Homologous Domains Mediate Distinct Gating Functions in EAG vs. Cyclic-Nucleotide Gated Channels

Abstract

EAG, ERG and ELK are members of the Ether-a-go-go voltage-gated potassium channel family. Each channel possesses a C-terminal domain that is homologous to the cyclic nucleotide-binding domain (CNBD) of CNG/HCN channels but insensitive to cyclic nucleotides. X-ray crystallography of EAG suggests the side chains of two residues, Y699 and L701, interact in a manner analogous to the two moieties of a cyclic nucleotide in the binding pocket of bona fide CNBDs. Using two-electrode voltage clamp of channel proteins expressed in Xenopus oocytes, we found that alanine substitutions of Y699 and L701 (“AA”) dramatically slowed activation and shifted the g-V curve by +15 mV. Mutagenesis of both residues were required to elicit an effect, as if the “apo” configuration required the loss of both side chains. Thus, the intrinsic ligand of the CNB homology domain (CNBhD) reduces the stability of the closed state relative to the open state. To determine whether the AA mutant phenotype represents a loss of function, we deleted the CNBhD. In contrast to the AA mutant, the deletion gave rise to faster activation kinetics but a more dramatic g-V shift of +40 mV. This finding indicates that, unlike HCN channel, in which the unliganded CNBD mimics the CNBD deletion, the AA mutant phenotype is not a loss of function of the CNBhD. Instead, both the intrinsically-liganded and apo conformations communicate with the gating machinery. The current properties can be recapitulated by a 7-state allosteric model, in which the AA mutant alters multiple transitions during channel gating whereas CNBhD deletion affects only the last opening step. We conclude that the EAG1 CNBhD serves a function mechanistically distinct from those of the corresponding domains in channels gated by cyclic nucleotides despite extensive sequence and structural homology.