Desir G V, Hamlin H A, Puente E, Reilly R F, Hildebrandt F, Igarashi P
Department of Medicine, Yale University School of Medicine, New Haven 06510.
Am J Physiol. 1992 Jan;262(1 Pt 2):F151-7. doi: 10.1152/ajprenal.1992.262.1.F151.
Epithelial voltage-gated potassium (K) channels have been well studied using electrophysiological methods, but little is known about their structures. We tested the hypothesis that some of these channels belong to the Shaker gene family, which encodes voltage-gated K channels in excitable tissues. From published sequences of Shaker proteins in Drosophila, rat, and mouse brain, we chose regions that were conserved between species. Based on these protein sequences, degenerate oligonucleotides flanking the putative voltage sensor (S4) were synthesized and used as primers for the polymerase chain reaction. Five Shaker-like cDNAs were amplified from rabbit kidney cortex and three from LLC-PK1, an epithelial cell line derived from pig kidney. Each partial-length rabbit kidney cDNA is approximately 850 base pairs (bp) long. The deduced amino acid sequences contain five putative transmembrane segments and are 79-97% identical to two Shaker isoforms expressed in rat brain (RBK1 and RBK2). Sequence similarity is greatest in the putative transmembrane segments S1-S5. Importantly, the S4 segment, the putative voltage gate is highly conserved in all 5 cDNAs. Southern analysis of rabbit genomic DNA suggests that each isoform is encoded by a different gene. The partial length LLC-PK1 cDNAs are 450-bp long, and the deduced amino acid sequences are 77-99% identical to the rabbit cDNAs. This is, to our knowledge, the first demonstration that Shaker-like genes are expressed in renal epithelial cells. These genes most likely encode voltage-gated K channels involved in renal epithelial K transport.
上皮电压门控钾（K）通道已通过电生理方法得到了充分研究，但对其结构却知之甚少。我们检验了这样一个假说，即这些通道中的一些属于Shaker基因家族，该家族在可兴奋组织中编码电压门控钾通道。根据果蝇、大鼠和小鼠脑中已发表的Shaker蛋白序列，我们选择了物种间保守的区域。基于这些蛋白质序列，合成了推测的电压感受器（S4）两侧的简并寡核苷酸，并用作聚合酶链反应的引物。从兔肾皮质中扩增出5个类似Shaker的cDNA，从LLC-PK1（一种源自猪肾的上皮细胞系）中扩增出3个。每个兔肾全长cDNA约850个碱基对（bp）长。推导的氨基酸序列包含5个推测的跨膜片段，与大鼠脑中表达的两种Shaker异构体（RBK1和RBK2）的同源性为79-97%。在推测的跨膜片段S1-S5中序列相似性最大。重要的是，推测的电压门控片段S4在所有5个cDNA中高度保守。兔基因组DNA的Southern分析表明，每个异构体由不同的基因编码。LLC-PK1的部分长度cDNA为450 bp长，推导的氨基酸序列与兔cDNA的同源性为77-99%。据我们所知，这是首次证明类似Shaker的基因在肾上皮细胞中表达。这些基因很可能编码参与肾上皮钾转运的电压门控钾通道。
