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Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin.

Hui K, Lipkind G, Fozzard HA, French RJ - J. Gen. Physiol. (2002)

Bottom Line: Strong charge-dependent effects emanate from this toxin surface.In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore.This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.

ABSTRACT
Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, mu-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in mu-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.

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Single-channel current block by μCTX derivatives depends on toxin residue-13 length. (A) Single sodium channel current traces in the presence of μCTX mutants with a neutral residue-13: 10.5 μM R13N or 25 μM R13W, at +60 mV and at −60 mV from one experiment for each mutant. As with the charge-substitution mutants, the residual currents for residue-13 size-changing mutants show outward rectification. General features of traces are as described in Fig. 2 A. (B) In all charge-conservative substitution groups, Fres(0 mV) is linearly dependent on the length of the substitution >3.7 Å. The R13A mutant shows an Fres(0 mV) almost identical to R13Q, but smaller than R13N (see discussion). Data averaged from five, six, eight, four, five, four, four, seven, and four experiments for R13D, R13E, R13A, R13N, R13Q, R13W, R13O, R13K, and R13R (wild type), respectively. Length was determined in Chem3D (Cambridge Soft) as the distance between the centers of Cα and the most distant heavy atom in the stretched out form of the amino acid side chain. (C) The steric attenuation of single-channel current, defined as the slope of the plot of Fres(0 mV) versus length-13 (B), for all charge-conservative substitution groups >3.7 Å is nearly identical at approximately −0.05 Å−1, summarizing the effect of length, independent of the influence of charge.
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Figure 3: Single-channel current block by μCTX derivatives depends on toxin residue-13 length. (A) Single sodium channel current traces in the presence of μCTX mutants with a neutral residue-13: 10.5 μM R13N or 25 μM R13W, at +60 mV and at −60 mV from one experiment for each mutant. As with the charge-substitution mutants, the residual currents for residue-13 size-changing mutants show outward rectification. General features of traces are as described in Fig. 2 A. (B) In all charge-conservative substitution groups, Fres(0 mV) is linearly dependent on the length of the substitution >3.7 Å. The R13A mutant shows an Fres(0 mV) almost identical to R13Q, but smaller than R13N (see discussion). Data averaged from five, six, eight, four, five, four, four, seven, and four experiments for R13D, R13E, R13A, R13N, R13Q, R13W, R13O, R13K, and R13R (wild type), respectively. Length was determined in Chem3D (Cambridge Soft) as the distance between the centers of Cα and the most distant heavy atom in the stretched out form of the amino acid side chain. (C) The steric attenuation of single-channel current, defined as the slope of the plot of Fres(0 mV) versus length-13 (B), for all charge-conservative substitution groups >3.7 Å is nearly identical at approximately −0.05 Å−1, summarizing the effect of length, independent of the influence of charge.

Mentions: Residue-13's binding interaction with Glu-758 on the channel is not only electrostatic (Chang et al. 1998), suggesting that its influence on conduction also may involve other factors. To test whether residue-13 can sterically inhibit the current, we substituted residue-13 with amino acids of variable length and volume (e.g., glutamine in Fig. 2 A, asparagine and tryptophan in Fig. 3 A, and alanine were used as neutral substitutions of different sizes). With the substitutions grouped by their nominal elementary charge, Fres(0 mV) showed a similar dependence on residue-13 length within each group (Fig. 3 B), as reflected in the “steric attenuation” for each charge group (Fig. 3 C). Shorter residue-13 length resulted in lesser block. A comparable correlation was seen when side chain volume, instead of length, was used as an index of side chain size. The only exception to this pattern was R13A, which showed a smaller Fres(0 mV) than expected for its size, based on the data for the other substitutions. In general, decreases in residue-13 length allow an increased residual current, but there may be no further increase when the substituent is made sufficiently small.


Electrostatic and steric contributions to block of the skeletal muscle sodium channel by mu-conotoxin.

Hui K, Lipkind G, Fozzard HA, French RJ - J. Gen. Physiol. (2002)

Single-channel current block by μCTX derivatives depends on toxin residue-13 length. (A) Single sodium channel current traces in the presence of μCTX mutants with a neutral residue-13: 10.5 μM R13N or 25 μM R13W, at +60 mV and at −60 mV from one experiment for each mutant. As with the charge-substitution mutants, the residual currents for residue-13 size-changing mutants show outward rectification. General features of traces are as described in Fig. 2 A. (B) In all charge-conservative substitution groups, Fres(0 mV) is linearly dependent on the length of the substitution >3.7 Å. The R13A mutant shows an Fres(0 mV) almost identical to R13Q, but smaller than R13N (see discussion). Data averaged from five, six, eight, four, five, four, four, seven, and four experiments for R13D, R13E, R13A, R13N, R13Q, R13W, R13O, R13K, and R13R (wild type), respectively. Length was determined in Chem3D (Cambridge Soft) as the distance between the centers of Cα and the most distant heavy atom in the stretched out form of the amino acid side chain. (C) The steric attenuation of single-channel current, defined as the slope of the plot of Fres(0 mV) versus length-13 (B), for all charge-conservative substitution groups >3.7 Å is nearly identical at approximately −0.05 Å−1, summarizing the effect of length, independent of the influence of charge.
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Related In: Results  -  Collection

