title: Molecular basis of isoform-specific micro-conotoxin block of cardiac, skeletal muscle, and brain Na+ channels.
abstract: mu-Conotoxins (mu-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 mu-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 (mu1, Na(v)1.4), human heart (hh1, Na(v)1.5), and rat brain (rb1, Na(v)1.1) 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 Na(v)1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Na(v)1.1 and Na(v)1.5. The Na(v)1.4-->Na(v)1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Na(v)1.4 numbering) reduced block of Na(v)1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Na(v)1.5--> Na(v)1.4 chimeric construct in which the Na(v)1.4 DII S5-P linker replaces the analogous segment in Na(v)1.5 showed enhanced mu-CTX block. However, these variant determinants are conserved between Na(v)1.1 and Na(v)1.4 and thus cannot explain their different sensitivities to mu-CTX. Comparison of their sequences reveals two variants at Na(v)1.4 positions 729 and 732: Ser and Asn in Na(v)1.4 compared with Thr and Lys in Na(v)1.1, respectively. The double mutation S729T/N732K rendered Na(v)1.4 more "brain-like" (30-fold downward arrow in block), and the converse mutation T925S/K928N in Na(v)1.1 reproduced the high affinity blocking phenotype of Na(v)1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in mu-CTX binding, thus shaping isoform-specific toxin sensitivity.
authors.affiliation: Institute of Molecular Cardiobiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA. ronaldli@jhmi.edu
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
authors.forename: Irene L
authors.initials: IL
authors.lastname: Ennis
authors.forename: Tian
authors.initials: T
authors.lastname: Xue
authors.forename: Hai M
authors.initials: HM
authors.lastname: Nguyen
authors.forename: Gordon F
authors.initials: GF
authors.lastname: Tomaselli
authors.forename: Alan L
authors.initials: AL
authors.lastname: Goldin
authors.forename: Eduardo
authors.initials: E
authors.lastname: Marbán
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000378:metabolism; D020916:Conotoxins / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D020033:Protein Isoforms / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics; D051381:Rats; D017386:Sequence Homology, Amino Acid; D015222:Sodium Channels / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics
chemical_list: D020916:Conotoxins; D020033:Protein Isoforms; D015222:Sodium Channels