Abstract

Nicotinic acetylcholine receptors are key molecules in cholinergic transmission in the nervous system. Because of their structural complexity, only a limited number of subtype-specific agonists and antagonists are available to study nicotinic receptor functions. To overcome this limitation, we used voltageclamp recordings to examine the effects of several frog skin alkaloids on acetylcholine-elicited currents in Xenopus laevis oocytes expressing major types of neuronal nicotinic receptors (α4β2, α7, α3β2, α3β4, and α4β4). We found that the 5,8-disubstituted indolizidine (-)-235B′ acted as a potent noncompetitive blocker of α4β2 nicotinic receptors (IC₅₀ = 74 nM). This effect was highly selective for α4β2 receptors compared with α3β2, α3β4, and α4β4 receptors. The inhibition of α4β2 currents by (-)-235B′ was more pronounced as the acetylcholine concentration increased (from 10 nM to 100 μM). Moreover, the blockade of α4β2 currents by (-)-235B′ was voltage-dependent (more pronounced at hyperpolarized potentials) and use-dependent, indicating that (-)-235B′ behaves as an open-channel blocker of this receptor. Several other 5,8-disubstituted indolizidines (5-n-propyl-8-n-butylindolizidines), two 5,6,8-trisubstituted indolizidines ((-)-223A and (+)-6-epi-223A), and a 1,4-disubstituted quinolizidine ((+)-207I) were less potent than (-)-235B′, and none showed selectivity for α4β2 receptors. The quinolizidine (-)-1-epi-207I and the tricyclic (+)-205B had 8.7- and 5.4-fold higher sensitivity, respectively, for inhibition of the α7 nicotinic receptor than for inhibition of the α4β2 receptor. These results show that frog alkaloids alter the function of nicotinic receptors in a subtype-selective manner, suggesting that an analysis of these alkaloids may aid in the development of selective drugs to alter nicotinic cholinergic functions.