Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels
Residues within the myristoylation motif determine intracellular targeting of the neuronal Ca2+ sensor protein KChIP1 to post-ER transport vesicles and traffic of Kv4 K+ channels
复制标题
DOI：
10.1242/jcs.00803
复制
发表时间：
2003-12-01
影响因子：
4
通讯作者：
Burgoyne, RD
中科院分区：
文献类型：
--
作者：
O'Callaghan, DW;Hasdemir, B;Burgoyne, RD
KChIPs （K+通道相互作用蛋白）通过改变通道特性和增加其细胞表面表达来调节a型Kv4钾通道的功能。我们探讨了影响本土化的因素。利用在HeLa细胞中表达的gfp变体融合蛋白靶向KChIP1和其他NCS蛋白。单独表达的ECFP-Kv4.2不保留在内质网中，而是到达高尔基复合体。在共表达ECFP-Kv4.2和KChIP1-EYFP的细胞中，这两种蛋白共定位，主要存在于质膜上。当KChIP1-EYFP单独表达时，它转而靶向点状结构。这与NCS蛋白NCS-1和hippocalcin的定位不同，NCS蛋白NCS-1和hippocalcin的定位是针对反式高尔基网络（TGN）和质膜的。每个NCS蛋白的膜定位都需要肉豆蔻化，而hippocalcin或KChIP1的最小肉豆蔻化基元足以将融合蛋白靶向TGN/质膜或点状结构。通过突变hippocalcin和NCS-1中3个保守碱性氨基酸对应的3、7、9位残基，证实了n端基序中存在靶向信息。这些位置上的残基决定了细胞内针对不同细胞器的靶向性。ECFP-Kv4.2有效转运至质膜需要肉豆蔻酰化和KChIP1的正确靶向。KChIP1(1-11)-EYFP的表达导致形成ERGIC-53和β - cop阳性的扩大结构。ECFP-Kv4.2也在这些结构中积累，表明KChIP1(1-11)-EYFP抑制了ERGIC外的交通。我们认为KChIP1通过其肉豆蔻酰化基序靶向er后转运囊泡，在局部Ca2+信号的影响下，它可以与Kv4通道相互作用并调节其到质膜的交通。
KChIPs (K+ channel interacting proteins) regulate the function of A-type Kv4 potassium channels by modifying channel properties and by increasing their cell surface expression. We have explored factors affecting the localisation. of Kv4.2 and the targeting of KChIP1 and other NCS proteins by using GFP-variant fusion proteins expressed in HeLa cells. ECFP-Kv4.2 expressed alone was not retained in the ER but reached the Golgi complex. In cells co-expressing ECFP-Kv4.2 and KChIP1-EYFP, the two proteins were co-localised and were mainly present on the plasma membrane. When KChIP1-EYFP was expressed alone it was instead targeted to punctate structures. This was distinct from the localisation of the NCS proteins NCS-1 and hippocalcin, which were targeted to the trans-Golgi network (TGN) and plasma membrane. The membrane localisation of each NCS protein required myristoylation and minimal myristoylation motifs of hippocalcin or KChIP1 were sufficient to target fusion proteins to either TGN/plasma membrane or to punctate structures. The existence of targeting information within the N-terminal motifs was confirmed by mutagenesis of residues corresponding to three conserved basic amino acids in hippocalcin and NCS-1 at positions 3, 7 and, 9. Residues at these positions determined intracellular targeting to the different organelles. Myristoylation and correct targeting of KChIP1 was required for the efficient traffic of ECFP-Kv4.2 to the plasma membrane. Expression of KChIP1(1-11)-EYFP resulted in the formation of enlarged structures that were positive for ERGIC-53 and beta-COP. ECFP-Kv4.2 was also accumulated in these structures suggesting that KChIP1(1-11)-EYFP inhibited traffic out of the ERGIC. We suggest that KChIP1 is targeted by its myristoylation motif to post-ER transport vesicles where it could interact with and regulate the traffic of Kv4 channels to the plasma membrane under the influence of localised Ca2+ signals.