Photoactivatable α-constoxins 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 cr-conotoxin GI were synthesized. All these compounds, similarly to native alpha -conotoxins, completely displaced the radioiodinated MT 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 (alpha Bgt). 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 alpha Bgt. Both equilibrium binding of benzophenone-derivatized a-conotoxins and their cross-linking could be totally abolished by physostigmine. The results obtained demonstrate that (a) specific binding sites for alpha -conotoxins and alpha Bgt 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 alpha Bgt or GI indicates that these antagonists induce structural alterations in the AChR outside their binding sites.