Molecular basis of isoform-specific μ-conotoxin block of cardiac, skeletal muscle, and brain Na+ channels

Ronald A. Li, Irene L. Ennis, Tian Xue, Hai M. Nguyen, Gordon F. Tomaselli, Alan L. Goldin, Eduardo Marbán

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

μ-Conotoxins (μ-CTXs) block skeletal muscle Na+ channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na+ channels. Although a number of conserved pore residues are recognized as critical determinants of μ-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (μ1, Nav1.4), human heart (hh1, Nav1.5), and rat brain (rb1, Nav1.1) Na+ channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Nav1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Nav1.1 and Nav1.5. The Nav1.4→Nav1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Nav1.4 numbering) reduced block of Nav1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Nav1.5→ Nav1.4 chimeric construct in which the Nav1.4 DII S5-P linker replaces the analogous segment in Nav1.5 showed enhanced μ-CTX block. However, these variant determinants are conserved between Nav1.1 and Nav1.4 and thus cannot explain their different sensitivities to μ-CTX. Comparison of their sequences reveals two variants at Nav1.4 positions 729 and 732: Ser and Asn in Nav1.4 compared with Thr and Lys in Nav1.1, respectively. The double mutation S729T/N732K rendered Nav1.4 more "brain-like" (30-fold ↓ in block), and the converse mutation T925S/K928N in Nav1.1 reproduced the high affinity blocking phenotype of Nav1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in μ-CTX binding, thus shaping isoform-specific toxin sensitivity.

Original languageEnglish (US)
Pages (from-to)8717-8724
Number of pages8
JournalJournal of Biological Chemistry
Volume278
Issue number10
DOIs
StatePublished - Mar 7 2003
Externally publishedYes

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Conotoxins
Muscle
Brain
Myocardium
Protein Isoforms
Skeletal Muscle
S 6
Rats
Mutation
Substitution reactions
Ions
Phenotype

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular basis of isoform-specific μ-conotoxin block of cardiac, skeletal muscle, and brain Na+ channels. / Li, Ronald A.; Ennis, Irene L.; Xue, Tian; Nguyen, Hai M.; Tomaselli, Gordon F.; Goldin, Alan L.; Marbán, Eduardo.

In: Journal of Biological Chemistry, Vol. 278, No. 10, 07.03.2003, p. 8717-8724.

Research output: Contribution to journalArticle

Li, Ronald A. ; Ennis, Irene L. ; Xue, Tian ; Nguyen, Hai M. ; Tomaselli, Gordon F. ; Goldin, Alan L. ; Marbán, Eduardo. / Molecular basis of isoform-specific μ-conotoxin block of cardiac, skeletal muscle, and brain Na+ channels. In: Journal of Biological Chemistry. 2003 ; Vol. 278, No. 10. pp. 8717-8724.
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abstract = "μ-Conotoxins (μ-CTXs) block skeletal muscle Na+ channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na+ channels. Although a number of conserved pore residues are recognized as critical determinants of μ-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (μ1, Nav1.4), human heart (hh1, Nav1.5), and rat brain (rb1, Nav1.1) Na+ channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Nav1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Nav1.1 and Nav1.5. The Nav1.4→Nav1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Nav1.4 numbering) reduced block of Nav1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more {"}cardiac-like.{"} Conversely, an Nav1.5→ Nav1.4 chimeric construct in which the Nav1.4 DII S5-P linker replaces the analogous segment in Nav1.5 showed enhanced μ-CTX block. However, these variant determinants are conserved between Nav1.1 and Nav1.4 and thus cannot explain their different sensitivities to μ-CTX. Comparison of their sequences reveals two variants at Nav1.4 positions 729 and 732: Ser and Asn in Nav1.4 compared with Thr and Lys in Nav1.1, respectively. The double mutation S729T/N732K rendered Nav1.4 more {"}brain-like{"} (30-fold ↓ in block), and the converse mutation T925S/K928N in Nav1.1 reproduced the high affinity blocking phenotype of Nav1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in μ-CTX binding, thus shaping isoform-specific toxin sensitivity.",
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AU - Goldin, Alan L.

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