Isolation And Purification Of A Novel Sodium Channel Modulator Of Scorpion Venom Polypeptide And Its Electrophysiological Function

Buthus martensii Karsch are widely distributed in China.The Loess Plateau in northern of Shaanxi is rich of scorpion due to its unique geographical environment.Scorpion,as a traditional Chinese medicine,has a long history of application,but little is known about its pharmacodynamic components and mechanism.Studies have shown that the medicinal value of scorpion mainly owes to the scorpion toxin which contains a large number of neurotoxins.Scorpion toxin can specifically act on sodium channel,affecting the gating properties of the sodium channel.The sodium channel always plays an important role in the physiological and pathological processes.Based on these,we measured the regulatory effect of the single active polypeptide components which were isolated and purified from scorpion venom by gel filtration chromatography and reversed-phase high performance liquid chromatography technology,on sodium channel with the whole cell patch clamp technology.The main research contents and results are as follows:(1)Isolation,Purification and Molecular Weight Determination of Scorpion Venom PolypeptideThree chromatographic peaks,which named H-1,H-2 and H-3 respectively,were obtained from scorpion crude toxin by isocratic elution with SephadexTM G-50 Fine gel column.28 peaks could be obtained from H-2 component by reversed-phase high performance liquid chromatography(RP-HPLC)Atlantis T3 column.14 single components were obtained from 6 components(H-2-10~H-2-15)by purified with RP-HPLC again.The collected and freeze-dried components were identified by electrospray ionization mass spectrometry(ESI-MS).H-2-11-2 and H-2-11-3 short-chain scorpion venom polypeptides with molecular weights of 3766 Da and 3765 Da were obtained.H-2-14-1,H-2-14-2 and H-2-15-1 long chain scorpion venom polypeptides with molecular weights of 7229.2 Da,7030 Da and 7028.5 Da were obtained respectively.Long chain scorpion venom polypeptides H-2-14-1 and H-2-15-1 were selected for identification on the modulatory function of sodium channel.(2)Regulation of H-2-14-1 on Nav1.5,Nav1.7 and Nav1.8The activation voltage,peak current and deactivation voltage of Nav1.5,Nav1.7 and Nav1.8 at different time were recorded by the whole cell patch clamp at 100 nM,200 nM and 500 nM H-2-14-1,respectively.The results showed that H-2-14-1 had no effect on Nav1.8 activation at each concentration,and the activation voltages were both-50 mV.H-2-14-1 at 100 nm has little effect on the peak current of Nav1.8.The peak current could reach the maximum value of-286 pA after 5 mins,while the H-2-14-1 reducing the deactivation voltage and accelerating the inactivation of Nav1.8.The 200 nM and 500 nM H-2-14-1 have the same effect,that is,the influence on the peak current of the channel is small.But it increased the Nav1.8 deactivation voltage,prolonged the channel opening time,and delayed its inactivation.100 nM,200 nM and 500 nM H-2-14-1 had the same regulation effect on Nav1.5 and Nav1.7,that is,the activation voltage was reduced and the activation was facilitated,the channel opening time was prolonged,the inactivation is delayed,and the channel peak current was increased.However,the time of the maximum peak current value for each concentration of H-2-14-1 with Nav1.5 and Nav1.7 were different.The time of the maximum peak current value for the three concentrations of H-2-14-1 with Nav1.5 appeared at 12 min,5 min and 9 min respectively,for Nav1.7 were at 5 min,7 min and 11 min.Therefore,H-2-14-1 has different regulatory effects on Nav1.8,Nav1.5 and Nav1.7 with sodium channel subtype selectivity and concentration dependence.(3)Regulation of H-2-15-1 on Nav1.5,Nav1.7 and Nav1.8In the whole cell patch clamp mode,the activation voltage,current and deactivation voltage of Nav1.5,Nav1.7 and Nav1.8 at different times were recorded at 100 nM,200 nM and 500 nM H-2-15-1,respectively.The results showed that three concentrations of H-2-15-1 could reduce the activation voltage of Nav1.7 and Nav1.5 and facilitate activation,but 100 nm H-2-15-1 shortened the Nav1.7 open time,accelerated deactivation,less impact on its peak current,the maximum peak current reached after 5 minutes of bathing,while 200 nM and 500 nMH-2-15-1 extended the Nav1.7 opening time,delaying its inactivation The peak current increases,and the maximum peak current is reached when the two baths are 9 min and 5 min.Three concentrations of H-2-15-1 extended the opening time of Nav1.5 and Nav1.8,delayed channel inactivation,and increased channel peak current,but different concentrations of H-2-15-1 and Nav1.5 The maximum peak current value of the Nav1.5 bath was different at 5 min,7 min and 5 min,and Nav1.8 at 9 min,9 min and 7 Min.However,H-2-15-1 had no effect on the activation of Nav1.8.Therefore,H-2-15-1 regulates Nav1.7 with sodium channel subtype selectivity and concentration dependence.In conclusion,the scorpion venom polypeptides H-2-14-1 and H-2-15-1 have different selectivity and concentration dependence on the sodium channel subtype.Thus,H-2-14-1 and H-2-15-1 could be used as potential tools for studying the modulatory mechanism of different sodium channel subtypes.The further study on the regulatory mechanism of H-2-14-1 and H-2-15-1 for sodium channel will provide important references for sodium channel modulators study.And it will also have important theoretical and practical value.