title: Alpha-conotoxin analogs with additional positive charge show increased selectivity towards Torpedo californica and some neuronal subtypes of nicotinic acetylcholine receptors.
abstract: Alpha-conotoxins from Conus snails are indispensable tools for distinguishing various subtypes of nicotinic acetylcholine receptors (nAChRs), and synthesis of alpha-conotoxin analogs may yield novel antagonists of higher potency and selectivity. We incorporated additional positive charges into alpha-conotoxins and analyzed their binding to nAChRs. Introduction of Arg or Lys residues instead of Ser12 in alpha-conotoxins GI and SI, or D12K substitution in alpha-conotoxin SIA increased the affinity for both the high- and low-affinity sites in membrane-bound Torpedo californica nAChR. The effect was most pronounced for [D12K]SIA with 30- and 200-fold enhancement for the respective sites, resulting in the most potent alpha-conotoxin blocker of the Torpedo nAChR among those tested. Similarly, D14K substitution in alpha-conotoxin [A10L]PnIA, a blocker of neuronal alpha7 nAChR, was previously shown to increase the affinity for this receptor and endowed [A10L,D14K]PnIA with the capacity to distinguish between acetylcholine-binding proteins from the mollusks Lymnaea stagnalis and Aplysia californica. We found that [A10L,D14K]PnIA also distinguishes two alpha7-like anion-selective nAChR subtypes present on identified neurons of L. stagnalis: [D14K] mutation affected only slightly the potency of [A10L]PnIA to block nAChRs on neurons with low sensitivity to alpha-conotoxin ImI, but gave a 50-fold enhancement of blocking activity in cells with high sensitivity to ImI. Therefore, the introduction of an additional positive charge in the C-terminus of alpha-conotoxins targeting some muscle or neuronal nAChRs made them more discriminative towards the respective nAChR subtypes. In the case of muscle-type alpha-conotoxin [D12K]SIA, the contribution of the Lys12 positive charge to enhanced affinity towards Torpedo nAChR was rationalized with the aid of computer modeling.
authors.affiliation: Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.
authors.forename: Igor E
authors.initials: IE
authors.lastname: Kasheverov
authors.forename: Maxim N
authors.initials: MN
authors.lastname: Zhmak
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Vulfius
authors.forename: Elena V
authors.initials: EV
authors.lastname: Gorbacheva
authors.forename: Dmitry Y
authors.initials: DY
authors.lastname: Mordvintsev
authors.forename: Yuri N
authors.initials: YN
authors.lastname: Utkin
authors.forename: René
authors.initials: R
authors.lastname: van Elk
authors.forename: August B
authors.initials: AB
authors.lastname: Smit
authors.forename: Victor I
authors.initials: VI
authors.lastname: Tsetlin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018118:Chloride Channels / Q000187:drug effects; D002942:Circular Dichroism; D020916:Conotoxins / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D021122:Protein Subunits; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014101:Torpedo / Q000378:metabolism*
chemical_list: D018118:Chloride Channels; D020916:Conotoxins; D021122:Protein Subunits; D011978:Receptors, Nicotinic