Photoactivatable alpha-conotoxins reveal contacts with all subunits as well as antagonist-induced rearrangements in the Torpedo californica acetylcholine receptor.

Azidobenzoyl (AzBz) and benzoylbenzoyl (BzBz) derivatives of alpha-conotoxin MI and L-benzoylphenylalanine (Bpa) analogs of alpha-conotoxin GI were synthesized. All these compounds, similarly to native alpha-conotoxins, completely displaced the radioiodinated MI or GI from the membrane-bound nicotinic acetylcholine receptor (AChR) of Torpedo californica. However, the GI(Bpa11) analog was considerably less potent than GI in competing with radioiodinated alpha-bungarotoxin (alphaBgt). Irradiation of iodinated AzBz derivatives bound to AChR resulted in labeling of all AChR subunits. The BzBz and Bpa derivatives gave lower levels of specific cross-linking but considerable labeling at additional sites that was enhanced, rather than suppressed, by an excess of native alpha-conotoxins or alphaBgt. Both equilibrium binding of benzophenone-derivatized alpha-conotoxins and their cross-linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for alpha-conotoxins and alphaBgt are overlapping but not identical, (b) each of the AChR subunits can be labeled with photoactivatable alpha-conotoxins and (c) enhancement of benzophenone-derivatized alpha-conotoxins cross-linking at additional (physostigmine-related) sites by alphaBgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.