The Subfamily-specific Assembly of Eag and Erg K+ Channels Is Determined by Both the Amino and the Carboxyl Recognition Domains |
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Authors: | Ting-Feng Lin I-Wen Lin Shu-Ching Chen Hao-Han Wu Chi-Sheng Yang Hsin-Yu Fang Mei-Miao Chiu Chung-Jiuan Jeng |
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Institution: | From the ‡Institute of Anatomy and Cell Biology, School of Medicine, and ;the ¶Brain Research Center, National Yang-Ming University, No. 155, Section 2, Li-Non Street, Taipei 12212, Taiwan and ;the §Department of Medical Research, National Taiwan University Hospital, Taipei 10051, Taiwan |
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Abstract: | A functional voltage-gated K+ (Kv) channel comprises four pore-forming α-subunits, and only members of the same Kv channel subfamily may co-assemble to form heterotetramers. The ether-à-go-go family of Kv channels (KCNH) encompasses three distinct subfamilies: Eag (Kv10), Erg (Kv11), and Elk (Kv12). Members of different ether-à-go-go subfamilies, such as Eag and Erg, fail to form heterotetramers. Although a short stretch of amino acid sequences in the distal C-terminal section has been implicated in subfamily-specific subunit assembly, it remains unclear whether this region serves as the sole and/or principal subfamily recognition domain for Eag and Erg. Here we aim to ascertain the structural basis underlying the subfamily specificity of ether-à-go-go channels by generating various chimeric constructs between rat Eag1 and human Erg subunits. Biochemical and electrophysiological characterizations of the subunit interaction properties of a series of different chimeric and truncation constructs over the C terminus suggested that the putative C-terminal recognition domain is dispensable for subfamily-specific assembly. Further chimeric analyses over the N terminus revealed that the N-terminal region may also harbor a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the intersubfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Our observations are consistent with the notion that both the N-terminal and the C-terminal recognition domains are required to sustain the subfamily-specific assembly of rat Eag1 and human Erg. |
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Keywords: | Electrophysiology Potassium Channel Protein Assembly Protein Domain Site-directed Mutagenesis Western Blot |
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