Correspondence to: David Naranjo, Instituto de Fisiología Celular, UNAM, Circuito Exterior, Ciudad Universitaria, 04510 México D.F., México., dnaranjo{at}ifcsun1.ifisiol.unam.mx (E-mail), Fax: 525-622-5607; (fax)
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Abstract |
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-Conotoxin-PVIIA (
-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors.
-PVIIA is a small, structurally constrained, 27-residue peptide that inhibits voltage-gated K channels. Three disulfide bonds shape a characteristic four-loop folding. The spatial localization of positively charged residues in
-PVIIA exhibits strong structural mimicry to that of charybdotoxin, a scorpion toxin that occludes the pore of K channels. We studied the mechanism by which this peptide inhibits Shaker K channels expressed in Xenopus oocytes with the N-type inactivation removed. Chronically applied to whole oocytes or outside-out patches,
-PVIIA inhibition appears as a voltage-dependent relaxation in response to the depolarizing pulse used to activate the channels. At any applied voltage, the relaxation rate depended linearly on the toxin concentration, indicating a bimolecular stoichiometry. Time constants and voltage dependence of the current relaxation produced by chronic applications agreed with that of rapid applications to open channels. Effective valence of the voltage dependence, z
, is ~0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 107108 M-1 s-1, consistent with diffusion-limited binding. Compatible with a purely competitive interaction for a site in the external vestibule, tetraethylammonium, a well-known K-pore blocker, reduced
-PVIIA's association rate only. Removal of internal K+ reduced, but did not eliminate, the effective valence of the toxin dissociation rate to a value <0.3. This trans-pore effect suggests that: (a) as in the
-KTx, a positively charged side chain, possibly a Lys, interacts electrostatically with ions residing inside the Shaker pore, and (b) a part of the toxin occupies an externally accessible K+ binding site, decreasing the degree of pore occupancy by permeant ions. We conclude that, although evolutionarily distant to scorpion toxins,
-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.
Key Words: pore blockade, patch clamp, Xenopus oocyte, Conus venom, Shaker K channel
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Introduction |
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Conotoxins are a family of small peptide toxins derived from the venom of marine snails of the Conus, a genus composed of ~500 predator species (-,
-, and
-conotoxins are specific for Ca, Na, and K voltage-gated channels, respectively. However, detailed knowledge of their mechanism of inhibition on voltage-gated channels is not yet clear.
-Conotoxin-PVIIA (
-PVIIA)1 is a 27-residue peptide component of the Conus purpurascens venom found to inhibit K channels. This peptide acts rapidly on its target, so it is proposed to have an important role in the quick excitotoxic prey immobilization after the venomous sting (
-PVIIA is ~10-residues shorter, it shows striking similarities to the space distribution of functionally relevant basic residues of charybdotoxin (CTX), a member of a well-characterized group of K channel pore blockers, the
-KTx, (
-PVIIA binds to the external vestibule of K channels. First, different pore splice variants bind
-PVIIA with dissimilar affinity (
-PVIIA competes with other putative pore blocker toxins (
-PVIIA, suggesting that this peptide also inhibits K channels by occluding the permeation pathway (
Gly, which enhances ~2,000-fold affinity for
-KTx (
-PVIIA (
-PVIIA seems to act differently whether or not Shaker K channels carries the N-type of inactivation domain, a portion of the protein that inactivates the open channel conformation from the intracellular side (
-PVIIA on Shaker K channels.
Using the well-known -KTx inhibition mechanism on K channels as a paradigm for comparison (
-PVIIA acts on open Shaker K channels. To our knowledge, together with
-PVIIA binding to Shaker K channel is consistent with a 1:1 stoichiometry and diffusion-limited association, (b) a well-known K-pore blocker, tetraethylammonium, reduces the toxin association, but not the dissociation rate, and (c) the peptide toxin interacts, perhaps electrostatically, with K+ from the intracellular side of the channel. We also argue that by binding to the external vestibule, the toxin reduces the occupancy of permeant ions inside the channel pore. Together, these results provide compelling evidence that
-PVIIA inhibits potassium currents by plugging the pore of K channels in way analogous to scorpion toxins (
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Methods |
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Peptide Synthesis
Boc-L amino acids were obtained from Novabiochem or the Peptide Institute, t-Boc-Val-OCH2-PAM-resin (substitution value 0.77 mmol g-1) was obtained from Perkin Elmer. 2-(1H-benzotriazol-1-yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) was obtained from Richelieu Biotechnologies. Other reagents were of peptide synthesis grade from Auspep.
