The Subfamily-Specific Assembly of Eag and Erg K+ Channels is Determined by Both the Amino and the Carboxyl Recognition Domains

Abstract

The ether-a-go-go family of Kv channels encompasses three subfamilies: Eag, Erg, and Elk. Members of different ether-a-go-go subfamilies fail to form hetero-tetramers. 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 generated various chimeric constructs between rat Eag1 (rEag1) and human Erg1 (hErg1) 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 contain a subfamily recognition domain. Importantly, exchanging either the N-terminal or the C-terminal domain alone led to a virtual loss of the inter-subfamily assembly boundary. By contrast, simultaneously swapping both recognition domains resulted in a reversal of subfamily specificity. Interestingly, introduction of the homologous hErg1 eag domain led to both a fast phase and a slow phase of channel inactivation in the rEag1 N-terminal chimeras. By contrast, the inactivation features were retained in the reverse hErg1 N-chimeras. Furthermore, an eag domain-lacking rEag1 deletion mutant also showed the fast phase of inactivation that was notably attenuated upon co-expression with the rEag1 eag domain fragment, but not with the hErg1 eag domain fragment. Overall, our findings 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 rEag1 and hErg1, and that the eag domain plays a critical role in regulating the inactivation gating of rEag1.