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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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Comparison between pulse and chronic applications of κ-PVIIA to outside-out patches. A and D show current records from two different outside-out patches elicited by 200-ms long voltage pulses from −60 to +50 mV, with intervals of 10 mV. Bath and pipette K+ concentration were 2 and 15 mM, respectively (see methods). (B) Rapid applications of 1 mM TEA+/500 nM κ-PVIIA to an outside-out patch ∼40 ms after the beginning of the activating voltage pulse. The shaded area (Pulse) indicates the duration of the toxin application. Records shown are the average of four individual records at each voltage. (Inset) A single record taken at 0 mV showing the inflection in the K currents produced by the presence of 1 mM TEA+ in the toxin-containing solution. The TEA+-induced inflection is lost in the average because of the variable latency of the solenoid valve. (E) Chronic application of 1 mM TEA+/500 nM κ-PVIIA to the outside-out patch. The solenoid valve was open while the acquisition lasted. C and F show point-by-point divisions of the leak-subtracted TEA+/toxin records by their respective leak-subtracted controls. Single-exponential fits to each resulting relaxation while the toxin was present were performed (thin lines shown alternately for clarity). The sections including current at holding potential and capacitative transient were eliminated. (F) Exponential functions were extrapolated to the beginning of the voltage pulse and converge to the same value. In A, B, D, and E, the dotted line indicates the zero current level.
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Figure 4: Comparison between pulse and chronic applications of κ-PVIIA to outside-out patches. A and D show current records from two different outside-out patches elicited by 200-ms long voltage pulses from −60 to +50 mV, with intervals of 10 mV. Bath and pipette K+ concentration were 2 and 15 mM, respectively (see methods). (B) Rapid applications of 1 mM TEA+/500 nM κ-PVIIA to an outside-out patch ∼40 ms after the beginning of the activating voltage pulse. The shaded area (Pulse) indicates the duration of the toxin application. Records shown are the average of four individual records at each voltage. (Inset) A single record taken at 0 mV showing the inflection in the K currents produced by the presence of 1 mM TEA+ in the toxin-containing solution. The TEA+-induced inflection is lost in the average because of the variable latency of the solenoid valve. (E) Chronic application of 1 mM TEA+/500 nM κ-PVIIA to the outside-out patch. The solenoid valve was open while the acquisition lasted. C and F show point-by-point divisions of the leak-subtracted TEA+/toxin records by their respective leak-subtracted controls. Single-exponential fits to each resulting relaxation while the toxin was present were performed (thin lines shown alternately for clarity). The sections including current at holding potential and capacitative transient were eliminated. (F) Exponential functions were extrapolated to the beginning of the voltage pulse and converge to the same value. In A, B, D, and E, the dotted line indicates the zero current level.

Mentions: 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 (Hamill et al. 1981). Outside-out patches were excised from the oocyte membrane and positioned near the outlet of a rapid perfusion system (Liu and Dilger 1991; Naranjo and Brehm 1993). In such a system, a single solenoid movement (225P071; NResearch) performs rapid exchange between two solution streams converging into the tip of the patch pipette. The solenoid simultaneously opens the path of one solution while it closes the path for the other. Solution exchange rate was complete in <5 ms. For most experiments described, patch pipettes (1–4 MΩ) 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 κ-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 Fig. 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).


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)

Comparison between pulse and chronic applications of κ-PVIIA to outside-out patches. A and D show current records from two different outside-out patches elicited by 200-ms long voltage pulses from −60 to +50 mV, with intervals of 10 mV. Bath and pipette K+ concentration were 2 and 15 mM, respectively (see methods). (B) Rapid applications of 1 mM TEA+/500 nM κ-PVIIA to an outside-out patch ∼40 ms after the beginning of the activating voltage pulse. The shaded area (Pulse) indicates the duration of the toxin application. Records shown are the average of four individual records at each voltage. (Inset) A single record taken at 0 mV showing the inflection in the K currents produced by the presence of 1 mM TEA+ in the toxin-containing solution. The TEA+-induced inflection is lost in the average because of the variable latency of the solenoid valve. (E) Chronic application of 1 mM TEA+/500 nM κ-PVIIA to the outside-out patch. The solenoid valve was open while the acquisition lasted. C and F show point-by-point divisions of the leak-subtracted TEA+/toxin records by their respective leak-subtracted controls. Single-exponential fits to each resulting relaxation while the toxin was present were performed (thin lines shown alternately for clarity). The sections including current at holding potential and capacitative transient were eliminated. (F) Exponential functions were extrapolated to the beginning of the voltage pulse and converge to the same value. In A, B, D, and E, the dotted line indicates the zero current level.
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Related In: Results  -  Collection

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Figure 4: Comparison between pulse and chronic applications of κ-PVIIA to outside-out patches. A and D show current records from two different outside-out patches elicited by 200-ms long voltage pulses from −60 to +50 mV, with intervals of 10 mV. Bath and pipette K+ concentration were 2 and 15 mM, respectively (see methods). (B) Rapid applications of 1 mM TEA+/500 nM κ-PVIIA to an outside-out patch ∼40 ms after the beginning of the activating voltage pulse. The shaded area (Pulse) indicates the duration of the toxin application. Records shown are the average of four individual records at each voltage. (Inset) A single record taken at 0 mV showing the inflection in the K currents produced by the presence of 1 mM TEA+ in the toxin-containing solution. The TEA+-induced inflection is lost in the average because of the variable latency of the solenoid valve. (E) Chronic application of 1 mM TEA+/500 nM κ-PVIIA to the outside-out patch. The solenoid valve was open while the acquisition lasted. C and F show point-by-point divisions of the leak-subtracted TEA+/toxin records by their respective leak-subtracted controls. Single-exponential fits to each resulting relaxation while the toxin was present were performed (thin lines shown alternately for clarity). The sections including current at holding potential and capacitative transient were eliminated. (F) Exponential functions were extrapolated to the beginning of the voltage pulse and converge to the same value. In A, B, D, and E, the dotted line indicates the zero current level.
Mentions: 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 (Hamill et al. 1981). Outside-out patches were excised from the oocyte membrane and positioned near the outlet of a rapid perfusion system (Liu and Dilger 1991; Naranjo and Brehm 1993). In such a system, a single solenoid movement (225P071; NResearch) performs rapid exchange between two solution streams converging into the tip of the patch pipette. The solenoid simultaneously opens the path of one solution while it closes the path for the other. Solution exchange rate was complete in <5 ms. For most experiments described, patch pipettes (1–4 MΩ) 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 κ-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 Fig. 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).

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