Stepwise synthesis (0.5 mmol scale, 0.649 g resin) was conducted manually using in situ BOC SPPS (
The folded product was obtained by dissolving reduced peptide (10 mg) in aqueous 0.33 M NH4OAc/0.5 M GnHCl (154 ml), with pH adjusted to 7.8 using 0.01 M NH4OH. The solution was stirred at 4°C for 5 d, in the presence of reduced and oxidized glutathione (molar ratio of peptide:GSH:GSSG was 1:100:10). Lowering the pH to 23 with TFA (5 ml) terminated the oxidation. The reaction mixture was loaded onto a preparative HPLC column (Vydac C18 column, 2.2 x 25 cm) (8 ml min-1) and washed with 0.1% TFA until all oxidation buffer had eluted. A 1% gradient (100% A to 80% B, 80 min) was applied and pure oxidized -PVIIA was isolated in 95% yield. Electrospray ionization mass spectra recorded on a PE Sciex API III triple quadrupole mass spectrometer were used to confirm the purity and molecular weights of synthetic peptides.
Heterologous Expression of Shaker K Channels
For electrophysiology, salts of analytical grade were purchased from Baker. Gentamicin, sodium pyruvate, EGTA, HEPES, N-methyl-D-glucamine (NMG), and BSA were from Sigma-Aldrich Química S.A. de C.V.
Females Xenopus laevis (Xenopus One) were anesthetized by immersion in ice. Ovarian lobes were surgically removed and collected in ND96 solution containing (mM): 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, pH 7.6, and 50 µg/ml gentamicin. Type II collagenase, 0.91.5 mg/ml, was used for digestion of connective tissue (Worthington Biochemical Corp.). After removing the enzyme, stage IVVI oocytes were isolated and manually defolliculated in a nominally Ca2+-free ND96 solution.
The inactivation-removed Shaker H4 (6-46) (
Electrophysiology
For whole-cell TEVC recordings, oocytes were positioned in the middle of an ~500-µl longitudinal recordings chamber (-PVIIA, 301,000 nM, added from a stock of 1 mM. Voltage pulse protocols and data acquisition were performed from a personal computer running pClamp 5.5 through a Digidata 1200B acquisition interface (Axon Instruments). Whole oocyte recordings were made with an OC-750C voltage clamp amplifier (Warner Instruments). Recording electrodes, with resistance of 0.31 M
were made with Ag/AgCl pellet assemblies (Axon Instruments) inside a capillary filled with a solution made of 3 M KCl, 5 mM EGTA, and 10 mM HEPES-KOH, pH 7.0. Voltage pulses of 50500 ms were applied from a holding potential of -90 mV, and usually ranged from -60 to 50 mV in 10-mV intervals. Because oocytes expressing outward currents >10 µA at 50 mV usually exhibited obvious slower rising times, experiments were performed in oocytes expressing 0.58 µA only.
For patch clamp recording, the vitelline membrane was removed after a 10-min incubation in a solution containing (mM): 200 K-aspartate, 10 KCl, 10 EGTA, and 10 HEPES, pH 7.4. After vitelline membrane removal, we followed conventional patch-clamp techniques () were filled with solutions consisting of (mM) 80 KF, 20 KCl, 1 MgCl2, 10 EGTA, and 10 HEPES-KOH, pH 7.4 (100-Kin). For the experiments with reduced internal K+ concentration (15-Kin), solution was 90 NMG-F, 10 KF, 1 MgCl2, 10 EGTA, and 10 HEPES-KOH, pH 7.4. External recording solution was 115 NaCl, 1 KCl, 0.2 CaCl2, 1 MgCl2, and 10 HEPES-NaOH, pH7.4 (1-Kex). For all patch clamp experiments shown in this paper, 500 nM
-PVIIA and 1 mM tetraethylammonium (TEA+) were added to the external patch clamp recording solution. The small inflection of the K current (~5%) produced by 1 mM TEA+ blockade was used to monitor the position of the patch pipette to obtain the optimal rate of solutions exchange (see Figure 4, inset). Because of intrinsic variation of the solenoid latency, the TEA+-produced inflection is not often visible in the averaged records. For experiments in nominally zero internal potassium (0-Kin), all potassium salts were replaced with NMG-F and the membrane patch was formed and pulled out in the 1-Kex recording solution. Then, the patch was moved to the perfusion system running a solution made of 16 NaCl, 100 KCl, 0.2 CaCl2, 1 MgCl2, and 10 HEPES-KOH, pH 7.6 (100-Kex).
