An Intrinsic Ligand Mediates N- and C-Terminal Interactions with the KCNH Gating Machinery

Abstract

EAG, ERG and ELK are members of the KCNH potassium channel family important in excitability. Two conserved domains in the cytosolic N and C termini uniquely characterize members of this family. The C-terminal domain has homology to the cyclic nucleotide-binding domain of CNG/HCN channels but is unaffected by cyclic nucleotides. At the tail end of this cyclic nucleotide-binding homology domain (CNBhD) is an intrinsic ligand comprising two highly conserved residues that loop back to occupy the cyclic nucleotide-binding pocket. The CNBhD interacts with the N-terminal domain, with one interface also occupied by the intrinsic ligand. We probed the intrinsic ligand and its effect on C- and N-terminal modulation of gating in hEAG1 channels. Using two-electrode voltage clamp of channels expressed in Xenopus oocytes, we found that substitution of the intrinsic ligand residues, Y699 and L701, with AA, SS and GG progressively shifted the midpoint of the conductance-voltage relationship to more depolarized potentials. The substitution with G, a residue unable to support a ligand-receptor interaction and thus considered fully unliganded, resulted in the greatest steady-state shift of channels into the closed state. Interestingly, deleting the CNBhD created the same phenotype as the GG mutant, suggesting the unliganded conformation confers a loss of function of the CNBhD's effects on gating. Both AA and GG mutants abolished in vitro binding of the PAS domain to the CNBhD in anisotropy measurements. Functionally, when combining GG/CNBhD deletion with mutations at PAS-CNBhD interfaces, we observed a non-additive phenotype that is indistinguishable from the GG or CNBhD deletion mutant. These findings suggest that both the intrinsic ligand and CNBhD are required for the proper function of PAS domain, and the ligand and binding pocket can adopt multiple conformations, each conferring a different gating behavior.