| BmK I, a site-3 voltage-gated sodium channel(VGSC)-specific modulator has been investigated to induce nociceptive behaviors in rat models. However, question remain unknown with regard to whether Nav1.8, a major VGSC subtype associating with inflammatory, and possibly neuropathic pain was involved in the rise and maintenance of BmK I-induced pain nociception. By animal behavioral testing methods and electrophysiological techniques, we study this question and explore the mechanism of Nav1.8 participated in the BmK I-induce pain rat models.1. BmK-induced inflammatory pain model was constructed in rat by plantar injection. Consistent with the previous study, spontaneous nociception, mechanical hyperalgeisa and decreased thermal threshold could considerably be observed in rat model after BmK I administration. In this research, the current density of Nav1.8 was found substantially increased in small DRG neurons acutely separated from the spinal cord of BmK I-treated rat at 2-hour time point. And Nav1.8 kinetic properties was altered that both the voltage-dependence activation and steady-state inactivation was hyperpolarized shifted. When applying A-803467, a selective Nav1.8 sodium channel blocker, at the spinal cord and the hind paw in rats before the injection of BmK I, the spontaneous nociceptive behaviors and mechanical hypersensitivity induced by BmK I were attenuated but the thermal hypersensitivity was unaffected. These results indicated that the increasing of Nav1.8 current density and the low activation threshold contributed to the enhancement of neuron excitability, thus involved in the pain-related behaviors. And the changes of Nav1.8 at 2-hour time point indicated primary sensory neurons were activated rapidly.2. As a site 3 VGSCs modulator, whether BmK I could regulate Nav1.8 had not be confirmed. Electrophysiology experiments revealed that BmK I could dose-dependently increase the transient and persistent currents of Nav1.8, largely hyperpolarized shift voltage-dependence activation that sensitize this sodium channel. Meanwhile, the study on steady-state inactivation, fast and slow inactivation was also showed a substantial hyperpolarized shifted after applying with BmK I, and both fast and slow inactivation was inhibited. All the clues present in this study clearly suggested that Nav1.8 expressed in small DRG neurons was the direct target of Bm K I, whose hyperexcitability due to the up-regulation and functional properties modulated by BmK I contribute to the hyperalgesia.3. Domain IV S3/S4 linker was demonstrated to be important region which determine the sensitivity of VGSCs to BmK I, while Nav1.8 was insensitivity to almost all test site 3 scorpion toxins via the insertion of four amino acids(Ser, Leu, Glu, Asn, SLEN) in this region. However, our results revealed BmK I have a prominent potency on Nav1.8. Thus two peptides were synthesized according to the DIV/S3-S4 linker in Nav1.8 and Nav1.5 as a positive control. The conditions of peptides binding to BmK I was investigated by surface Plasmon resonance(SPR), revealed that Nav1.8 could bind with BmK I in this region, and have higher association rate constant and lower disassociation rate constant by contrast with Nav1.5, while the capacity in Nav1.8 was less than Nav1.5. These results revealed that the ability of Nav1.8 binding with BmK I is not deprived by the insertion of these amino acids, which indicated the mechanism of Nav1.8 interacting with BmK I was different with other site 3 toxins. |
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