Butler Amy S, Lindesay Sarah A, Dover Terri J, Kennedy Matthew D, Patchell Valerie B, Levine Barry A, Hope Anthony G, Barnes Nicholas M
Cellular and Molecular Neuropharmacology Research Group, School of Experimental and Clinical Medicine, The Medical School, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Neuropharmacology. 2009 Jan;56(1):292-302. doi: 10.1016/j.neuropharm.2008.08.017. Epub 2008 Aug 22.
Amongst the family members of Cys-loop LGICs, the atypical ability of the 5-HT3A subunit to form functional homomeric receptors allowed a direct investigation of the role of the C-terminus. Deletion of the three C-terminal amino acids (DeltaGln453-DeltaTyr454-DeltaAla455) from the h5-HT3A subunit prevented formation of a specific radioligand binding site as well as expression within the cell membrane. Removal of merely the C-terminal residue (DeltaAla455) reduced specific radioligand binding (to 4+/-1% relative to the wild-type; cells grown at 37 degrees C) and also cell membrane expression; these reductions were less evident when the DeltaAla455 expressing cells were grown at 27 degrees C (specific radioligand binding levels 27+/-5% relative to wild-type also grown at 27 degrees C). Mutation of the h5-HT3A C-terminal amino acid, alanine, for either glycine (Ala455Gly), valine (Ala455Val) or leucine (Ala455Leu) reduced specific radioligand binding levels by 24+/-23%, 32+/-12% and 88+/-1%, respectively; the latter mutant also displaying reduced membrane expression. In contrast, mutation to alanine of the two amino acids preceding the C-terminal alanine (Gln453Ala and Tyr454Ala) had no detrimental effects on specific radioligand binding or cell membrane expression levels. The present study demonstrates an important role for the C-terminus in the formation of the functional h5-HT3A receptor. The partial restoration of 5-HT3 receptor binding and cell membrane expression when cells expressing C-terminal mutant 5-HT3A subunits were grown at a lower temperature (27 degrees C) suggests that the C-terminus stabilises the 5-HT3 receptor allowing subunit folding and subsequent maturation.
在半胱氨酸环配体门控离子通道（Cys-loop LGICs）家族成员中，5-HT3A亚基形成功能性同聚体受体的非典型能力使得能够直接研究C末端的作用。从h5-HT3A亚基中删除三个C末端氨基酸（ΔGln453-ΔTyr454-ΔAla455）可阻止特异性放射性配体结合位点的形成以及细胞膜内的表达。仅去除C末端残基（ΔAla455）会降低特异性放射性配体结合（相对于野生型为4±1%；细胞在37℃下生长）以及细胞膜表达；当表达ΔAla455的细胞在27℃下生长时，这些降低不太明显（相对于同样在27℃下生长的野生型，特异性放射性配体结合水平为27±5%）。将h5-HT3A C末端氨基酸丙氨酸分别突变为甘氨酸（Ala455Gly）、缬氨酸（Ala455Val）或亮氨酸（Ala455Leu），可使特异性放射性配体结合水平分别降低24±23%、32±12%和88±1%；后一种突变体也表现出膜表达降低。相比之下，将C末端丙氨酸之前的两个氨基酸突变为丙氨酸（Gln453Ala和Tyr454Ala）对特异性放射性配体结合或细胞膜表达水平没有不利影响。本研究证明了C末端在功能性h5-HT3A受体形成中的重要作用。当表达C末端突变5-HT3A亚基的细胞在较低温度（27℃）下生长时，5-HT3受体结合和细胞膜表达的部分恢复表明C末端稳定了5-HT3受体，允许亚基折叠并随后成熟。
