hoth, M., Heinemann, S.H., Stu¨hmer, W., Schro¨ ter, K.-H., and Ruppersberg, J.P. (1991). Swapping of func- We thank Zhe Lu for helpful discussions. K. J. S. was supported by tional domains in voltage-gated Kϩ channels. Proc. R. Soc. Lond. an NRSA (GM17387) from the National Institutes of Health. This [Biol.] 245, 101–107. Hanatoxin Modifies the Gating of a Kϩ Channel 673

Swartz, K.J., and MacKinnon, R. (1995). An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron 15, 941–949. Swartz, K.J., and MacKinnon, R. (1997). Mapping the receptor site for Hanatoxin, a gating modifier of voltage-dependent Kϩ channels. Neuron 18, this issue. Swenson, R.P., and Armstrong, C.M. (1981). Kϩ channels close more slowly in the presence of external Kϩ and Rbϩ. Nature 291, 427–429.