| Summary: | 碩士 === 國立陽明大學 === 解剖學及細胞生物學研究所 === 100 === EAG K+ channel superfamily includes three subfamilies: Eag, Erg, and Elk. Each a-subunit contains six transmembrane segments (S1-S6), cytoplasmic N- and C-terminus. The cytoplasmic C-terminus comprises three important domains, C-linker, CNBHD (cyclic nucleotide binding homology domain), and CAD (carboxyl assembly domain). The assembly of four a-subunits into tetramers is a prerequisite for producing functional K+ channels. Four same a-subunits can form a homotetramer, while the combination of different a-subunits can form a heterotetramer. However, a-subunits belonged to different subfamilies can’t assemble to form functional K+ channels. Although there have been many studies investigating the assembly mechanism of the EAG K+ channels in the past, the major protein region responsible for the efficiency and specificity of the channel assembly remains unclear. The purpose of this study is to re-examine the channel assembly domains of the EAG K+ channel family, including two Eag K+ channel subunit isoforms, rEag1 (rat Eag1), and rEag2 (rat Eag2), and human Erg subfamily hErg (human Erg1).
Firstly we applied co-immunoprecipitation experiments to identify structural domains essential for the formation of homotetramers in rEag1 and rEag2. By study-ing various rEag1 and rEag2 truncation mutants, we demonstrated that mutants lacking either CAD, CNBHD, C-linker, or the S6 segment can still interact with their WT counterpart. By contrast, hErg truncation mutants lacking proximal C-terminus failed to interact with hErg-WT, suggesting that the proximal C-terminus of hErg may contribute to its assembly domain.
Next we focused on the protein region involved in the formation of heterotetramers. Since rEag1 and rEag2 are members of the same EAG channel sub-family, they are expected to form intra-subfamily heterotetramers. Accordingly, we found that rEag2 truncation mutants lacking the S6 segment and the entire cyto-plasmic C-terminus are still capable of interacting with rEag1 WT. In contrast, no in-ter-subfamily heterotetramers are expected to form between rEag1 and hErg, mem-bers of two different EAG subfamilies. Interestingly, a rEag1 chimera containing the post-CNBHD region of hErg can interact with both rEag1-WT and hErg-WT, thereby breaking the subfmaily-specificity of channel assembly. Overall, our co-immunoprecipitation data suggest that subsets of certain critical sequences within the S6 segment-C-terminus region of rEag1/rEag2/hErg K+ channels may determine the specificity of channel assembly.
We also investigated the interaction between hErg and its binding partner, 14-3-3 protein. 14-3-3 protein family consists of seven isoforms. Except for the sigma isoform, all six 14-3-3 isoforms can interact with hErg, indicating that there was no isoform specificity in the interaction between hErg and 14-3-3 protein. Interestingly, when we co-expressed 14-3-3 protein and hErg in HEK293T cells, compared to myc vector alone, there was 4- to 6-fold increase in the expression level of hErg proteins. These results imply that over-expression of 14-3-3 protein can enhance the hErg protein expression via certain molecular mechanisms. It is worth further investigation in the future.
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