Toxin Resistance Mechanism Of Scorpion Channels And The Function Of New Type Toxin

Scorpions have been on earth for more than four hundred million years. During natural evolution, they developed a unique venom system for capturing prey and defending against predators. A lot of research showed the toxicity of the venoms containing different types of neurotoxins targeting different ion channels, from those in invertebrates to those in vertebrates. Because it has been a huge challenge to clone functional scorpion ion channels, researchs about the resistance or insensitivity of scorpions to its own venoms are far from enough. The first genome of the scorpion M martensii which was reported in 2013 rekindled the hope of unveiling the scorpion ion channel and then the mechanism of scorpion insensitivity to its own venom and toxin.With the assistance of bio informatics methods, the sequence information of different types of scorpion ion channels were discovered in this work based on the genome of the scorpion Mesobuthus martensii and therefore we obtained different scorpion channels one after another through molecular cloning, such as voltage-gated potassium channel MmKv3, calcium-activated potassium channel MmSK1, voltage-gated sodium channel MmNav2, members of subfamily C of transient receptor potential cation channel MmTRPC-1 and MmTRPC-2, member of subfamily V of transient receptor potential cation channel MmTRPV-1, member of subfamily A of transient receptor potential cation channel MmTRPA-1 and members of subfamily M of transient receptor potential cation channel MmTRPM-1 and MmTRPM-2. These scorpion ion channel genes lay the foundation for the study of scorpion resistance to its own venom and toxin.We developed the research on the resistance of the functional scorpion ion channels to toxins based on the discovery of scorpion channel. First, we carried out the interactions between scorpion voltage-gated potassium channel MmKvl and scorpion toxins in the present thesis. The channel kinetic experiments showed that MmKvl was a classical voltage-gated potassium channel with a voltage-dependent fast activation and slow inactivation. Electrophysiology experiments showed that a 1:2,000 dilution of scorpion M. martensii venom inhibited 71.5 ± 1.0% of hKvl.3 channel current, but only 16.1 ± 0.8% of MmKv1. Moreover,1 nM toxin BmKTX-D33H could block 71.5 ± 1.1% of the hKvl.3 currents; however,100 nM toxin BmKTX-D33H could only block 16.1 ± 0.8% of the MmKvl currents. Those significant differences of current inhibition effect indicated a remarkable insensitivity of MmKv1 channel to both scorpion venom and toxin. Moreover, MmKv1-TF, the chimeric channels of MmKv1 (the Turret and Filter regions were identical with the corresponding domains of the hKvl.3 channel), showed comparable inhibition with human Kv1.3 channel by scorpion venoms in same concentration which revealed that the extracellular pore region (include turret and filter regions) of MmKvl channel were critical for the toxin insensitivity of MmKvl. When two basic residues (Arg399 and Lys403) in the MmKv1 turret region and Arg425 in the MmKv1 filter were simultaneously substituted with the corresponding residues from hKvl.3, the mutated MmKv1-R399T/K403S/R425H channel exhibited similar sensitivity to both scorpion venom and toxin as hKv1.3, which revealed the determining role of these three basic residues in the toxin insensitivity of MmKvl channel More strikingly, a similar triad sequence structure is present in all Shaker-like channels from venomous invertebrates, which suggested a possible convergent functional evolution of these channels to enable them to resist their own venoms. This paper also studied calcium-activated potassium channel MmSKl from Mesobuthus martensii based on the research of scorpion channel MmKvl. Different from the unique structure of MmKvl channel, MmSK1 channel have remarkable similar sequence and 3D structure with human SK3 channel, but it also exhibited a remarkable insensitivity to both scorpion venom and toxin. It was found that a 1 mg/ml of freeze-dried scorpion M. martensii venom inhibited the MmSK1 and hSK3 channels by 31.1 ± 2.3% and 80.8 ± 0.6%, respectively. Moreover,100 nM scorpion toxin ScyTx inhibited the MmSK1 and hSK3 channels by 23.8 ± 3.1% and 77.2 ± 5.2%, respectively. Structure analysis showed that MmSKl channel with a more compact structure compared to human SK3 channel, which may leads to its resistance to toxin. Together, these findings first illustrated the mechanism by which scorpions are insensitive to own venoms at the ion channel receptor level and enriched our knowledge of the insensitivity of scorpions and other venomous animals to their own venoms.This paper also developed research on the interaction between the new type scorpion toxin and potassium channels in view of the general character of potassium channel toxins. Ascaris-type peptides had been identified as potent protease inhibitors possessing similar properties with toxins acting on potassium channel. In this paper, we found SjAPI-2 had 62 amino acid residues, including 10 cysteine residues, and it have two acidic residues regionally distributed, and 10 basic residues widely distributed. Pharmacological studies confirmed that SjAPI-2 was a selective KCNQ1 potassium channel inhibitor with weak effects on other potassium channels, such as Kv1.1, Kv1.2, Kvl.3, SKCa2, SKCa3, and IKCa channels. Concentration-dependent studies showed that SjAPI-2 inhibited the KCNQ1 potassium channel with an IC50 of 771.5 ± 169.9 nM. To the best of our knowledge, SjAPI-2 is the first neurotoxin with a unique Ascaris-type fold, providing novel insights into the divergent evolution of neurotoxins from venomous animals.