Screening And Indentification Of Peptide Drug Acting On ASIC1a

Acid-sensing ion channels?ASICs?,which are activated by extracelluar H⁺,are H⁺-gated cation channels,it contains six subunits and are widely expressed in the peripheral and the central nervous systems.In recent years,ASIC1a has been shown to have important functions in pain and learning memory.Meanwhile,ASIC1a also involves some pathological processes,such as epilepsy,glioblastoma,ischemic stroke and so on.Ion channel stable expressing cell lines combined with calcium fluorescence imaging technology and automatic patch clamps will undoubtedly facilitate the screening of modulators.Therefore,we have constructed stable cell lines forthe study of ASICs that is a hot topic in ion channels.We tested current density of each generation via the patch clamps technique and observed the green fluorescent expressional intensity of each generation through inverted phase contrast microscope to detect the cell lines'stability,and the results showed that the number of ion channels expressed in the stable cell lines did not changesignificantly,statistically,and the intensity of fluorescence was stable.It confirmed that two kinds of ion channel stable cell lines,ASIC1a,ASIC3,have been successfully constructed.On the basis of ASIC1a stable cell line,we screened modulators from venom ofpoisonous animals.We found that PPCV-21.8 isolated from the venom of Psalmopoeus pulcher by reverse-phrase high-performance liquid chromatography,acts as an inhibitor or activator under different conditions.Due to its low content and failure to be purified to a single component for identification,but combined with the Spider venom gland cDNA library data analysis,suggesting that the amino acid sequence obtained from cDNA library analysis is the key component of PPCV-21.8.It consists of 40 amino acid residues,containing 3 pairs of disulfide bonds.PPCV-21.8 directly activated ASIC1 channel.It apparently increased affinity for H⁺of ASIC1a,which leading to channel desensitization at pH7.4,In the presence of 5?M PPCV-21.8,the pH₅₀ of activation was significantly?P<0.01?leftward shifted by 0.36 pH units to pH 7.07±0.059.And then we explored the effect of PPCV-21.8 on the other ASICs subtypes,We found that 18?M PPCV-21.8 had no effect on ASIC2a and slightly activated ASIC3 current,about 32.67±4.0%,and strongly activated ASIC1b with an EC₅₀ 1.4?M.Moreover,18?M PPCV-21.8accelerated the speed of ASIC1b opening??=0.162±0.029s without toxin,?=0.042±0.005s with toxin,P<0.01,n=5?and significantly prolonged the channel desensitization??=0.579±0.107s without toxin,?=2.13±0.265s,with toxin,P<0.01,n=5?.Long-term incubation of 15 nM PPCV-21.8?about 200 s?can inhibit ASIC1a current,the inhibition rate is 93.89±1.9%.60 nM PPCV-21.8 does not affect other ASICs subtypes.The most important thing is high concentration PPCV-21.8 have no effect on voltage-gated sodium channel,so the toxin is safe to human.In summary,we found that PPCV-21.8 is an inhibitor of ASIC1a with high activity and strong selectivity,which can be used as a drug lead molecule in the treatment of pain,stroke and other diseases and also contributes to the study of the structure and function of ASICs,so that,we can further enhancing the understandment the relationship between ASICs and physiological and pathological conditions and explore potency of ASICs to be an important target for disease treatment.