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For whole oocyte and outside-out currents, off-line leak subtraction was carried out before performing point-by-point division of current records. Curve fitting, statistics, and figure preparation were carried out with Microcal Origin 3.5 (Microcal Inc.).
Elements of Analysis of a Voltage-dependent Blockade with Bimolecular Stoichiometry
Figure 10 describes the 1:1 toxin binding equilibrium, where Sh, T, and Sh·T correspond to a conducting empty channel, the toxin, and a nonconducting channeltoxin complex, respectively. The rate constants for association and dissociation are kon and koff, respectively. This scheme predicts that in response to a step-like perturbation, the toxin-binding will relax exponentially to a new equilibrium. The relaxation rate, measured as the reciprocal of the relaxation time constant, is a function of the toxin concentration as following:
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(1) |
which defines a straight line with slope kon, and koff as intercept. Meanwhile, the fraction of unblocked channels in the new equilibrium is given by:
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(2) |
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Together, Equation 1 and Equation 2 provide a system with two equations and two unknowns, kon and koff. Thus, by fitting the values of and
to the macroscopic relaxation in response to each voltage step, a pair of values for kon and koff is obtained. The magnitude of effective valence for the voltage dependence of each rate constant, z, was calculated from the expression:
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(3) |
where V is the membrane potential in millivolts, and k(0) is the rate constant at zero applied potential. Because the voltage dependence of macroscopic relaxation depends nonlinearly on the voltage dependence of both kon and koff to describe it, we preferred to use the expression:
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(4) |
where (0) is the time constant at zero applied potential and Vs is the membrane voltage, in millivolts, that produces an e-fold increase in
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Results |
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Whole cell TEVC recordings in oocytes expressing the inactivation removed Shaker (6-46) K channels (
-PVIIA (Figure 1). In this set of experiments, the toxin was applied continuously to the recording solution superfusing the oocyte. Figure 1A and Figure B, shows K currents elicited by 200-ms depolarizing steps between -60 and +50 mV at 10-mV intervals in the presence and absence of 100 nM
-PVIIA. The apparent effect of 100 nM
-PVIIA on the K channels is to make activation kinetics more complex by the introduction of a second, and slower, phase in the rising of the currents elicited by voltage steps (Figure 1 B). Also, the conductancevoltage relationship measured at the end of the pulse is shifted to the right and is less steep in the presence of the toxin (Figure 1 C). This voltage-dependent effect is in contradiction with the nearly voltage independent affinity of
-PVIIA on the inactivating Shaker H4 observed previously (
Results described in Figure 1 may suggest that the toxin effect on Shaker K channels is to modify the gating mechanism. The toxin would bind preferentially to the closed channel conformation, delaying the rate of activation. Such a mechanism has been proposed for hanatoxin, a component of the spider venom, on other K channels as Kv2.1 (
Figure 2 A shows a point-by-point division of the traces in the presence of the toxin by the control traces at pulse voltages positive to -20 mV shown in Figure 1. As done before by
DoseResponse Experiments
A simple bimolecular stoichiometry predicts that the rate of relaxation should increase linearly to the toxin concentration. Figure 3 A shows records obtained with a 100-ms pulse to +40 mV with 0, 30, 100, and 300 nM -PVIIA. In Figure 3 B, the point-by-point divisions of the traces by their controls at zero toxin are shown. With this operation, the concentration dependence of the blockade at holding potential becomes apparent. From these measurements, a dissociation constant at holding potential of 65 ± 11 nM is obtained (mean ± SEM for four different experiments, Figure 3 B, inset). This value is in close agreement with the 60 nM measured by
-PVIIA inhibition to the N-inactivating Shaker H4 by
As is apparent from Figure 3A and Figure B, the rate of relaxation increases at higher toxin concentrations. Figure 3 C shows that the rate of relaxation, measured as the reciprocal of the time constant, increases linearly with the concentration of -PVIIA at all voltages. Linear regressions to the data show that the intercept changes with the pulse voltage; meanwhile, the slope is almost invariant. This linearity is consistent with a bimolecular stoichiometry for the interaction between
-PVIIA and the Shaker K channel as shown by Figure 10 in METHODS. For such a scheme, Equation 1 states that the slope is kon and the intercept is koff.
