Research On The Inhibition Of TREK-1 Channel By Peptide 7901 From Ixodes Scapularis

TREK-1 channel is a member of TERK channels,a subfamily of the Two pore domain K+ channel(K2P channel).TREK-1 channel exists primarily as a dimer.TREK-1 channel is highly expressed in the central nervous system and cardiomyocytes,and participates in physiological processes such as pain perception,mood regulation,anesthesia reaction,cardiac mechano-electrical feedback,and vasodilation.Since TREK-1 channel is involved in many important physiological processes,it is associated with the development of many diseases,such as ischemic cerebrovascular disease,epilepsy,depression,and others.TREK-1 channel can be regulated by a variety of chemical and physical stimuli.In recent years,it has been found that some small molecule compounds and small linear peptide spadin can function on TREK-1 channel.However,peptides that have a spatial structure functioning on the channel have not been discovered.There are abundant active substances in Ixodes scapularis.More and more researchers have been focusing on the study of active substances from ticks.At present,these active substances have been found to have some important functions such as anticoagulation and antibacterial,and are valuable treasures for research.The mature peptide 7901 from Ixodes scapularis has 89 amino acid residues and forms four pairs of intramolecular disulfide bonds.In our study,recombinant protein of peptide 7901 was obtained by prokaryotic expression.Then,a series of separation and purification was done,such as His-Tag affinity chromatography,enterokinase digestion,Sephadex G-75 gel separation,RP-HPLC.lastly,the purity of peptide 7901 containing four pairs of disulfide bonds was determined by MALDI-TOF-MS.Using patch clamp technique we found that peptide 7901 can inhibit TREK-1 channel with an IC50 value of 23.46 μM.In order to further explore the selectivity of peptide 7901 on other ion channels,we tested the function of the peptide on voltage-gated sodium channels,potassium channels,and calcium and chloride ion channels.The peptide was found to have no effect on Nav1.3,Nav1.4,Nav1.5,Nav1.7,Kv1.5,Kv2.1,Kv4.1,Cav3.1,TMEM16A channels.It was found that the affinity of peptide 7901 to TREK-1 channel was much higher than that to other K2P channel,such as KCNK3,KCNK9,KCNK18.We found that the peptide could inhibit TREK-1 channel in a voltage-independent manner.The peptide could function on the channel in a rapid wash-on and wash-off rate.And the peptide could inhibit the open state activated by arachidonic acid.Futhermore,it was found that peptide 7901 could inhibit TREK-1 channel more strongly in a weakly acidic environment compared to that at a pH 7.4,and the affinity of peptide 7901 to the TERK-1 channel increased 5 fold.Peptide 7901 could inhibit TREK-1 channel at the same way,though the site H142 effecting protonation of TREK-1 channel was changed to other redidues containing different charge properties.In our study,peptide 7901 was found to inhibit TREK-1 channel in a different way from fluoxetine,suggesting that peptide 7901 might have different binding sites.Further,we would explore the binding sites.In our research,peptide 7901 was obtained firstly by prokaryotic expression and it could selectively inhibit TREK-1 channel.The affinity of peptide 7901 to TREK-1 channel is significantly increased under a weak acidic condition.The manner in which peptide 7901 and fluoxetine function on the channel was different.These findings suggest that peptide 7901 might play an important role of the study on the structure and function of TREK-1 channel.