Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-L247T nicotinic acetylcholine receptor.
The effects of the native alpha-conotoxin PnIA, its synthetic derivative [A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L]PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nm, respectively. Rates of onset of inhibition were similar for PnIA and [A10L]PnIA; however, the rate of recovery was slower for [A10L]PnIA, indicating that the increased potency of [A10L]PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nm. In contrast, [A10L]PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [A10L]PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.
Department of Physiology, CMU, 1 rue Michel Servet, CH-1211 Geneva 4, Switzerland. hogg1@etu.unige.ch
Ron C
RC
Hogg
Gene
G
Hopping
Paul F
PF
Alewood
David J
DJ
Adams
Daniel
D
Bertrand
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002645:Chickens; D020916:Conotoxins / Q000235:genetics* / Q000494:pharmacology*; D005260:Female; D066298:In Vitro Techniques; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
D020916:Conotoxins; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins; C089038:alpha-conotoxin PnIA; D064569:alpha7 Nicotinic Acetylcholine Receptor