α4/7-conotoxin Lp1.1 is a novel antagonist of neuronal nicotinic acetylcholine receptors

Abstract

Cone snails comprise approximately 500 species of venomous molluscs which have evolved the ability to generate multiple toxins with varied and exquisite selectivity. α-Conotoxin is a powerful tool for defining the composition and function of nicotinic acetylcholine receptors which play a crucial role in excitatory neurotransmission and are important targets for drugs and insecticides. An α4/7 conotoxin, Lp1.1, originally identified by cDNA and genomic DNA cloning from Conus leopardus, was found devoid of the highly conserved Pro residue in the first intercysteine loop. To further study this toxin, α-Lp1.1 was chemically synthesized and refolded into its globular disulfide isomer. The synthetic Lp1.1 induced seizure and paralysis on freshwater goldfish and selectively reversibly inhibited ACh-evoked currents in Xenopus oocytes expressing rat α3β2 and α6α3β2 nAChRs. Comparing the distinct primary structure with other functionally related α-conotoxins could indicate structural features in Lp1.1 that may be associated with its unique receptor recognition profile.

Atypical α-Conotoxin LtIA from Conus litteratus Targets a Novel Microsite of the α3β2 Nicotinic Receptor

“…The sequence of ␣-conotoxin LtIA (GCCARAACAGIHQELC) was based on a cDNA clone (LeD2P) from C. litteratus from Hainan, China (Table 1) (17). A highly similar cDNA sequence was also reported from Conus leopardus (18). Clones of rat ␣2-␣7 and ␤2-␤4 as well as mouse muscle ␣1␤1␦⑀ cDNAs were kindly provided by S. Heinemann (Salk Institute, San Diego).…”

Characterization of a novel α4/4-conotoxin, Qc1.2, from vermivorous <italic>Conus quercinus</italic>

“…A two-step oxidation protocol was used to selectively fold the peptide to its natural global disulfide isomer as described previously [12]. Briefly, the disulfide bridge between Cys3 and Cys13 was closed by air oxidation.…”

“…Oocytes of Xenopus laevis frogs were prepared and injected with capped RNA to elicit expression of mouse fetal skeletal-muscle and various rat neuronal nAChR subtypes as described previously in detail [12][13][14][15][16].…”

Chemical synthesis and characterization of two α4/7-conotoxins

“…The molecular mass of the purified linear peptide was characterized by a Q-trap mass spectrometer (Applied Biosystems, Foster City, USA). Thereafter, a two-step oxidation was used to prepare folded Mr1.1 and Lp1.4 with site-direct disulfide formation [21]. The first disulfide bond between Cys3 and Cys16 whose protection groups were simultaneously removed during peptide cleavage from resin was formed by air oxidation.…”

“…Electrophysiological recordings from nAchRs exogenously expressed in Xenopus laevis oocytes Oocytes of X. laevis frogs were prepared and injected with capped RNA (cRNA) to elicit expression of mouse fetal skeletal muscle and various rat neuronal nAChR subtypes as described previously [21,22]. cRNAs were prepared using mMESSAGE mMACHINE in vitro RNA transcription kit (ABI) with either T7 or SP6 promoter and recovered by lithium chloride precipitation.…”