The subfamily-specific mechanism of EAG assembly
| Autor: | I-Wen Lin, 林宜玟 |
|---|---|
| Rok vydání: | 2013 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 101 EAG potassium (K+) channels belong to the superfamily of voltage-gated K+ channels. Based on the amino acid sequence of pore-forming α subunits, the EAG K+ channel family can be divided into three subfamilies: Eag, Erg, and Elk. A prerequisite for the formation of a functional K+ channel is the assembly of four α subunits into a tetramer, and only α subunits of the same EAG subfamily can form functional tetramers. It is still not clear which domain structures are responsible for the subfamily-specific assembly of EAG channels. In this thesis, we set out to explore the mechanism of the subfamily-specific assembly of rEag1 and hErg, which belong to different EAG subfamilies and therefore cannot assemble into a tetramer. We used recombinant DNA technology to construct a series of different chimeras between rEag1 and hErg, and employed co-immunoprecipitation (co-IP) to examine whether these chimeras could interact with one another. We found that chimeras involving either post-CNBHD or N-terminus were able to interact with α subunits of opposite subfamilies. We speculate that the post-CNBHD and N-terminal regions of rEag1 and hErg potassium channels probably contain specific recognition domains for EAG α subunit assembly in ER: only α subunits with the same recognition domain(s) are able to proceed with the tetramer assembly process. Both wild-type rEag1 and hErg show double protein bands on immunoblots, suggesting that they are glycosylated proteins. When we examined the pattern of rEag1- or hErg-chimeras on immunoblot, they showed different degrees of glycosylation. The glycosylation of rEag1 became severely disrupted if the C-linker, as well as the post C-linker region, was replaced. Similarly, hErg chimeras containing CNBHD, as well as post-CNBHD region, from rEag1 displayed defective glycosylation. These data suggest that C-terminal regions may play an important role in the protein maturation process of EAG K+ channels. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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