Splice variants of a ClC-2 chloride channel with differing functional characteristics
Splice variants of a ClC-2 chloride channel with differing functional characteristics
复制标题
DOI：
10.1152/ajpcell.2000.279.4.c1198
复制
发表时间：
2000-10-01
影响因子：
5.5
通讯作者：
Sepúlveda, FV
中科院分区：
文献类型：
--
作者：
Cid, LP;Niemeyer, MI;Sepúlveda, FV
我们在豚鼠肠上皮cDNA文库中鉴定了两个ClC-2克隆，其中一个在NH 2末端有30 bp的缺失。使用包含缺失的引物的PCR得到两种产物，进一步用特异性引物扩增，证实其真实性。相应的基因组DNA序列给出了三个外显子和两个内含子的结构。发生在外显子之一内的内部供体位点解释了缺失，与选择性剪接一致。变体gpClC-2和gpClC-2 Delta 77-86在HEK-293细胞中的表达产生具有相似激活特征的内向整流氯电流。然而，失活在gpClC-2 Delta 77-86中以更快的动力学发生。定点诱变表明，蛋白激酶C介导的磷酸化共识网站gpClC-2德尔塔77-86中丢失的是不负责观察到的变化。携带缺失的变异体存在于大多数检查的组织中，并且在近端结肠、肾脏和睾丸中更丰富。在其NH 2-末端结构域中修饰的C1 C-2的剪接变体的存在可能在其相对表达水平不同的组织中具有功能性后果。
We identified two ClC-2 clones in a guinea pig intestinal epithelial cDNA library, one of which carries a 30-bp deletion in the NH2 terminus. PCR using primers encompassing the deletion gave two products that furthermore were amplified with specific primers confirming their authenticity. The corresponding genomic DNA sequence gave a structure of three exons and two introns. An internal donor site occurring within one of the exons accounts for the deletion, consistent with alternative splicing. Expression of the variants gpClC-2 and gpClC-2 Delta 77-86 in HEK-293 cells generated inwardly rectifying chloride currents with similar activation characteristics. Deactivation, however, occurred with faster kinetics in gpClC-2 Delta 77-86. Site-directed mutagenesis suggests that a protein kinase C-mediated phosphorylation consensus site lost in gpClC-2 Delta 77-86 is not responsible for the observed change. The deletion-carrying variant is found in most tissues examined, and it appears more abundant in proximal colon, kidney, and testis. The presence of a splice variant of ClC-2 modified in its NH2-terminal domain could have functional consequences in tissues where their relative expression levels are different.