Figure 3 D plots the voltage dependence of both rate constants for the data plotted in C. It is clear that most of the voltage dependence resides in koff (). At zero voltage, koff is 17 ± 1 s-1, with a z = 0.58 ± 0.08. Meanwhile, the apparent second-order rate constant is nearly voltage independent. The rate constant kon has a zero-voltage value of 70 ± 9 µM-1 s-1 with z
= 0.08 ± 0.16. This value agrees very well with that of 61 µM-1 s-1 for charybdotoxin, a toxin whose binding to potassium channels is proposed to be diffusion limited (
Rapid Application of -PVIIA to Outside-Out Patches
Because the use of voltage-step protocols is inherent to the study of Shaker K channels, to differentiate between the effects of the voltage or the open probability of the channels on -PVIIA binding, chronic bath application to whole oocytes are inadequate. We compared chronic and rapid applications of the toxin to membrane patches expressing macroscopic currents in the outside-out configuration (
Outside-out patches were separated from the oocyte following standard techniques (
To separate gating from toxin kinetics in the chosen interval of pipette voltages, toxin was applied ~40 ms after the beginning of the test pulse. Visual clues are not usually enough to position the pipette tip where solution exchange is optimally fast. To monitor the optimal positioning of the membrane patch in the rapid application system, together with the toxin, we added 1 mM TEA+ to the test solution. Because this TEA+ concentration produces a fast blockade of a small but measurable fraction of the current, we could monitor the solution exchange rate by monitoring the speed of the inflection produced by the TEA+ effect on K currents on individual records (see Figure 4 B, inset). Figure 4 B shows an average of four runs of -PVIIA/TEA+. The rapid TEA+ inflection is not visible in averaged records because of inherent variable latency of the solenoid valve (<5 ms). At all voltages, 1 mM TEA+/500 nM
-PVIIA pulse applications produced a decrease in the current in which the kinetics of the onset and offset of toxin blockade are faster as the voltage is made more positive. As with the whole oocyte recordings, we made point-by-point division of toxin current records by control records at each voltage. As a result of such an operation, we also avoided possible distortions on toxin kinetics resulting from the apparent inactivation, as seen by the complete recovery in the current records taken at more positive voltage pulses (Figure 4 C). These normalized records show voltage-dependent on and off kinetics in addition to a voltage-dependent steady state inhibition. Single exponential fits applied to each normalized trace are shown as solid lines over some traces. A general result from single exponential fits to the normalized traces is shown in Figure 5 (open symbols). This figure plots averages of data pooled from a total of 13 outside-out patches with pulse applications of
-PVIIA. Because we did not find significant difference in the toxin kinetics between experiments obtained with 100-Kin (n = 7) or 15-Kin (n = 6) solutions in the patch pipette (not shown, see Table 1 and DISCUSSION), these data also represent the merging together of these two experimental conditions.
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If the toxin inhibition is produced merely by toxin binding instead of inducing, or preventing, a conformational change, the kinetics of the response to a voltage step in the presence of the toxin should be identical to that induced by pulse applications of toxin at a constant voltage. To the patch whose current record is shown in Figure 4 D, 1 mM TEA+/500 nM -PVIIA was chronically applied, a protocol equivalent to that of the whole-oocyte experiments (Figure 4 E). As in the whole oocyte experiments, the apparent time course of the current activation is also delayed in these conditions. A point-by-point division of these traces by the controls of Figure 4 D is shown in F. This family of normalized traces shows that the voltage dependence of the relaxation and steady state inhibition is similar to those with the toxin pulse protocol shown in Figure 4 C. Thin lines in Figure 4 F correspond to exponential fits to some of the normalized traces. Average time constant and steady state inhibition for 11 pooled patches are plotted as solid symbols in Figure 5 (n = 4 with 100-Kin and 7 with 15-Kin in the pipette).
