Roles of cloned chloride channels in kidney chloride transport

为了确定我们分离的三种氯通道在肾内的定位，我们用合成肽作为抗原制备了大鼠ClC-K_1、-K_2、-3抗血清。我们获得了ClC-K1和ClC-K2的特异性抗血清。用这些抗血清进行免疫组织化学研究，以确定ClC-K1和ClC-K2在肾内的定位。ClC-K1存在于亨利氏袢的细上升支的顶端和基底侧质膜中，ClC-K2存在于连接小管的顶端质膜中。这些发现将有助于阐明ClC-K1和ClC-K2在肾脏中的生理作用。接下来，我们研究了ClC-K1和ClC-3的结构与功能关系。在体外突变的带正电荷的氨基酸在第一个细胞外环的ClC-K1到带负电荷的氨基酸大大增强了在非洲爪蟾卵母细胞中表达的氯电流，这表明该地区的重要性。使用ClC-K1和-K2的嵌合体氯离子通道的进一步研究正在进行中。对于ClC-3，在CHO细胞中建立了稳定表达的细胞系，并且膜片钳分析揭示了ClC-3的Ca调节性质。最后，我们分离了人ClC-K1和ClC-K2 cDNA，以研究这些基因在某些人类肾脏疾病中的参与。利用这些cDNA，我们进一步分离了人ClC-K1和-K2基因，并表征了基因结构，包括外显子-内含子结构。我们制备了覆盖所有外显子和外显子-内含子边界的PCR引物组，并建立了通过直接测序确定患者ClC-K2基因序列的系统。用FISH方法对ClC-K1、ClC-K2和ClC-K3进行了染色体定位。

To determine the intrarenal localization of three chloride channels isolated by us, we prepared antisera for rat ClC-K1, -K2, -3 using synthetic peptide as antigens. We obtained specific antisera to ClC-K1 and ClC-K2. Using these antisera, we performed immunohistochemical study to localize the intrarenal localizaton of ClC-K1 and ClC-K2. ClC-K1 is present in the both apical and basolateral plasma membranes of the thin ascending limb of Henle's loop, and ClC-K2 is present in the apical plasma membrane of the connecting tubules. These findings will shed light on the physiological roles if ClC-K1 and -K2 in the kidney.Next, we studied structure- function relationship of ClC-K1 and ClC-3. The in vitro mutations of positively charged amino acids in the first extracellular loop of ClC-K1 into negatively charged amino acids greatly enhanced the chloride current expressed in Xenopus oocytes, suggesting the importance of this region. Further studies using chimera chloride channels of ClC-K1 and -K2 are in progress. As for ClC-3, a stably expressing cell line was established in CHO cells, and patch clamp analysis revealed the Ca-regulated nature of ClC-3. Finally, we isolated human ClC-K1 and -K2 cDNAs to study the involvements of these genes in certain human kidney diseases. Using these cDNA,we further isolated human ClC-K1 and -K2 genes and characterized the gene structure including exon-intron structures. We prepared PCR primer sets to cover all exons and exon-intron boundaries and could established the system to determine the sequences of ClC-K2 gene from patients by direct sequencing. Also, we determined the chromosomal localization of ClC-K1, -K2 and -3 by FISH method.