大鼠脑组织Nav1.5钠通道的基因克隆及分布分析

Cloning and Distribution Analysis of Nav1.5 Sodium Channel in Rat Brain

为了阐明大鼠脑组织Nav1.5钠通道α亚单位的编码基因、分子特性及其在不同发育阶段各脑叶的分布差异,应用逆转录聚合酶链反应(RT-PCR)方法,对大鼠脑组织Nav1.5钠通道α亚单位进行了克隆(命名为rN1),并比较其在不同发育阶段各脑叶的分布情况.rN1基因编码2016个氨基酸残基,序列分析显示,其与rH1氨基酸相似性为96.53%,与hNbR1相似性为96.13%.在DI-S3～S4发现与rH1不同的一个新的外显子(第7外显子),同时发现DⅡ～Ⅲ选择性剪切了第20外显子(53个氨基酸残基)的异构体(命名为rN1-2).分布结果显示,大鼠脑组织Nav1.5钠通道α亚单位在不同发育阶段各脑叶分布有明显的差异,研究证实,Nav1.5钠通道在大鼠脑组织显著表达,存在脑叶的分布差异,而且随着脑组织的发育,其表达逐渐趋于稳定,实验证实Nav1.5钠通道基因编码了一个新的外显子而且其表达范围更加广泛.

To clarify the gene and molecule characters of the tetrodotoxin-resistant （TTX-R） voltage -gate sodium channel and the distribution in different lobe of brain in different developmental stage, reverse transcriptase- polymerase chain reaction （RT-PCR） was used to clone the full sequence ofNav1.5 sodium channel （designated as rN1） α subunit in rat brains and the distribution was compared in different lobe of brain in different developmental stage. The open reading frame（ORF） of rN1 encodes 2016 amino acid residues and sequence analysis indicated that rN1 is highly homologous with 96.53% amino acids identity to rat cardiac Navl.5 sodium channel （rill） and 96.13% amino acids identity to human neuroblastoma Navl.5 sodium channel （hNbRl）. It has all the structural features of a voltage-gated sodium channel and the presence of a cysteine residue （C373） in the pore loop region of domain Ⅰ suggests that this channel is TTX resistant. A new exon （exon7） that distinct from rN1 was found in D Ⅰ -S3-S4. In addition, an alternative splicing isoforms that deleted 53 amino acids（exon20） was found in the loop between D Ⅱ-Ⅲ in rN1 （designated as rNI-2）. Distribution result demonstrated that rN1 expressed discrepancy in different ages and lobe in brain. The expression level ofrN1 was gradually more stable in adult than neonatal. These results suggest that Nav1.5 has a newly identified exon for alternative splicing and is more widely expressed than previously thought.