Functional Properties of the “Unliganded” CNBhD in EAG K Channels

Abstract

Members of the ether-a -go-go family are voltage-gated K channels possessing a C-terminal domain with homology to cyclic nucleotide-gated channels (CNG/HCN) (1). Despite this similarity, these channels are not directly modulated by cyclic nucleotides (2,3). Recent X-ray crystal structures their cyclic nucleotide binding homology domains (CNBhD's) have a structure corresponding to the binding pocket of HCN channels occupied with tyrosine (Y) and leucine (L) side chains on a re-entrant beta strand (4,5). Thus, the structure appears self-liganded. Functional analysis using double-alanine mutagenesis indicates these residues are involved in gating in both zebrafish ELK and human EAG1 (4,5). To further investigate whether the EAG1 CNBhD is in the “liganded” or “unliganded” state, we evaluated kinetics of activation using two-electrode voltage clamp and excised macropatch recordings from oocytes. Ten-to-90% rise time of activation increased more than 3-fold in Y699A/L701A (“unliganded”) mutants. Single alanine mutations failed to slow activation, suggesting the side chain at either position is sufficient to mediate the liganded conformation. Single or double aspartate substitutions phenocopied the slow activation of double-alanine mutant. Modifying a binding-pocket cysteine with MTSET, or deleting the ligand-containing loop, similarly slowed activation. Despite removing the intrinsic ligand, cyclic nucleotides failed to modulate the mutant channel in excised macropatches or bind to the mutant protein domain in fluorescent studies in vitro. Like activation, deactivation is also slowed. These findings support the conclusion that the double-alanine mutation creates an unliganded binding pocket, and suggest the role of the CNBhD is to reduce the energy barrier between closed and open states of the channel.1. Warmke et al. (1994) PNAS 91:34382. Robertson et al. (1996) Neuropharmacology 35:8413. Brelidze et al. (2009) JBC 284:279894. Brelidze et al. (2012) Nature 481:5305. Marques-Carvalho et al. (2012) JMB 423:34