Designs And Syntheses Of α-conotoxins Which Target At Nicotinic Acetylcholine Receptor α3β2 Subtype And The Functional Investigations Of This Subtype

Nicotinic acetylcholine receptor (nAchR) is widely distributed in the muscle, central nervous system and peripheral nervous system, which regulates neural and muscular conduction and participates in learning, feeling, nicotine addiction, nerve disorder and parkinsonism. nAchR is one of the most popular ion channels and membrane proteins. The latest research demonstrated that the nAChRα3β2 subtype is related to analgesia.Based on the structure and action mechanism ofα-conotoxins AnIB that targets nAChRα3β2 subtype, a series of newlyα-conotoxin mutants were designed and synthesized in the present study. By one-step and two-step oxidation, we got eight purified peptides and determined their analgesic activity using partial sciatic nerve ligation model (PSL model). In addition, we synthesized AnIB[A11G] fluorescence analog to determine the distribution ofα3β2 subtype in mouse brain.For the further study of the activity and selectivity of AnIB mutants toα3β2 subtype, we have constructed the two-electrode voltage clamp platform and cloned ten nAChR subunits from rat brains and lungs into T7 promoter vectors pcDNA3.1(+) and pCI. The results were summarized as follows:1. Eight mutants were synthesized by one-step or two-step oxidation (three by one-step oxidation, five by two-step oxidation). The folding efficiency of conotoxins are significantly affected by the mutated residues.2. One mutant, a chimera of AnIB and Vc1.1(GCC SDPR CAGNNQDYC-NH2, Z-1), displayed higher analgesic activity compared with Vc1.1 in PSL model. Others showed similar or lower activities.3. The 5-TAMRA- AnIB [A11G] fluorescent analog was mainly distributed on mice brain hippocampus, few in striatum and litter or not in cerebellar and other regions. while the control group 5-TAMRA has no selectivity except cerebral cortex. This distribution ofα3β2 in brain corresponds with its analgesic function.4. Ten nAChR subunits were cloned by overcoming several difficulties such as low levels of mRNA, high contents of GC bases, high homology of ten subunits, long sequence and high-fidelity. These subunits were inserted into the expression vector pcDNA3.1(+) and pcI. cDNAs, and transcribed in vitro. The cRNA was then microinjected into the oocyte for recording. The two-electrode voltage clamp experiment is now being ongoing.