Figure 5A and Figure B, summarizes results of the kinetic analysis of the experiments of -PVIIA applications to outside out patches. In Figure 5 A, time constants for the onset of inhibition and recovery after toxin removal for the pulse protocol experiments are plotted as open symbols; meanwhile, plotted as solid symbols are the time constants of the relaxations in the experiments with chronic application as shown in Figure 4 F. Figure 5 B plots the steady state inhibition for pulse (
) and chronic () applications. Two lines of evidence strongly suggest that the rate-limiting step for the relaxation of the inhibition is the association of the toxin with the channel and not a conformational change from a closed to open state. (a) Although the levels of steady state fractional current reached with both protocols are significantly different, in average, the systematic difference between both curves accounts for <3% of the inhibition. On the other hand, for both protocols of
-PVIIA applications, no significantly different values for the time constant of onset relaxation are detected, indicating that both the voltage pulse in the presence of
-PVIIA and the toxin pulse applied to the open channels promote a nearly identical perturbation on the blockade equilibrium, as expected by a most simple voltage-dependent blocking mechanism. Such a mechanism only requires toxin binding with 1:1 stoichiometry to produce inhibition. (b) Although the toxin binds with moderately higher affinity to the closed state (
on = 29 ± 3 ms, with Vs = 141 ± 58 mV. For pulse application, the zero-voltage onset time constant is
on = 27 ± 2 ms, with Vs = 80 ± 17 mV, while for the recovery it is
off = 73 ± 2 ms with Vs = 56 ± 4 mV.
Rate Constants
In a bimolecular scheme as presented in METHODS, the time constant of the macroscopic relaxation is governed by Equation 1. In the absence of toxin, koff can be calculated directly from the reciprocal of the recovery time constant, off in the measurements of toxin removal. Also, we have two additional experimental conditions in which the relaxation in the presence of the toxin is seen, chronic and pulse application onset. For a 1:1 stoichiometry, these two types of macroscopic relaxations should reach a steady state level given by Equation 2.
From the values plotted in Figure 5, we calculated both kon and koff for each pipette potential by using Equation 1 and Equation 2. Results from such calculations are summarized in Figure 6. The small open and filled symbols correspond to the calculations of kon and koff, respectively. There is a good agreement in the values of koff measured directly from pulse experiments (Figure 6, small solid circles) with those calculated from the two-equation system regardless of the experimental protocol. As in the whole oocyte experiments of Figure 3, most of the voltage dependence appears to be in koff. An overall average calculated from all three experimental data sets is plotted as the big circles in Figure 6. The solid line is a single exponential fit to the calculated rate constants. At zero voltage, the second order rate constant, kon, is 43 ± 3 µM-1 s-1 with z = 0.11 ± 0.05. This value for kon is only 40% smaller than that measured on the whole-oocyte experiments and is also consistent with a diffusion-limited association (
= 0.53 ± 0.09.
The near perfect agreement between the patch clamp and two-electrode voltage clamp experiments reinforce the idea that the -PVIIA exerts its inhibition effect on the open K channel by binding to an external site only. Thus, the relaxation of the macroscopic K currents observed in oocytes under chronically applied toxin is analogous to the slow hook seen previously when tetraethylammonium ions dissociate from squid axon potassium channels before rapid deactivation in response to repolarization (
Competition with TEA+
Although there is no compelling evidence that -PVIIA plugs the pore of K channels, there are several lines of evidence that
-PVIIA interacts with the Shaker external vestibule. Transference of the H5 segment from a
-PVIIAsensitive K channel to an insensitive channel also transfers toxin sensitivity (
-PVIIA binds to the Shaker vestibule, similar effects are expected. With two-electrode voltage clamp,
-PVIIA binding was studied in the presence of TEA+ 010 mM, added on top of the recording solution. Figure 7 shows a summary of such experiments. In the same fashion as we did for the results shown in Figure 3, we made a point-by-point division of records taken in
-PVIIA/TEA+ solution from those taken in TEA+ only (not shown). Applying Equation 1 and Equation 2 to the time constants and steady state inhibition obtained from the single exponential fits to the relaxations, apparent kon and koff for the effect of TEA+ were obtained. TEA+ decreases the apparent association rate with little effect on the dissociation rate. This effect seems to be specific for TEA+ because raising Na+ concentration to 10 mM does not significantly affect either kon or koff (Figure 7, solid lines). Together, these results indicate that the effect of TEA+ on
-PVIIA binding is not due to the increased ionic strength, but rather to a specific exclusion of the toxin from the external vestibule of Shaker.
