Molecular-biological and pharmacological analysis of regulating sites of glycine receptor function in Xenopus oocytes using novel compounds

我们以前曾报道过吗啡衍生物之一的二甲双胍（DM）对甘氨酸诱导的大鼠脑单个神经元电流有抑制作用。本研究旨在阐明甘氨酸受体功能的调节位点，并进一步了解DM和止咳药对其他神经受体和通道的作用。[结果]1）DM、士的宁、可待因和咖啡因对表达甘氨酸受体α_1-和α_2a-亚单位的爪蟾卵母细胞的甘氨酸诱导电流均有抑制作用。DM<50S>和可待因的IC_1在α_1中分别为5.2 × 10 ~（13）<-5>M和2.2 × 10 ~（13<-4>）M，在α_2中分别为1.2 × 10 ~（13）<-5>M和5.5 × 10 ~（13<-5>）M。这两种药物对α_2的抑制作用强于α_1，而士的宁和咖啡因则相反。2)在新合成的11个化合物中，有3个化合物对甘氨酸诱导的表达α_1亚基的非洲爪蟾卵母细胞电流有抑制作用。3)在α_1突变体Y161 F、F159 Y/Y161 F和S267 I中，DM的抑制作用与野生型相比没有改变。另一方面，士的宁的行动是加强在双突变体，F159 Y/Y161 F比野生型。4)在急性分离的大鼠中缝背核神经元膜片钳技术中，DM不仅能抑制5-HT诱导的K^+电流（IC_1为1.43 × 10 ~（-6）M），而且<-5><50>在无5-HT存在的情况下，DM也能不可逆地抑制胞内GTPγS激活的K^+电流。结果提示：（1）吗啡烷结构可能是甘氨酸受体功能改变所必需的化学结构的一部分;（2）DM在甘氨酸受体上可能存在不同于Str作用的位点。

We have previously reported that dextromethorphan (DM), one of morphan derivatives, inhibited glycine-induced currents in single neuron acutely dissociated from rat brain. This study was designed to clarify the regulating site of glycine receptor function and to get further information about the action of DM and antitussives on other neuronal receptors and channels.【Results】1) DM, strychnine, codeine and caffeine inhibited the glycine-induced cur-rents in Xenopus oocytes expressing α_1-or α_2a-subunit of glycine receptor. IC_<50S> for DM and codeine were 5.2x10^<-5>M and 2.2x10^<-4>M, respectively, in the α_1, and 1.2x10^<-5>M and 5.5x10^<-5> M, respectively in the α_2. Both drugs showed more potent inhibitory action on the α_2 than α_1, while the reverse was true for strychnine and caffeine. 2) Out of newly synthesized 11 compounds, which have a morphan structure in the molecule, three compounds inhibited the currents induced by glycine in Xenopus oocytes expressing the α_1 subunit. 3) In the mutants, Y161F, F159Y/Y161F and S267I, of the α_1, the inhibitory action of DM was not changed, compared with the action in the wild type. On the other hand, the action of strychnine was potentiated in the double mutant, F159Y/Y161F than in the wild type. 4) In patch-clamp study using acutely dissociated dorsal raphe neurons of rats, DM inhibited not only the K^+ currents induced by 5-HT with 1.43x10^<-5> M of the IC_<50>, but also the currents irreversibly activated by intracellular GTPγS even in the absence of 5-HT.The results sugest that (1) the morphan structure might be a part of chemical structure essential for modifing the function of glycine receptors, (2) DM may have a site on the glycine receptor different from the site where Str acts.