Eldstrom Jodene, Doerksen Kyle W, Steele David F, Fedida David
Department of Physiology, University of British Columbia, 2146 Health Sciences Mall, V6T 1Z3, Vancouver, BC, Canada.
FEBS Lett. 2002 Nov 20;531(3):529-37. doi: 10.1016/s0014-5793(02)03572-x.
We have investigated the interactions of prototypical PDZ domains with both the C- and N-termini of Kv1.5 and other Kv channels. A combination of in vitro binding and yeast two-hybrid assays unexpectedly showed that PDZ domains derived from PSD95 bind both the C- and N-termini of the channels with comparable avidity. From doubly transfected HEK293 cells, Kv1.5 was found to co-immunoprecipitate with the PDZ protein, irrespective of the presence of the canonical C-terminal PDZ-binding motif in Kv1.5. Imaging analysis of the same HEK cell lines demonstrated that co-localization of Kv1.5 with PSD95 at the cell surface is similarly independent of the canonical PDZ-binding motif. Deletion analysis localized the N-terminal PDZ-binding site in Kv1.5 to the T1 region of the channel. Co-expression of PSD95 with Kv1.5 N- and C-terminal deletions in HEK cells had contrasting effects on the magnitudes of the potassium currents across the membranes of these cells. These findings may have important implications for the regulation of channel expression and function by PDZ proteins like PSD95.
我们研究了典型的PDZ结构域与Kv1.5及其他Kv通道的C端和N端之间的相互作用。体外结合和酵母双杂交试验的组合意外地表明，源自PSD95的PDZ结构域以相当的亲和力结合通道的C端和N端。在双重转染的HEK293细胞中，发现Kv1.5与PDZ蛋白共免疫沉淀，而不管Kv1.5中是否存在典型的C端PDZ结合基序。对相同HEK细胞系的成像分析表明，Kv1.5与PSD95在细胞表面的共定位同样独立于典型的PDZ结合基序。缺失分析将Kv1.5中的N端PDZ结合位点定位到通道的T1区域。在HEK细胞中，PSD95与Kv1.5 N端和C端缺失的共表达对这些细胞膜上钾电流的大小有相反的影响。这些发现可能对像PSD95这样的PDZ蛋白调节通道表达和功能具有重要意义。