Internal Potassium Alters Binding to Shaker
To determine whether -PVIIA inhibit ionic conduction by occluding the ionic pathway, we analyzed the effect of altering permeant ion concentration in the opposite side of the pore. This strategy had shown that the binding of CTX to the MaxiK-channel is very sensitive to the intracellular K+ (
-PVIIA occludes the conduction pathway, it is not very sensitive to K occupancy in the pore, or the pore occupancy is not very different in these two distinct conditions. As in the MaxiK-channel, it had already been suggested that in the Shaker K channel there are at least one micromolar affinity potassium binding sites inside the K channel pore (
-PVIIA and 1 mM TEA+. Because, at any voltage, this type of record displayed <10% of inactivation (not shown), the rate of dissociation was measured by fitting single exponential functions directly to the time course of the inward currents recovery. Average values (mean ± SEM) for four such experiments are plotted in Figure 8 B. For the sake of comparison, the best fit to the average dissociation rate made with in whole oocyte TEVC at 100 mM external K+ is shown by dotted lines (Figure 8, and see Table 1). In agreement with the results that
-PVIIA on K channel is physically blocking the ion conduction pathway (
-PVIIA Protects the Shaker Pore from Collapsing
As with CTX (-PVIIA binding to Shaker (Figure 8). The most economical interpretation for this residual voltage dependence is that a positive charge located in the pore-occluding side chain of the toxin interacts intimately with the narrowest part of the K channel pore, getting located inside the electric field. An electrical distance of 0.20.25 coincides with an externally located cationic binding site that is present in the pore of Shaker. A binding site detected at approximately the same electrical distance can accommodate mono- and divalent cations. NH4+, Cs+, Rb+, and K+ bind to this site with low millimolar or high micromolar affinity (
-PVIIA in the absence of internal permeant ions (Figure 9). Outside-out patches were recorded in 0-Kin//100-Kex condition. From a holding potential of -90 mV, the channels were opened by a pulse to -40 mV to ensure a strong driving force for K+ to flow inwardly. First, we determined that an application of nominally zero K+ solution (100 mM NMG+) to the patch pipette that exposed 58 ms before the voltage pulse ended was necessary to observe any measurable amount of current reduction between consecutive pulses (shaded area in Figure 9 A). For an interpulse interval of 5 s, this reduction indicates the population of channels visiting any of the occupancy-dependent nonconducting states. Because Shaker K channels show little rectification between +40 and -40 mV, we expected the external binding site to be empty in <500 µs (Baukrowitz and Yellen, 1996). Thus, the amount of time needed to observe current reduction might represent incomplete wash out of the high K+ solution. In any case, this figure represents an upper limit to the time needed to empty the pore, and is 2030-fold shorter than the residence time of the bound toxin at -40 mV in this experimental condition (~150 ms, see Figure 8 B). After the 40th pulse, <40% of the current remains (Figure 9 C,
). On the other hand, Figure 9 B shows an experiment in which, instead of exposing the open channel to zero potassium, we applied
-PVIIA to the patch with the intention of occluding the external exit of the pore. In such a case, the K channel pore should be emptied into the internal solution. At 150 ms after channel opening, a 150-ms pulse of 1 mM TEA+/500 nM
-PVIIA that blocked ~60% of the channels was applied (Figure 9, shaded area). While the toxin was still being applied, the channels were forced to close by stepping the voltage back to -90 mV. Thus, given that the toxin blocking time is more than two orders of magnitude longer than the time needed to empty the pore (Table 1), closing the channels while they are still blocked should drive a measurable fraction into any occupancy-dependent nonconducting state. Figure 9 B shows a set of 10 traces representing one of every four acquisitions from one of such an experiment. If the toxin does not protect the channel, ~40% of the current is expected to have disappeared at the 40th pulse (Figure 9 C and legend). However, in four experiments like this, no reduction of the current was detected at even the 60th pulse, suggesting that the channel pore cannot collapse while the toxin is bound. Because both types of nonconducting states require or are facilitated by pore vacancy, while
-PVIIA is bound to the channel, the pore behaves as if occupancy were preserved. The simplest interpretation to this result is that a part of the toxin impedes pore collapse. Thus, we suggest that a component of
-PVIIA occupies the most externally located K+ binding site of Shaker pore, resulting in a protection of the channel. In normal physiological conditions, such occupancy should reduce the average number of permeant ions residing in the pore.
