Molecular Cloning Research On Novel Channel Modulators From Scorpion Venom And Scorpion Sodium Channel

In this study, the several cDNA cloning or genomic DNA of modulators specific on voltage-gated sodium channel and a ligand of KCa, as well the cDNA cloning of sodium channel of scorpion BmK have been investigated. The research includes mainly four parts.1. cDNA cloning of two novel (-like toxin named as BmK (1 and BmK (2 According to a relative conserved fragment of (-scorpion toxins, a degenerate primer was designed and synthesized, two full-length cDNAs encoding the precursors of two novel putative (-like toxins were then amplified from the total cDNAs of venomous glands of the Chinese scorpion Buthus martensi Karsch using 3' RACE and 5' RACE (rapid amplification of cDNA ends). They were named as BmK (1 and BmK (2. The precursors are both composed of 85 amino acid residues, including a putative signal peptide of 19 residues and a mature toxin of 66 residues, respectively. The predicted amino acid sequences of these two toxins show a homology of 82% with each other, and of 55% - 70% with other ( or (-like toxins. Regarded to the length of B-loop between (2 and (3 strand same as classic ( and (-like toxin as well as the sequence homology, these two toxins are attributed to (-like toxin group.2. cDNA cloning of a unique long-chained scorpion toxin named as BmK abT According to the partial N-terminal sequence of native BmK abT, adegenerate primer was assigned for 3' RACE, the whole cDNA was overlapped by the 3' and 5' RACE results. The molecular weight diversity between the putative native peptide and that deduced from cDNA sequence suggested a basic residue Lys at C-terminal was cut during post-translation modification, and then the peptide was amidated at the C-terminal. Electrophysiological recording proved 10 μM BmK abT could prolong the action potential and increase the amplitude of the peak Na+ currents of rat DRG nerve, which accorded with the physical characterization of (-toxin. Furthermore, the B-loop between (2 and (3 - strand of BmK abT was the same as classic (-toxin. However, alignment of amino acid sequence revealed that BmK abT had the most homology of 46.2% with Ts VII, a ( toxin from the new world in family of long-chained scorpion toxins. The N'-terminal 6 residues (fuction domain) of BmK abT was in common with two (-toxin from the new world. All above on deduced BmK abT is a transitional member between (- and (-type toxins.3．Gene cloning and analysis of martentoxin, a novel ligand for Calcium activated potassium channel According to the N-terminal sequence of native martentoxin, a degenerate primer was assigned for 3' RACE, the whole cDNA was overlapped by the 3' and 5' RACE results. Afterwards, the whole genomic DNA of martentoxin was directly got by PCR using the specific primer originated from the 3' and 5' UTR. One intron located in the signal peptide was found, and another intron was found in the 5' UTR, which was never reported before. Two introns were ended with gt in the 5'-terminal and ag in the 3'-terminal, which was the same as the other scorpion toxins. Amino acid sequence alignments found martentoxin has the most homology with Lqh 15-1 and TmTX (higher than 73%), however, lower than 50% with other potassium channel ligands. Biosensor and electrophysiological recording revealed martentoxin acted on Ca++ activated K+ channel of RACC, after elution, the recovering of K+ channels acted by martentoxin was faster and more entire than those acted by ChTX. The results thus suggested that martentoxin might form a new group with unique structural and functional properties in the family of K+ channel-blocking ligands, which will be useful probes to facilitate characterization of the composition, electrical signaling of K+ channels.4. cDNA cloning of sodium channel from scorpion nerve fibers The total RNA was extracted from scorpion nerve fibres and reverse transcripted to cDNA. Analyzing the sodium channel amino acid sequence from different species (mammal, amphibian, fish, mollusc and insect), it was found that thehomologous fragments focused on the segm...