BmK I, a site-3-specific modulator of VGSCs (voltage-gated sodium channels) from the Chinese scorpion Buthus martensi Karsch, can induce spontaneous nociception and hyperalgesia and generate epileptiform responses in rats, which is attributed to the modulation of VGSCs in the neural system. However, which VGSC subtype is targeted by BmK I remains to be identified. Using two-electrode voltage-clamp recording, we studied the efficacy and selectivity of BmK I to three neuronal VGSCs co-expressed with the auxiliary β1 subunit in Xenopus oocytes. Results revealed that BmK I induced a large increase in both transient and persistent currents in mNav1.6α/β1 (where m indicates mouse), which correlated with a prominent reduction in the fast component of inactivating current. In comparison, BmK I-increased currents of rNav1.2α/β1 (where r indicates rat) and rNav1.3α/β1 were much smaller. The EC50 values of BmK I for rNav1.2α/β1 (252±60 nM) and mNav1.6α/β1 (214±30 nM) were similar and roughly half of that for rNav1.3α/β1 (565±16 nM). Moreover, BmK I only accelerated the slow inactivation development and delay recovery of mNav1.6α/β1 through binding to the channel in the open state. Residue-swap analysis verified that an acidic residue (e.g. Asp1602 in mNav1.6) within the domain IV S3-S4 extracellular loop of VGSCs was crucial for the selectivity and modulation pattern of BmK I. Our findings thus provide the molecular determinant explaining the divergent and intriguing behaviour of neuronal VGSCs in response to site-3-specific modulators, indicating that these subtypes play different roles in BmK I-induced hyperexcitablity in rat models.

BackgroundBmK 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.Principal Findings2 µM BmK IT2 strongly shifted the activation of DmNav1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNav1.2, rNav1.3 and mNav1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu896, Leu899, Gly904 in extracellular loop Domain II S3–S4 of DmNav1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNav1 could be conferred by Domain III. Analysis of subsequent DmNav1 mutants highlighted the residues in Domain III pore loop, esp. Ile1529 was critical for recognition and binding of BmK IT2.Conclusions/SignificanceIn this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNav1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Nav1.2, Nav1.3 and Nav1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.

This study provides direct evidence that BmK AS facilitates steady-state activation and inhibits slow inactivation by stabilizing both the closed and open states of the Na(v)1.3 channel, which might result from an integrative binding to two receptor sites on the voltage-gated sodium channels. These results may shed light on therapeutics against Na(v)1.3-targeted pathology.