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PLoS One
2011 Jan 07;61:e14510. doi: 10.1371/journal.pone.0014510.
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Localization of receptor site on insect sodium channel for depressant β-toxin BmK IT2.
He H
,
Liu Z
,
Dong B
,
Zhang J
,
Shu X
,
Zhou J
,
Ji Y
.
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BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized.2 µM BmK IT2 strongly shifted the activation of DmNa(v)1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNa(v)1.2, rNa(v)1.3 and mNa(v)1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu(896), Leu(899), Gly(904) in extracellular loop Domain II S3-S4 of DmNa(v)1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNa(v)1 could be conferred by Domain III. Analysis of subsequent DmNa(v)1 mutants highlighted the residues in Domain III pore loop, esp. Ile(1529) was critical for recognition and binding of BmK IT2.In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNa(v)1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Na(v)1.2, Na(v)1.3 and Na(v)1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.
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Figure 1. Effect of BmK IT2 on wild-type VGSCs expressed in Xenopus oocytes.A. Current responses of rNav1.2, rNav1.3, mNav1.6 and DmNav1 channels to a test voltage of â50 mV, where channels were closed under control conditions (gray traces). Black traces represented currents in the presence of 2 µM BmK IT2 without a prepulse (âPP, upper panel) and with a prepulse (+PP, lower panel). The scale bar in figure 1A covered all four embodied currents. B. Normalized conductance plotted as a function of voltage for the indicated channel subtypes. C. Current-voltage curves for the indicated channel types. âª, control conditions; âµ, 2 µM BmK IT2 without a prepulse (âPP); â, 2 µM BmK IT2 with a prepulse (+PP).
Figure 2. Analysis of mutations in DII of DmNav1.A. Sequence comparison of extracellular loops DII S1âS2 and DII S3âS4 among wild-type VGSCs. B-E. Normalized conductance-voltage (GâV) curves of DmNav1 mutants DmD838C, DmE896C, DmL899C and DmG904N in the absence (âª) and presence (â) of 2 µM BmK IT2. All the currents were recorded after applying a prepulse of â10 mV for 25 ms.
Figure 3. Schematic composition of DmNav1-Nav1.2 domain chimeras and effect of BmK IT2 on four chimeric channels.A. Cartoons illustrating the construction of channel chimeras. The channel domains of rNav1.2 were shown in grey, while the domains from DmNav1 were shown in black. B. Normalized conductance-voltage plotted for chimeras ChD1-ChD4 before (âª) and after (â) application of 2 µM BmK IT2, with a prepulse (PP).
Figure 4. Analysis of DmNav1-Nav1.2 DIII SS2 loop chimeras.A. Sequences of SS2 loop in DIII of wild-type VGSCs. B. Diagram illustrating the composition of the SS2 loop chimeras L(Dm)Nav1.2 and L(1.2)DmNav1 (Nav1.2 SS2 loop: grey; DmNav1 SS2 loop: black). CâD. Effect of BmK IT2 on voltage-dependent activation of L(Dm)Nav1.2 and L(1.2)DmNav1 with a prepulse (PP) of â10 mV for 25 ms. âª, control conditions; â, 2 µM BmK IT2.
Figure 5. Site-directed mutations introduced in DIII SS2-S6 loop of DmNav1.AâI. Normalized conductance-voltage curves for the indicated mutant channels before (âª) and after (â) application of 2 µM BmK IT2, with a prepulse (+PP) in all cases.
Figure 6. Schematic presentation of domain arrangement and key residues involved in BmK IT2-DmNav1 interaction.Schematic BmK IT2 structural model (in amino residue) was constructed by Swiss-model Workspace (http://swissmodel.expasy.org) based on the known structure of the depressant β-toxin LqhIT2 (>80% similarity in sequence) (PDB accession 2i61A). Key residues involved in BmK IT2 interaction with DmNav1 were highlighted in red and indicated with sequence numbers on extracellular loop of DII (yellow) and DIII (blue).
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