Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture?

Gating modifiers of voltage-gated sodium channels (Na_vs) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion β-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa_v1.2a) and skeletal muscle (rNa_v1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4^{F14A/E15A/E28R}, of rNa_v1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4^{F14A/E15A/E28R} was tested using an rNa_v1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4 ^{F14A/E15A/E28R} and residues whose equivalents in the rat brain channel rNa_v1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na_vs, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa_v1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na_vs resembles that of voltage-gated potassium channels.