Threonine-for-leucine mutation within domain M2 of the neuronal α ₇ 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 α ₇ subunit. Acetylcholine (AcCho) elicited large currents (I(AcChu)) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC ₅₀) of 56 μM and a Hill coefficient (nH) of 1.2. The inhibition of IAacCHO BY %HT 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 α ₇ subunit cDNA. In contrast, 5HT elicited membrane currents (I5(HT)) in oocytes injected with cDNA encoding an α ₇ mutant subunit with a threonine-for-leucine-247 substitution (L247T α ₇). I(5HT) was inhibited by the potent nicotinic receptor blockers α-bungarotoxin (100 nM) and methyllycaconitine (1 μM). 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 μM and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T α 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 α ₇ nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.