Structure And Function Of JZTX-50,A Novel Selective Inhibitor Of K_v 1.1

Kv1.1 belonging to the Kv1 or mammalian Shaker-family is encoded by the gene KCNA1.It can be expressed on HEK293T cell membrane and generates the delayed outward Rectifier potassium channel currents which can be detected by patch-clamp.Channel Kv1.1 is widely expressed throughout the nervous system,especially the central nervous system(CNS).In general,most forms of neuronal Kv1 channels are thought to contain at least one Kv1.1 and/or Kv1.2 subunit,and the Kv1.1 is regarded as one target for various CNS disorders.In fact,it is of great importance in controlling the neuroexcitability.Various mutations of Kv1.1 are demonstrated to be associated with diseases such as epilepsy,spinal cord injury and multiple sclerosis.In this study,I separated and purified a selective inhibitor of Kv1.1 screened from the crude of Spider Chilobrachys jingzhao who mainly distribute in Guangxi Province.Then,a series of experiments were performed.By the combination of Reversed-Phase High Performance Liquid Chromatography(HPLC),ion exchange chromatography and mass spectrometry identification technique,I got a peptide which could inhibit the current of Kv 1.1 expressed on HEK293T by transient transfection.The relative molecular weight of this peptide was identified to be 3897.0.Edman degradation sequencing revealed the sequence information.The peptide consists of 35 amino acids,and they are GRCIEEGKWCPKKAPCCGRLECKGPSPKQKKCTRP.It turns to be jingzhao toxin 50(JZTX-50),which contains 3 pairs of S-S and is proposed to be typical ICK motif.JZTX-50 is a novel inhibitor of Kv 1.1.Electrophysiological experiments conducted by using patch clamp indicate that JZTX-50 inhibits the current of Kv 1.1 obviously on the concentration of 50 nmol/L.Further studies demonstrate the inhibit effect of JZTX-50 to Kv 1.1 has great concentration dependence,and the IC50 is 38.15 nmol/L.JZTX-50 won’t make the steady-state activation curve shift a lot,so it makes no great difference to the activation dynamic characteristics of Kv1.1.To test the selectivity of JZTX-50 to Kv1.1,I applied JZTX-50 to different subtypes of potassium channel.It delighted me that JZTX-50 has no effect on Kv 1.2,Kv 1.3,Kv 2.1,Kv 2.2,Kv 4.2 and Kv4.3,and only a little inhibition on currents of Kv 1.4 and Kv7.1 on really high concentration.Moreover,I conducted tests where JZTX-50 was applied to currents of sodium channel and calcium channel on acutely-isolated rat Dorsal Root ganglion(DRG)cells.Taking the effect of JZTX-50 on different sodium channel subtypes expressed on HEK293T,JZTX-50 rarely has no influence in their currents or just a little inhibition effect.All the experimental results suggest that JZTX-50 is a novel selective inhibitor of Kv1.1,and it makes no big difference on the activation kinetics characteristics of Kv 1.1.So,JZTX-50 has potential to be developed into a tool reagent for the study of Kv1.1.It may also make great contribution to the treatment of diseases,such as multiple sclerosis.