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A marine snail neurotoxin shares with scorpion toxins a convergent mechanism of blockade on the pore of voltage-gated K channels.

García E, Scanlon M, Naranjo D - J. Gen. Physiol. (1999)

Bottom Line: 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 alpha-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, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biomédicas, Universidad de Colima, 28045 Colima, México.

ABSTRACT
kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-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 kappa-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, kappa-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, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) 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 kappa-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 alpha-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, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.

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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: . 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+ .
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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: . 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+ .

Mentions: To determine whether κ-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+ (MacKinnon and Miller 1988; Park and Miller 1992b). The rationale for these experiments is that occupancy of the pore by permeant cations coming from the internal side of the channel will repel electrostatically the highly positively charged toxin (+4). In the MaxiK-channel, an approximately fourfold increase in the toxin residence time is expected when the internal K+ concentration is lowered from 110 to ∼10 mM. As with the Ca-activated K channel, half saturation concentration for K conductance in Shaker K channels is near 300 mM, suggesting the presence of a low affinity K-binding site (Eisenmann et al. 1986; Heginbotham and MacKinnon 1993). To our surprise, in outside out patches, preliminary experiments showed a minor effect, either on the association or the dissociation rates, produced by the reduction of the intracellular K+ concentration from 100 to 15 mM (Table ). This result suggests that, if κ-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 (Neyton and Miller 1988a; Baukrowitz and Yellen 1995). Thus, we substituted all the potassium by the nonpermeant cation NMG+ in the patch pipette solution, and we formed and pulled outside-out patches from the oocyte membrane in the 1-Kex recording solution. We attempted to reduce contamination of the patch pipette with K+, and thus minimized occupancy of the pore from internal potassium ions. After obtaining a stable outside-out patch, the pipette was positioned in the rapid perfusion system applying an external solution containing 100 mM K+. Under these conditions, 0-Kin//100-Kex and high levels of channel expression, voltage pulses activated inward currents that frequently produced positive feedback responses characteristic of inadequate clamp. This latter effect was avoided by doing recordings with small currents (<200 pA). Fig. 8 A shows 400-ms records with pulse application of a solution that, in addition to the 100 mM KCl, contained 500 nM κ-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 Fig. 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 (Fig. 8, and see Table ). In agreement with the results that Goldstein and Miller 1993 previously obtained for CTX in the nominal absence of internal K+, both the amplitude of the zero-voltage dissociation rate and its effective valence were reduced by half. Such a trans-pore effect of potassium ions is by itself indicative that the mechanism of inhibition κ-PVIIA on K channel is physically blocking the ion conduction pathway (Bezanilla and Armstrong 1972; MacKinnon and Miller 1988). These results also suggest that, as in CTX, the side chain of a basic residue interacts with permeant ions residing in the narrowest region of the pore.


A marine snail neurotoxin shares with scorpion toxins a convergent mechanism of blockade on the pore of voltage-gated K channels.

García E, Scanlon M, Naranjo D - J. Gen. Physiol. (1999)

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: . 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+ .
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Related In: Results  -  Collection

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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: . 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+ .
Mentions: To determine whether κ-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+ (MacKinnon and Miller 1988; Park and Miller 1992b). The rationale for these experiments is that occupancy of the pore by permeant cations coming from the internal side of the channel will repel electrostatically the highly positively charged toxin (+4). In the MaxiK-channel, an approximately fourfold increase in the toxin residence time is expected when the internal K+ concentration is lowered from 110 to ∼10 mM. As with the Ca-activated K channel, half saturation concentration for K conductance in Shaker K channels is near 300 mM, suggesting the presence of a low affinity K-binding site (Eisenmann et al. 1986; Heginbotham and MacKinnon 1993). To our surprise, in outside out patches, preliminary experiments showed a minor effect, either on the association or the dissociation rates, produced by the reduction of the intracellular K+ concentration from 100 to 15 mM (Table ). This result suggests that, if κ-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 (Neyton and Miller 1988a; Baukrowitz and Yellen 1995). Thus, we substituted all the potassium by the nonpermeant cation NMG+ in the patch pipette solution, and we formed and pulled outside-out patches from the oocyte membrane in the 1-Kex recording solution. We attempted to reduce contamination of the patch pipette with K+, and thus minimized occupancy of the pore from internal potassium ions. After obtaining a stable outside-out patch, the pipette was positioned in the rapid perfusion system applying an external solution containing 100 mM K+. Under these conditions, 0-Kin//100-Kex and high levels of channel expression, voltage pulses activated inward currents that frequently produced positive feedback responses characteristic of inadequate clamp. This latter effect was avoided by doing recordings with small currents (<200 pA). Fig. 8 A shows 400-ms records with pulse application of a solution that, in addition to the 100 mM KCl, contained 500 nM κ-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 Fig. 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 (Fig. 8, and see Table ). In agreement with the results that Goldstein and Miller 1993 previously obtained for CTX in the nominal absence of internal K+, both the amplitude of the zero-voltage dissociation rate and its effective valence were reduced by half. Such a trans-pore effect of potassium ions is by itself indicative that the mechanism of inhibition κ-PVIIA on K channel is physically blocking the ion conduction pathway (Bezanilla and Armstrong 1972; MacKinnon and Miller 1988). These results also suggest that, as in CTX, the side chain of a basic residue interacts with permeant ions residing in the narrowest region of the pore.

Bottom Line: 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 alpha-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, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biomédicas, Universidad de Colima, 28045 Colima, México.

ABSTRACT
kappa-Conotoxin-PVIIA (kappa-PVIIA) belongs to a family of peptides derived from a hunting marine snail that targets to a wide variety of ion channels and receptors. kappa-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 kappa-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, kappa-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, zdelta, is approximately 0.55 and resides primarily in the rate of dissociation from the channel, while the association rate is voltage independent with a magnitude of 10(7)-10(8) 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 kappa-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 alpha-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, kappa-PVIIA shares with them a remarkably similar mechanism of inhibition of K channels.

Show MeSH
Related in: MedlinePlus