Research On The Screening, Design And Function Identification Of Scorpion Toxin Peptides Interacting With KCNQ1 Channel

Potassium ion channels are closely related to many physiological and pathological progresses. Among various kinds of potassium ion channels, KCNQ subfamlily is one of the voltage-gated potassium channels (Kv). It comprises five members:KCNQ1 (also known as Kv7.1), KCNQ2 (also known as Kv7.2), KCNQ3 (also known as Kv7.3), KCNQ4 (also known as Kv7.4) and KCNQ5 (also known as Kv7.5). Among all five KCNQ subtypes, four subtypes are related to human diseases. Numerous loss-of-function mutations in Kv7.1 subunits have been identified in long QT syndrome (LQTS), in which the QT interval is prolonged; Many loss-of-function mutations in KCNQ2 or KCNQ3 are associated with a rare hereditary human epilepsy called benign familial neonatal convulsions. Furthermore, loss-of-function mutations in Kv7.4 are associated with autosomal dominant deafness. Importantant progresses have been made in the physiological and pathological functions of KCNQ channels, but the structure and function relationship research of these channels remains obviously unclear, and one of important reasons is the lacking of peptide modulators binding KCNQ channels.Scorpion is one of the most ancient arthropods, and has survived on earth over 400 million years. Scorpions are used as an important medicinal material in Chinese traditional medicine. The research of modern biochemistry and molecular biology technology showed that there are thousands of bioactive peptides in scorpion venoms, and it is a great challenge to search the targets of so many kinds of peptides from the venom of scorpion. Based on the research progresses of KCNQ1 channel and scorpion toxins, computational and experimental methods were used to screen and design peptide modulators specially binding KCNQ1 channel.First, we analysised the KCNQ1 channnel structure features of the regions that interact with peptides. Sequence alignments of the pore region of potassium channels Kvl.x, SKCal-3, BKCa showed that there is a conserved Asp317 residue, which likely makes the pore region of these channels form positively charged potential and permit the recoginition of toxin peptide with many basic residues. KCNQ1 channel is different from the above channels, and there is a positively charged Lys318 residue near Asp317 in the pore region of KCNQ1 channel, which may result in the insensitive of the basic scorpion toxins towards KCNQ1 channel. Besides these basic toxin peptide in the venom of scorpion, there is another kind of toxin peptides with unknown function that contain many negatively charged residues in the molecules (named "acidic potassium channel toxins"). In the light of the features of KCNQ1 channel and acidic scorpion toxins, KCNQ1 channel could be the potential target of acidic scorpion toxin peptides.Second, we screened and idenfied a series of acidic potassium channel toxins that possibly interact with KCNQ1 channel. All selected toxin peptides were expressed with a GST fusion protein in Rosetta (DE3) strains using the vector of pGEX-6P-1.The recombinant GST-fusion proteins were purified by GST affinity chromatography, treated with enterokinase or Factor Xa. The six recombinant toxin peptides were purified by RP-HPLC, and successfully identified with MALDI-TOF-MS. Subsequently, electrophysiological experiments were carried to identify the interaction of the six toxin peptides with KCNQ1 channel. The results showed that 10μM recombinant toxin peptides Im 104, Lmk2,23,32, BmP01, Pbtx1 can inhibit KCNQ1 channel current about 29.1%,23.4%,15.8%, 33.0%,9.7%,30.7%, respectively. And 10μM recombinant toxin peptide Im104, Lmk2,23,32, BmP01, Pbtx1 can also inhibit Iks current (formed by KCNQ1+mink) about 37.7%,27.7%,4.2%,22.0%,4.8%,21.4%, respectively. Overall, the six toxin peptides presented weak effect towards KCNQ1 channel current and Iks current, and only 10μM toxin peptides had significant effects on channel currents. In order to improve the activity of peptide Im104 towards KCNQ1 channel, we determinated its spatial structure. The 15N labeled rIm104 peptide was prepared with M9 minimal medium, and its structure was successfully determined by NMR technique. The results showed that rIm104 had a stable conformation in the solution with classical disulfide bridges (C1-C4, C2-C5 and C3-C6). The second structures of Im104 were not apparent, and only a short a-helix was formed. So, Im104 is more flexible than the traditional basic scorpion peptides towards potassium channels.And then, we designed a new mutant peptide and identified its effect on KCNQ1 channel. On the basis of the different activity towards KCNQ1 channel from acidic potassium channel toxins and basic potassium channel toxins, we considered structure-property features of both KCNQ1 channel pore and turret, and proposed a new idea to design the potent peptide inhibitor: (1) keep the designed peptides slightly alkaline;(2) make the designed peptides a polarized molecule;(3) design some special residues to enhance the interaction between the peptide and channel turret region. Based on these three strategies, a new toxin peptide T1 was designed with a Kv1.3 channel potent toxin peptide ADWX-1 (PDB code:2K4U) as a template by changing six basic residues (K6A, K8N,H9E, K15N, K18G, R23E). The recombinant peptide T1 was expressed in E.coli and successfully identified with MALDI-TOF-MS. Electrophysiological experiments further indicated that the recombinant T1 peptide inhibited KCNQ1 channel current with IC50 value of 4.6±1.9μM. From a KCNQ1 channel unsensitive peptide ADWX-1 to a sensitive designed T1, it indicated that our original idea was reasonable. In the future research we will select more suitable molecular templates to design potent and selective inhibitor binding with KCNQ1 channel. In summary, we tried to obtain a potent KCNQ1 peptide modulator in this research. We firstly selected six novel toxin peptides from the scorpion toxin peptides libraries, and identified their effects on KCNQ1 current. The electrophysiological experiments indicated that all the six toxin peptide showed weak activity to KCNQ1 channel. Based on the former results, we proposed a new strategy to design the potent inhibitor of KCNQ1 channel and designed a new peptide T1 (IC50=4.6±1.9μM). The primary successful design of T1 peptide indicated that it is feasible to design the more potent and selective inhibitor targeting KCNQ1 channel.