Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals
Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals
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DOI：
10.1111/j.1469-7793.1999.0323m.x
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发表时间：
1999-09-01
影响因子：
5.5
通讯作者：
Lazdunski, M
中科院分区：
文献类型：
--
作者：
Sakai, H;Lingueglia, E;Lazdunski, M
1.简并聚合酶链反应（PCR）同源性筛选程序应用于大鼠脑cDNA，以确定新的基因属于阿米洛利敏感的Na+通道和简并蛋白（NC/DEG）家族的离子通道。鉴定了编码与已经克隆的家族成员相关但明显不同的蛋白质的单个基因（18-30%氨基酸序列同一性）。系统发育分析将该蛋白质与包括哺乳动物酸敏感离子通道和Phe-Met-Arg-Phe-酰胺（FMRFamide）激活的Na+通道的配体门控通道组联系起来。cRNA注入非洲爪蟾卵母细胞或瞬时转染考克斯细胞后功能获得性突变体的表达诱导了大的组成性电流。激活的通道是阿米洛利敏感的（IC 50，1.31 μ M），并显示出低电导率（9-10 pS）和对Na+相对于K+的高选择性（各自渗透率的比率，PNa+ / Pk+大于或等于10），所有这些都是NaC/DEG通道行为的特征。北方印迹和逆转录聚合酶链反应（RT-PCR）分析显示其mRNA主要在小肠、肝脏（包括肝细胞）和脑中表达。该通道被称为脑-肝-肠阿米洛利敏感性Na+通道（BLINaC）。秀丽隐杆线虫退化蛋白中相应的功能获得性突变是线虫遗传性神经变性的原因。除了BLINaC的生理功能仍有待建立，在这种新的哺乳动物变性蛋白样通道的突变可能是遗传性神经变性和肝脏或肠道病理的病理生理学的重要性。
1. A degenerate polymerase chain reaction (PCR) homology screening procedure was applied to rat brain cDNA in order to identify novel genes belonging to the amiloride-sensitive Na+ channel and degenerin (NC/DEG) family of ion channels. A single gene was identified that encodes a protein related to but clearly different from the already cloned members of the family (18-30% amino acid sequence identity). Phylogenetic analysis linked this protein to the group of ligand-gated channels that includes the mammalian acid-sensing ion channels and the Phe-Met-Arg-Phe-amide (FMRFamide)-activated Na+ channel.2. Expression of gain-of-function mutants after cRNA injection into Xenopus laevis oocytes or transient transfection of COX cells induced large constitutive currents. The activated channel was amiloride sensitive (IC50, 1.31 mu M) and displayed a low conductance (9-10 pS) and a high selectivity for Na+ over K+ (ratio of the respective permeabilities, PNa+ / Pk+ greater than or equal to 10), all of which are characteristic of NaC/DEG channel behaviour.3. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed a predominant expression of its mRNA in the small intestine, the liver (including hepatocytes) and the brain. This channel has been called the brain-liver-intestine amiloride-sensitive Na+ channel (BLINaC).4. Corresponding gain-of-function mutations in Caenorhabditis elegans degenerins are responsible for inherited neurodegeneration in the nematode. Besides the BLINaC physiological function that remains to be established, mutations in this novel mammalian degenerin-like channel might be of pathophysiological importance in inherited nenrodegeneration and liver or intestinal pathologies.