Palma E, Mileo A M, Eusebi F, Miledi R
Istituto Pasteur-Fondazione Cenci Bolognetti, Rome, Italy.
Proc Natl Acad Sci U S A. 1996 Oct 1;93(20):11231-5. doi: 10.1073/pnas.93.20.11231.
A study was made of the effects of 5-hydroxytryptamine (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 (IAcCho) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC50) of 56 microM and a Hill coefficient (nH) of 1.2. The inhibition of IAcCho 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 (I5HT) in oocytes injected with cDNA encoding an alpha(7) mutant subunit with a threonine-for-leucine-247 substitution (L247T alpha(7)). I5HT was inhibited by the potent nicotinic receptor blockers alpha-bungarotoxin (100 nM) and methyllycaconitine (1 microM). Furthermore, the characteristics of I5HT, including its voltage dependence, were similar to those of IAcCho. The 5HT dose-I5HT response gave an apparent dissociation constant EC50 of 23.5 microM and a Hill coefficient nH 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.
对注射编码野生型鸡α(7)亚基的cDNA后，5-羟色胺（5HT）对非洲爪蟾卵母细胞中表达的同聚体神经元烟碱样受体（nAcChoR）的影响进行了研究。乙酰胆碱（AcCho）引发的大电流（IAcCho）被5HT以可逆且剂量依赖的方式降低，半数抑制浓度（IC50）为56微摩尔，希尔系数（nH）为1.2。5HT对IAcCho的抑制是非竞争性且不依赖电压的，这种行为与通道阻断机制不相符。单独的5HT在注射野生型α(7)亚基cDNA的卵母细胞中不引发膜电流。相反，5HT在注射编码α(7)突变亚基（亮氨酸247被苏氨酸取代，即L247T α(7)）的cDNA的卵母细胞中引发膜电流（I5HT）。I5HT被强效烟碱样受体阻断剂α-银环蛇毒素（100纳摩尔）和甲基lycaconitine（1微摩尔）抑制。此外，I5HT的特性，包括其电压依赖性，与IAcCho相似。5HT剂量-I5HT反应的表观解离常数EC50为23.5微摩尔，希尔系数nH为1.7，在有AcCho存在时未被改变。同样，L247T α(7)对AcCho的表观亲和力及其协同性也不受5HT影响，表明5HT与AcCho之间缺乏相互作用。这些结果表明，5HT是神经元α(7) nAcChoR的强效非竞争性拮抗剂，但在位于通道结构域M2的高度保守的亮氨酸残基247发生突变后，它变成了非竞争性激动剂。
