Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23

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Abstract
Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N‐hydroxysuccinimide esters of p‐azidobenzoic, p‐azidotetraflourobenzoic, p‐benzoylbenzoic and p‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzoic acids. The ion‐exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross‐linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross‐linking yield (up to 16 % of the bound toxin) was observed for azidobenzoyl‐Lys23‐CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group : the azido derivatives labelled the γ and δ subunits, benzoylbenzoyl derivative labelled the α and δ subunits, while p‐[3‐(trifluoromethyl)‐3H‐diazirin‐3‐yl]benzoyl derivative reacted with α, γ and δ subunits. The cross‐linking experiments in the presence of varying concentrations of (+)‐tubocurarine demonstrated that the Lys23‐attached diazirinyl group contacts the δ and α subunits in one ligand‐binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23‐diazirinyl are with γ and α subunits. This means that the central loop in the two CTX molecules binds at the α/γ and α/δ interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X‐ray structure of CTX, showed that this value does not exceed 13 Å. The results obtained favor the disposition of the ligand‐binding sites at the subunit interfaces, with the distance between α and δ, or α and γ subunits at these sites being not more than 13 Å.

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