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.
Kasheverov
I
I
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
Rozhkova
A
A
Zhmak
M
M
Utkin
Y
Y
Ivanov
V
V
Tsetlin
V I
VI
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins / Q000494:pharmacology; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008969:Molecular Sequence Data; D010777:Photochemistry; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014101:Torpedo
D002038:Bungarotoxins; D020916:Conotoxins; D007457:Iodine Radioisotopes; D021122:Protein Subunits; D011978:Receptors, Nicotinic; C029960:conotoxin GI; C036155:conotoxin MI