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Discussion |
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Here we present evidence for a mechanism of inhibition of a peptide component of the venom of the predator marine snail Conus on K channels. The 27-residue -PVIIA inhibits K+ currents by binding to the external vestibule of the Shaker K channel pore with a 1:1 stoichiometry at a rate consistent with a diffusion-limited manner (
-KTx, the best characterized of all peptide toxin specific for K-channels (
Effect of Internal K+ on Toxin Unbinding
As with -KTx,
-PVIIA voltage dependence of the binding equilibrium to the channel resides in the dissociation rate (see Figure 3 and Figure 6). For CTX bound to the external vestibule of the MaxiK-channel, the effective valence of the dissociation rate is 1.0 (
vanishes. Thus, the voltage dependence seems to arise exclusively from the electrostatic repulsion between K+ residing inside the pore and the positively charged toxin (
, indicating that the
-amino group of Lys27 interacts electrostatically with K+ residing in the pore (see
-PVIIA/Shaker agree qualitatively and quantitatively with those of CTX/Shaker. Replacement of the internal potassium with the nonpermeant NMG+ reduces, but not eliminate, the effective valence of the
-PVIIA unbinding. For
-PVIIA, the value z
is reduced from ~0.55 in 100 mM internal K+ to 0.29 in nominally zero internal K+ (Figure 8). This trans-pore effect suggests that, as in CTX, a fraction of the voltage dependence of
-PVIIA unbinding arises from electrostatic interaction with permeant ions in the conduction pathway. Thus, we postulate that, as with
-KTx, a positively charged residue in the surface
-PVIIA interacts with permeant ions inside the Shaker K channel pore.
At pH 7.6, -PVIIA net charge is ~4. It contains two Asp, three Lys, and three Arg residues, and it is not amidated as many conotoxins are (
-PVIIA that occludes the pore, we favor a Lys instead of an Arg residue to be the best candidate. First, based on the structural mimicry between CTX and
-PVIIA on the space position of a Lys associated with a key aromatic residue to conform a functional dyad present at all K channel pore peptide blockers known (
promoted by the internal K+ replacement by nonpermeant cations suggests a similar electrostatic interaction.
Electrostatic Repulsion of the Toxin and K+ Permeation
In the MaxiK-channel, the internally accessible binding site that destabilizes CTX binding has an affinity for K+ in the 13-M range ( = 1) is that a K+ would have to travel across the whole transmembrane electric field to interact with CTX. In other words, the internal cation has to permeate all the way across the pore to bind a site near the external end to electrostatically repel CTX. In agreement with the idea that equates permeation with occupancy of an external site, half-maximal K+ concentration for K conductance of the MaxiK-channel is near 300 mM (
of 1.0 for the toxin destabilization observed in the MaxiK-channel implies a net translocation of one charge across the permeation pathway. Also, it accounts for the close agreement between half-maximal K+ concentration for K conductance and toxin destabilization effect. Thus, in this view, the magnitude z
is determined by coupling between the binding of K+ to the internal site and occupancy of the most external one.
