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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