Kv channel interacting proteins (KChIPs) are Ca2+-binding proteins with four EF-hands and well-known to modulate Kv4.2 channel gating. The present study is carried out to investigate the molecular mechanism related to regulate the interaction of KChIP2.2 and KChIP4a with Kv channel. In comparison with KChIP4a, the interaction of KChIP2.2 with Kv4.2 was more obvious in the absence of Ca2+ or Mg2+. However the binding of KChIP2.2 and KChIP4a toward Kv4.2 increased with increasing Ca2+ and Mg2+ concentration. Nevertheless, no individual regions within KChIP2.2 and KChIP4a could exclusively fulfill the interaction between KChIPs mutants and Kv channel. Fluorescence measurement showed that KChIP2.2 possessed both high affinity and low affinity Ca2+-binding sites, but only low affinity Ca2+-binding site was observed with
KChIP4a. However, both of them have only one Mg2+-binding site. Studies on the truncated mutants revealed that the EF-hand 4 of KChIP2.2 was related to high affinity binding with Ca2+, and the integrity of molecular structure of KChIP2.2 and KChIP4a was important for Ca2+ -and Mg2+-binding. The thermal stability of KChIP2.2 and KChIP4 was found to be differentiately affected by Ca2+ and Mg2+. Proteolytic digestion and thiol reactivity assays also supported that Ca2+ and Mg2+-induced conformational change of KChIP2.2 was differed from KChIP4a. Moreover, in cells
co-transfected with Kv4.2 cDNA, it was formed that KChIP2.2 trafficking to the cell surface was increased by elevating intracellular Ca2+ concentration, but no noticeable change was observed for KChIP4a. Taken together, these results suggest that the conformational changes of KChIP2.2 and KChIP4a differently induced by Ca2+ and Mg2+ affect their binding with Kv channel and/or cellular distribution.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0629105-114704 |
| Date | 29 June 2005 |
| Creators | Chen, Ching-Ping |
| Contributors | Long-sen Chang, Chung-chang Chang, Wen-chun Hung |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | Cholon |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0629105-114704 |
| Rights | withheld, Copyright information available at source archive |
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