Although MaxiK-channel's conductance is ~20-fold higher than that of Shaker K channels, in addition to their high K+ selectivity, they share many ionic conduction properties. Shaker K channel is also a multiple-occupancy channel (-PVIIA off rate to have a similar dependence on the internal K+, as CTX does in the MaxiK-channel. By reducing the internal K+ from 100 to 15 mM, we anticipated a fivefold increase in the
-PVIIA residency time. To our surprise, we found that the toxin dissociation rate was nearly identical to that of 100 K+ (Table 1). In contrast to the CTX/MaxiK-channel system, in
-PVIIA/Shaker, the toxin-unbinding rate seems to be rather insensitive to the occupation of a internally accessed binding site. To observe any trans-pore effect on
-PVIIA unbinding rate, we had to replace K+ completely with the nonpermeant NMG+. Although the contaminating K+ in our internal NMG+ solutions may be enough to keep busy a high affinity binding site in the Shaker pore, thus accounting for the residual z
, the overall voltage dependence of the toxin unbinding is smaller than in the MaxiK-channel. In such an experimental condition, instead of 1.0 as in the MaxiK-channel, only half of the total effective valence of the dissociation rate is accounted for in the replacement of the internal K+. This low voltage sensitivity attributable to the interaction with internal K+ may not correspond specifically to charge delocalization in the
-PVIIA basic residue that occludes the pore. Instead, because in Shaker the interaction with K+ also accounts for a small fraction of the total voltage dependence of CTX, this low z
(together with the low sensitivity to internal K+) may be a reflection of the manner in which the toxin interacts with permeant ions inside the pore of Shaker. The net charge translocation associated with the occupancy of the externally located binding site is 0.30.4 of an electronic charge. Thus, it seems that in the MaxiK-channel the electrostatic repulsion coupling between the occupancy of the most internal and external binding sites is three- to fivefold stronger than in Shaker. This difference could represent an average greater charge separation of the ions inside the Shaker pore. Although in physiological conditions both types of channels seem to be occupied simultaneously by four cations (
-amino group of the toxins occupies the most external site, at an electrical distance of 0.20.3, effectively reducing the number of available sites, and then K+ occupancy. The experiments outlined in Figure 9 agree with this interpretation, suggesting that
-PVIIA occupies the most external binding site in Shaker pore. Thus, the electrostatic repulsion can occur only from a more internally located site in the pore. Consequently, the net charge translocation associated with electrostatic interaction with the toxin is less than in the MaxiK-channel because the toxins,
-PVIIA or CTX, reduce the net K+ occupancy of the pore. In this scheme, the elements for a mechanism of destabilization of a toxin bound to the external vestibule seems to be the same as that for ionic permeation: the exit rate depends on electrostatic repulsion and single-file multiple occupancy.
Fast Prey Immobilization
Conus purpurascens preys on teleost fish. It has been proposed that, upon injection of the Conus venom into the prey, -PVIIA is a central player in the excitotoxic cabal, which causes a massive depolarization of excitable tissue that quickly immobilizes the prey (
-PVIIA behaves as part of an optimized prescription to promote the excitotoxic paralysis of C. purpurascens prey. We have found that the mode of action of
-PVIIA is nearly identical to the mechanism of action of distantly related scorpion peptide toxins of the
-KTx family: first, both bind with 1:1 stoichiometry to the pore of K-channels and, second, the only action that the peptide toxin performs is to occlude the permeation pathway with a tethered substrate analogue that interrupts K+ transport. The high association rate of
-PVIIA to Shaker is consistent with a diffusion-limited, but aided by through-space electrostatic effects, proteinprotein interaction (
-KTx from scorpions and BgK from sea anemone (
In the classical example of structural and functional convergence, the catalytic site of the bacterial subtilisin and the mammalian serine proteinase share a similar space topology. Although dissimilar in their overall construction, the side-chain atoms forming the functional catalytic triad, Ser/His/Asp, are in almost identical relative space positions. The breakdown of the peptide bond proceeds by the same basic mechanism (
Thus, some peptide toxins seem to have had a convergence to a common mechanism of action on K channels. They bind with 1:1 stoichiometry to the most conserved structural locus of the channel protein: the pore. Such a precise mechanism appears to constrain the possible ways in which K channelspecific peptide toxin can be constructed.
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Acknowledgements |
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We thank Miguel Hernandez for technical help and frog care and other members of the laboratory IFC 204-O for support. We are grateful to A. Hernández-Cruz, F. Cifuentes, and F. Gómez-Lagunas for insightful discussions, and to Ramón Latorre, Osvaldo Alvarez, and Christopher Miller for critical reading of the manuscript. Our recognition to Paul Brehm, Irwin Levitan, and Christopher Miller for providing equipment that was essential to develop this project.
This work was partially funded by DGAPA-IN200397 and CONACYT-25247N to D. Naranjo, and by the Juan García Ramos Fund and FOMES-SEP 1997 to E. García.
Submitted: March 2, 1999; Revised: May 18, 1999; Accepted: May 19, 1999.
1used in this paper: CTX, charybdotoxin; -PVIIA,
-conotoxin-PVIIA; MaxiK-channel, large conductance Ca-activated K channel; NMG+, N-methyl-D-glucamine in ionic form; TEA+, tetraethylammonium; TEVC, two-electrode voltage clamp; TFA, trifluoroacetic acid
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References |
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