Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) a ntagonists, are emerging as important probes of the role played by differen t nAChR subtypes in cell function and communication, In this study, the nat ive alpha-conotoxins PnIA and PnIB were found to cause concentration-depend ent inhibition of the ACh-induced current in all rat parasympathetic neuron s examined, with IC50 values of 14 and 33 nM, and a maximal reduction in cu rrent amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nM and a maximally effective concentration caused 91% block, [A10L]PnIA was the m ost potent inhibitor, reducing the ACh-induced current in similar to 80% of neurons, with an IC50 value of 1.4 nM and 46% maximal block of the total c urrent, The residual current was not inhibited further by alpha-bungarotoxi n, but was further reduced by the cu-conotoxins PnIA or PnIB, and by mecamy lamine. H-1 NMR studies indicate that PnIA, PnIB, and the analogues, [A10L] PnIA and [N11S]PnIA, have identical backbone structures. We propose that po sitions 10 and II of PnIA and PnIB influence potency and determine selectiv ity among alpha 7 and other nAChR subtypes, including alpha 3 beta 2 and al pha 3 beta 4, Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conope ptides.