Structure And Function Of The Fs48,A New Potassium Channel Blocker From Xenopsylla Cheopis

In nature,blood-sucking arthropods,with a wide-range of distribution and an abundance in species,generally require periodical blood-sucking to accomplish their life cycles.This phenomenon might take a toll on host’s health and cause disease transmission.Doing the blood-sucking process,which might be inhibited by immune,hemostatic and inflammatory systems in mammal host,the salivary gland of blood-sucking arthropods tends to secret various substances that possesses anti-inflammatory,antinociceptive,immunomodulatory and hemostatic activities to suppress the host defense systems.The high efficiency and diversity in pharmacological capabilities of these substances lead to the bright prospect in research and new drug development.xenopsylla cheopis are widely distributed in China,and FS family peptides have been reported to be existed in their salivary glands in previous studies.In recent study,we investigated Fs48,one of the members in FS family,is a protein containing 55 amino acids with a molecular weight of 6.325 kDa.Our study demonstrated that it is an ion channel inhibitor and the main results are as follows:1)E.coli BL21(DE3)expression system and AKTA purification system were used for expression and purification Fs48,and the function of Fs48,with more than 95%of purity,was studied.2)Patch clamp assay has showed the Fs48 failed to block the Nav channel and Cav channel on DRG,but has high affinity on Kv channel.Subsequently,subtype screening on HEK293T cell model by patch clamp technique showed that Fs48 has specific ion channel block activity on Kv1.1 and Kv1.3 with IC50 of 1.21±0.40 μM and 0.816±0.12 μM,respectively.It was found by ion channel kinetics assay that the manner of Fs48 binding to Kv1.1 and 1.3 channels is a reversible binding and Fs 48 did not change its activation process.Homologous modeling and molecular docking techniques revealed that the mode of action of Fs48 is to form an electrostatic interaction with Kv channel S5-S6 subunit and constitute a three pairs of hydrogen bonds with its conserved domain.According to the above results,we consider that the blockage of Fs48 on Kv1.1 and Kv1.3 channels is physical inhibition rather than combination with voltage receptor.3)Western blot,RT-qPCR,siRNA interference,ELISA,and patch clamp were used to determine that Fs48 have no effect on the physiology of un-activated Raw264.7 cells but affect the Kv1.3 channel on its cell membrane to suppress the activation of MAPKs signaling pathway to inhibit the activation process of LPS-stimulated Raw264.7 cells,such as cytokine mRNA transcription and secretion,thereby exposing the anti-inflammatory capability.By using inflammation and nociception related animal experiments,Fs48 has shown to be able to reduce the second-phase inflammatory pain rather than the first-phase neuropathic pain in formalin test.Moreover,Fs48 also inhibited neutrophil chemotaxis,inflammatory factor secretion,and MPO activity in mice paw edema induced by carrageenan.4)Through Western blot,siRNA interference,Ca2+fluorescent probe,Elisa and patch clamp,Fs48 was strongly proved that could inhibit Kvl.3 channel on Jurkat T cell membrane without affecting the physiological activities of Jurkat T cells.Fs48 is able to reduce K+efflux in Jurkat cells,inhibit hyperpolarization of cell membranes and reduce the permeation into cytoplasm of extracellular Ca2+,suppress Jurkat T cell NF-κB p65 nuclear translocation,thereby inhibiting PMA/ionomycin stimulated Jurkat T cell activation and cytokine production.Animal experiment in rats confirmed that Fs48 be able to ameliorate OVA inducted-delayed hypersensitivity in rats.These above results strongly proved that Fs48 possesses immunomodulatory activity in both vivo and vitro experiments.In conclusion,our study demonstrated that Fs48,a novel potassium channel inhibitor,possesses a favorable anti-inflammatory,antinociceptive and immunomodulatory capabilities in vivo and in vitro.It appears to be a potential molecular template for treatment of inflammatory pain and autoimmune diseases.Furthermore,our study also become a part of research in potassium channel and understanding the molecular mechanism of xenopsylla cheopis.