Threonine-for-leucine mutation within domain M2 of the neuronal a7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist

A study was made of the effects of 5-hydroxy-tryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken alpha(7) subunit. Acetylcholine (AcCho) elicited large currents (I-AcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 mu M and a Hill coefficient (n(H)) of 1.2. The inhibition of I-AcCho by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism, 5HT alone did not elicit membrane currents in oocytes injected with the wild-type alpha(7) subunit cDNA, In contrast, 5HT elicited membrane currents (I-5HT) in oocytes injected with cDNA encoding an cv mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)), I-5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 mu M). Furthermore, the characteristics of I-5HT, including its voltage dependence, were similar to those of I-AcCho. The 5HT dose-I-5HT response gave an apparent dissociation constant EC(50) of 23.5 mu M and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho, Similarly, the apparent affinity of L247T alpha(7) for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal alpha(7) nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.