cDNA cloning and endogenous ligand identification of the novel ligand-gated ion-channel receptors

5HT3C是5 -羟色胺门控嗜离子受体的新成员。通过对5HT3C cDNA与人类基因组数据库的序列同源性检索，在同一染色体3q27.1上又发现了3个受体基因5HT3D、3E和3L1。这四个预测的基因由9个外显子组成，与先前已知的5 -羟色胺受体5HT3A和3B具有外显子-内含子组织的共同特征。人体组织RT-PCR分析显示，5HT3C mRNA在胎盘、肺和肾中高表达，5HT3D mRNA在肺和胰腺中高表达。cDNA克隆结果显示，在肾脏和胰腺中分别表达了缺乏5HT3C外显子2、3、4和6的备选剪接形式和缺乏5HT3D外显子3的一个剪接形式。为了比较这些mRNA在各组织中的表达水平，使用从外显子7和外显子9的保守序列设计的共同引物进行PCR-RFLP分析。在脑、肺和肝脏中，5HT3C转录本占主导地位，而5HT3D信息主要在心脏表达。5HT3D和5HT3L1在骨骼肌中共表达，5HT3C、5HT3D和5HT3L1在胎盘、肾脏和胰腺中共表达。未检测到5HT3E转录本的表达，因此假设该基因为假基因。此外，5HT3D和5HT3L1的外显子7和外显子8交界处的其他剪接变异体在胰腺中表达。综上所述，这些结果表明，新鉴定的离子通道受体通过各种剪接变异体的寡聚化在不同组织中执行或调节许多生理功能。

5HT3C is the new member of serotonin-gated ionotropic receptor. From the sequence homology search for 5HT3C cDNA against human genomic database, three additional receptor genes, designated 5HT3D, 3E and 3L1, were identified on the same chromosome 3q27.1. These four predicted genes are composed of nine exons and share common features in exon-intron organization with previously known serotonin receptors 5HT3A and 3B. RT-PCR analysis in human tissues demonstrated that 5HT3C mRNA was highly expressed in placenta, lung and kidney, whereas 5HT3D mRNA was seen in lung and pancreas. The cDNA cloning revealed that alternative splicing form lacking exon 2,3,4 and 6 of 5HT3C and one lacking exon 3 of 5HT3D were expressed in kidney and pancreas, respectively. To compare the expression levels of these mRNA in each tissue, PCR-RFLP assay was performed by using common primers designed from the conserved sequences in exon 7 and exon 9. In brain, lung and liver, 5HT3C transcript was dominant, while 5HT3D message was mainly expressed in heart. Coexpression of 5HT3D and 5HT3L1 was demonstrated in skeletal muscle while 5HT3C, 5HT3D and 5HT3L1 were co-expressed in placenta, kidney and pancreas. The expression of 5HT3E transcript could not be detected, and hence, this gene was assumed as a pseudogene. Furthermore, other splicing variants at the junction of exon 7 and exon 8 of 5HT3D and 5HT3L1 were expressed in pancreas. Taken together, these results suggest that novel identified ion-channel receptors execute or modulate many physiological functions in diverse tissues through oligomerization of various splicing variants.