Cloning and functional expression of the mouse epithelial sodium channel
Cloning and functional expression of the mouse epithelial sodium channel
复制标题
DOI：
10.1152/ajprenal.1999.277.1.f121
复制
发表时间：
1999-07-01
影响因子：
4.2
通讯作者：
Kleyman, TR
中科院分区：
文献类型：
--
作者：
Ahn, YJ;Brooker, DR;Kleyman, TR
上皮钠通道（ENaC）在肾皮质集管、远端结肠和肺的钠的经上皮重吸收中起主要作用。ENaC由三个结构相关的亚基组成，称为α -、β -和γ ENaC。我们之前分离并测序了编码部分小鼠α -、β -和γ ENaC的cdna （α -、β -和γ mENaC）。这些cDNA用于筛选oligo- dt引物小鼠肾脏cDNA文库。分离出全长β mENaC和部分长度α和γ mENaC克隆。随后通过cDNA末端的5′快速扩增（5′- race） PCR获得全长α和γ mENaC cDNA。将小鼠α -、β -和γ - ENaC crna注射到爪蟾卵母细胞中，可表达酰胺敏感（K-i = 103 nM）、Na+选择性电流，单通道电导为4.7 pS。Northern印迹显示，α -、β -和γ - mENaC在肺和肾中表达。有趣的是，在肝脏中检测到α mENaC，尽管转录本大小为9.8 kb和3.1 kb，与在其他组织中观察到的3.2 kb信息大小不同。采用5′-RACE PCR从小鼠肝脏中分离出部分cDNA克隆。发现其序列与amENaC几乎相同。为了开始确定α mENaC中可能对天然异聚通道组装很重要的区域，使用α mENaC编码预测的细胞质NH2末端的结构进行了一系列功能实验。野生型α -、β -和γ mENaC与α mENaC的细胞内NH2端共注射可消除非洲爪蟾卵母细胞中酰胺敏感电流，这表明α mENaC的NH2端参与亚基组装，当其过量存在10倍时，在ENaC的功能性表达中起主要的负作用。
The epithelial sodium channel (ENaC) plays a major role in the transepithelial reabsorption of sodium in the renal cortical collecting duct, distal colon, and lung. ENaCs are formed by three structurally related subunits, termed alpha-, beta-, and gamma ENaC. We previously isolated and sequenced cDNAs encoding a portion of mouse alpha-, beta-, and gamma ENaC (alpha-, beta-, and gamma mENaC). These cDNAs were used to screen an oligo-dT-primed mouse kidney cDNA library. Full-length beta mENaC and partial-length alpha- and gamma mENaC clones were isolated. Full-length alpha- and gamma mENaC cDNAs were subsequently obtained by 5'-rapid amplification of cDNA ends (5'-RACE) PCR. Injection of mouse alpha-, beta-, and gamma ENaC cRNAs into Xenopus oocytes led to expression of amiloride-sensitive (K-i = 103 nM), Na+-selective currents with a single-channel conductance of 4.7 pS. Northern blots revealed that alpha-, beta-, and gamma mENaC were expressed in lung and kidney. Interestingly, alpha mENaC was detected in liver, although transcript sizes of 9.8 kb and 3.1 kb differed in size from the 3.2-kb message observed in other tissues. A partial cDNA clone was isolated from mouse liver by 5'-RACE PCR. Its sequence was found to be nearly identical to amENaC. To begin to identify regions within alpha mENaC that might be important in assembly of the native heteroligomeric channel, a series of functional experiments were performed using a construct of alpha mENaC encoding the predicted cytoplasmic NH2 terminus. Coinjection of wild-type alpha-, beta-, and gamma mENaC with the intracellular NH2 terminus of alpha mENaC abolished amiloride-sensitive currents in Xenopus oocytes, suggesting that the NH2 terminus of alpha mENaC is involved in subunit assembly, and when present in a 10-fold excess, plays a dominant negative role in functional ENaC expression.