Novel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channels

Ronald A. Li, Irene L. Ennis, Patricio Vélez, Gordon F. Tomaselli, Eduardo Marbán

Research output: Contribution to journalArticle

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Abstract

μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nM) and E765D (IC50 = 22.0 ± 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nM) and E765K (IC50 = 4202.7 ± 1088.0 nM) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.

Original languageEnglish (US)
Pages (from-to)27551-27558
Number of pages8
JournalJournal of Biological Chemistry
Volume275
Issue number36
DOIs
StatePublished - Sep 8 2000
Externally publishedYes

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Conotoxins
Inhibitory Concentration 50
Muscle
Rats
Skeletal Muscle
Cysteine
S 6
Restoration
Electrostatics
Static Electricity
Association reactions
Kinetics
methanethiosulfonate ethylammonium
(2-sulfonatoethyl)methanethiosulfonate
Electric potential
Mutation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Novel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channels. / Li, Ronald A.; Ennis, Irene L.; Vélez, Patricio; Tomaselli, Gordon F.; Marbán, Eduardo.

In: Journal of Biological Chemistry, Vol. 275, No. 36, 08.09.2000, p. 27551-27558.

Research output: Contribution to journalArticle

Li, Ronald A. ; Ennis, Irene L. ; Vélez, Patricio ; Tomaselli, Gordon F. ; Marbán, Eduardo. / Novel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channels. In: Journal of Biological Chemistry. 2000 ; Vol. 275, No. 36. pp. 27551-27558.
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abstract = "μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nM) and E765D (IC50 = 22.0 ± 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nM) and E765K (IC50 = 4202.7 ± 1088.0 nM) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.",
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T1 - Novel structural determinants of μ-conotoxin (GIIIB) block in rat skeletal muscle (μ1) Na+ channels

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AU - Ennis, Irene L.

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AU - Tomaselli, Gordon F.

AU - Marbán, Eduardo

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N2 - μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nM) and E765D (IC50 = 22.0 ± 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nM) and E765K (IC50 = 4202.7 ± 1088.0 nM) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.

AB - μ-Conotoxin (μ-CTX) specifically occludes the pore of voltage-dependent Na+ channels. In the rat skeletal muscle Na+ channel (μ1), we examined the contribution of charged residues between the P loops and S6 in all four domains to μ-CTX block. Conversion of the negatively charged domain II (DII) residues Asp-762 and Glu-765 to cysteine increased the IC50 for μ-CTX block by -100-fold (wild-type = 22.3 ± 7.0 nM; D762C = 2558 ± 250 nM; E765C = 2020 ± 379 nM). Restoration or reversal of charge by external modification of the cysteine-substituted channels with methanethiosulfonate reagents (methanethiosulfonate ethylsulfonate (MTSES) and methanethiosulfonate ethylammonium (MTSEA)) did not affect μ-CTX block (D762C: IC(50, MTSEA+) = 2165.1 ± 250 nM; IC(50, MTSES-) = 2753.5 ± 456.9 nM; E765C: IC(50, MTSEA+) = 2200.1 ± 550.3 nM; IC(50, MTSES-) = 3248.1 ± 2011.9 nM) compared with their unmodified counterparts. In contrast, the charge-conserving mutations D762E (IC50 = 21.9 ± 4.3 nM) and E765D (IC50 = 22.0 ± 7.0 nM) preserved wild-type blocking behavior, whereas the charge reversal mutants D762K (IC50 = 4139.9 ± 687.9 nM) and E765K (IC50 = 4202.7 ± 1088.0 nM) destabilized μ-CTX block even further, suggesting a prominent electrostatic component of the interactions between these DII residues and μ-CTX. Kinetic analysis of μ-CTX block reveals that the changes in toxin sensitivity are largely due to accelerated toxin dissociation (k(off)) rates with little changes in association (k(on)) rates. We conclude that the acidic residues at positions 762 and 765 are key determinants of μ-CTX block, primarily by virtue of their negative charge. The inability of the bulky MTSES or MTSEA side chain to modify μ-CTX sensitivity places steric constraints on the sites of toxin interaction.

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