Hu Xiang-Qun, Zhang Li, Stewart Randall R, Weight Forrest F
Laboratory of Molecular and Cellular Neurobiology, National Institute on Alcohol Abuse and Alcoholism/NIH, Park Building Room 150, Bethesda, MD 20892-8115, USA.
J Biol Chem. 2003 Nov 21;278(47):46583-9. doi: 10.1074/jbc.M308974200. Epub 2003 Sep 11.
Ligand-gated ion channels are integral membrane proteins that mediate fast synaptic transmission. Molecular biological techniques have been extensively used for determining the structure-function relationships of ligand-gated ion channels. However, the transduction mechanisms that link agonist binding to channel gating remain poorly understood. Arginine 222 (Arg-222), located at the distal end of the extracellular N-terminal domain immediately preceding the first transmembrane domain (TM1), is conserved in all 5-HT3A receptors and alpha7-nicotinic acetylcholine receptors that have been cloned. To elucidate the possible role of Arg-222 in the function of 5-HT3A receptors, we mutated the arginine residue to alanine (Ala) and expressed both the wild-type and the mutant receptor in human embryonic kidney 293 cells. Functional studies of expressed wild-type and mutant receptors revealed that the R222A mutation increased the apparent potency of the full agonist, serotonin (5-HT), and the partial agonist, 2-Me-5-HT, 5- and 12-fold, respectively. In addition, the mutation increased the efficacy of 2-Me-5-HT and converted it from a partial agonist to a full agonist. Furthermore, this mutation also converted the 5-HT3 receptor antagonist/very weak partial agonist, apomorphine, to a potent agonist. Kinetic analysis revealed that the R222A mutation increased the rate of receptor activation and desensitization but did not affect rate of deactivation. The results suggest that the pre-TM1 amino acid residue Arg-222 may be involved in the transduction mechanism linking agonist binding to channel gating in 5-HT3A receptors.
配体门控离子通道是介导快速突触传递的整合膜蛋白。分子生物学技术已被广泛用于确定配体门控离子通道的结构-功能关系。然而，将激动剂结合与通道门控联系起来的转导机制仍知之甚少。精氨酸222（Arg-222）位于第一个跨膜结构域（TM1）之前的细胞外N端结构域的远端，在所有已克隆的5-HT3A受体和α7-烟碱型乙酰胆碱受体中都是保守的。为了阐明Arg-222在5-HT3A受体功能中的可能作用，我们将精氨酸残基突变为丙氨酸（Ala），并在人胚肾293细胞中表达野生型和突变型受体。对表达的野生型和突变型受体的功能研究表明，R222A突变分别使完全激动剂5-羟色胺（5-HT）和部分激动剂2-甲基-5-HT的表观效力提高了5倍和12倍。此外，该突变增加了2-甲基-5-HT的效力，并将其从部分激动剂转变为完全激动剂。此外，该突变还将5-HT3受体拮抗剂/非常弱的部分激动剂阿扑吗啡转变为强效激动剂。动力学分析表明，R222A突变增加了受体激活和脱敏的速率，但不影响失活速率。结果表明，TM1前的氨基酸残基Arg-222可能参与了将激动剂结合与5-HT3A受体通道门控联系起来的转导机制。