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Figure 3: Single-channel current block by μCTX derivatives depends on toxin residue-13 length. (A) Single sodium channel current traces in the presence of μCTX mutants with a neutral residue-13: 10.5 μM R13N or 25 μM R13W, at +60 mV and at −60 mV from one experiment for each mutant. As with the charge-substitution mutants, the residual currents for residue-13 size-changing mutants show outward rectification. General features of traces are as described in Fig. 2 A. (B) In all charge-conservative substitution groups, Fres(0 mV) is linearly dependent on the length of the substitution >3.7 Å. The R13A mutant shows an Fres(0 mV) almost identical to R13Q, but smaller than R13N (see discussion). Data averaged from five, six, eight, four, five, four, four, seven, and four experiments for R13D, R13E, R13A, R13N, R13Q, R13W, R13O, R13K, and R13R (wild type), respectively. Length was determined in Chem3D (Cambridge Soft) as the distance between the centers of Cα and the most distant heavy atom in the stretched out form of the amino acid side chain. (C) The steric attenuation of single-channel current, defined as the slope of the plot of Fres(0 mV) versus length-13 (B), for all charge-conservative substitution groups >3.7 Å is nearly identical at approximately −0.05 Å−1, summarizing the effect of length, independent of the influence of charge.
Mentions: Residue-13's binding interaction with Glu-758 on the channel is not only electrostatic (Chang et al. 1998), suggesting that its influence on conduction also may involve other factors. To test whether residue-13 can sterically inhibit the current, we substituted residue-13 with amino acids of variable length and volume (e.g., glutamine in Fig. 2 A, asparagine and tryptophan in Fig. 3 A, and alanine were used as neutral substitutions of different sizes). With the substitutions grouped by their nominal elementary charge, Fres(0 mV) showed a similar dependence on residue-13 length within each group (Fig. 3 B), as reflected in the “steric attenuation” for each charge group (Fig. 3 C). Shorter residue-13 length resulted in lesser block. A comparable correlation was seen when side chain volume, instead of length, was used as an index of side chain size. The only exception to this pattern was R13A, which showed a smaller Fres(0 mV) than expected for its size, based on the data for the other substitutions. In general, decreases in residue-13 length allow an increased residual current, but there may be no further increase when the substituent is made sufficiently small.

Bottom Line: Strong charge-dependent effects emanate from this toxin surface.In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore.This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.

ABSTRACT
Pore-blocking toxins are valuable probes of ion channels that underlie electrical signaling. To be effective inhibitors, they must show high affinity and specificity and prevent ion conduction. The 22-residue sea snail peptide, mu-conotoxin GIIIA, blocks the skeletal muscle sodium channel completely. Partially blocking peptides, derived by making single or paired amino acid substitutions in mu-conotoxin GIIIA, allow a novel analysis of blocking mechanisms. Replacement of one critical residue (Arg-13) yielded peptides that only partially blocked single-channel current. These derivatives, and others with simultaneous substitution of a second residue, were used to elucidate the structural basis of the toxin's blocking action. The charge at residue-13 was the most striking determinant. A positive charge was necessary, though not sufficient, for complete block. Blocking efficacy increased with increasing residue-13 side chain size, regardless of charge, suggesting a steric contribution to inhibition. Charges grouped on one side of the toxin molecule at positions 2, 12, and 14 had a weaker influence, whereas residue-16, on the opposite face of the toxin, was more influential. Most directly interpreted, the data suggest that one side of the toxin is masked by close apposition to a binding surface on the pore, whereas the other side, bearing Lys-16, is exposed to an aqueous cavity accessible to entering ions. Strong charge-dependent effects emanate from this toxin surface. In the native toxin, Arg-13 probably presents a strategically placed electrostatic barrier rather than effecting a complete steric occlusion of the pore. This differs from other well-described channel inhibitors such as the charybdotoxin family of potassium channel blockers and the sodium channel-blocking guanidinium toxins (tetrodotoxin and saxitoxin), which appear to occlude the narrow part of the pore.

Show MeSH
Related in: MedlinePlus