Dhulipala P D, Wang Y X, Kotlikoff M I
Department of Animal Biology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104-6046, USA.
Gene. 1998 Jan 30;207(2):259-66. doi: 10.1016/s0378-1119(97)00647-1.
ATP acts as a fast excitatory neurotransmitter by binding to a large family of membrane proteins, P2X receptors, that have been shown to be ligand-gated, non-selective cation channels. We report the cloning of a full-length and alternatively spliced form of the human P2X4 gene. Clones were identified from a human stomach cDNA library using a rat P2X4 probe. Nucleotide sequence analysis of positive clones identified the full-length human P2X4 cDNA, which codes for a 388-residue protein that is highly homologous (82%) to the rat gene, and an alternatively spliced cDNA. In the alternatively spliced cDNA, the 5'-untranslated region and the first 90 amino acids in the coding region of full-length human P2X4 are replaced by a 35 amino acid coding sequence that is highly homologous with a region of chaperonin proteins in the hsp-90 family. The open reading frames of the full-length and splice variant clones were confirmed by in vitro translation. Northern analysis indicated expression of the full-length P2X4 message in numerous human tissues including smooth muscle, heart, and skeletal muscles. Alternatively spliced RNAs were identified in smooth muscle and brain by RT-PCR and confirmed by RNAse protection assays using a 710 bp anti-sense RNA probe that spanned the alternatively spliced and native P2X4 regions. Injection of full-length, but not alternatively spliced, cRNA into Xenopus oocytes resulted in the expression of ATP gated non-selective cation currents.
ATP通过与一大类膜蛋白P2X受体结合而作为一种快速兴奋性神经递质，这些受体已被证明是配体门控的非选择性阳离子通道。我们报告了人类P2X4基因全长及选择性剪接形式的克隆。使用大鼠P2X4探针从人胃cDNA文库中鉴定出克隆。对阳性克隆的核苷酸序列分析确定了全长人类P2X4 cDNA，其编码一个由388个氨基酸残基组成的蛋白质，该蛋白质与大鼠基因高度同源（82%），以及一个选择性剪接的cDNA。在选择性剪接的cDNA中，全长人类P2X4编码区的5'-非翻译区和前90个氨基酸被一个35个氨基酸的编码序列取代，该序列与hsp-90家族中伴侣蛋白的一个区域高度同源。全长和剪接变体克隆的开放阅读框通过体外翻译得到证实。Northern分析表明全长P2X4信息在包括平滑肌、心脏和骨骼肌在内的多种人类组织中表达。通过RT-PCR在平滑肌和脑中鉴定出选择性剪接的RNA，并使用跨越选择性剪接和天然P2X4区域的710 bp反义RNA探针通过RNA酶保护试验进行了证实。将全长而非选择性剪接的cRNA注射到非洲爪蟾卵母细胞中导致了ATP门控的非选择性阳离子电流的表达。
