Abstract
Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 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(v)1.2a 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(v)s, 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(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.
Highlights
Among the superfamily of voltage-gated ion channels, sodium channels (Navs)3 play a central role in the generation of action potentials in excitable cells and are the target of a large variety of neurotoxins, drugs, and insecticides [1, 2]
Scorpion -toxins are short polypeptides reticulated by four disulfide bonds that target Navs and modulate their gating properties [17]
The G/V relations of rNav1.4E1251N and rNav1.4H1257K were affected by Css4E15A, but with lower potency (EC50 ϭ 1.91 and 5.3 M, respectively) (Tables 1 and 2 and Fig. 4B). These results suggest that Leu-653 and Gly-658, and to a lesser extent Glu-1251 and His-1257, are involved in the interaction of Css4E15A with
Summary
Voltage-gated sodium channel; Css, Centruroides suffusus suffusus -toxin 4; hypoPP, hypokalemic periodic paralysis. Scorpion -toxins (e.g. the anti-mammalian Css and Css from Centruroides suffusus suffusus) are short polypeptides reticulated by four disulfide bonds that target Navs and modulate their gating properties [17] They are typified by the shift they induce in the voltage dependence of channel activation to more negative membrane potentials upon binding to receptor site 4 [17], shown to be associated with Domain 2 of the Nav (18 –21). Four conserved residues in D2/S1-S2 and S3-S4 have been implicated in the interaction of the -toxin Css with mammalian Navs [19, 20], and two residues in D3/SS2-S6 have recently been linked to the preference of the -toxin Tz1, from the scorpion Tityus zulianus, for the muscle channel rNav1.4 over the brain channel rNav1.2a [22] These data, and the fact that Css is more potent at hNav1.2a than hNav1.4 [23], have suggested that the set of residues comprising receptor site 4 on both channels are not identical.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
