Engineering the hERG1 Selectivity Filter into the NaK Pore Domain

Abstract

Comparison of the hERG1 selectivity filter sequence and pore helix with a variety of prokaryotic ion channels revealed an unusually high sequence identity (63%) with the non-selective cation channel NaK (∼19 amino acids). Taking advantage of this remarkable similarity, we have used NaK as a template to carry out a structural analysis of a hERG1-like NaK selectivity filter in K+. To engineer the NaK filter into that of hERG1, we substituted two critical residues, V59S in the pore helix and D66F at the selectivity filter (equivalent to positions Ser620 and Phe627 in hERG1, respectively). The final construct shares 73% sequence identity with the hERG1 selectivity filter, equivalent to the closest orthologue of hERG1, the bovine ether-a-go-go channel (bEAG1). Crystals of this engineered hERG-NaK diffracted to 2.8 Å resolution and were solved by molecular replacement using closed NaK as template. The crystal structure of hERG1-like NaK in 100 mM K+ revealed a striking similarity to the all canonical K+ channel filters in the conductive conformation (RMSD= 0.5 Å with the KcsA filter) and shows three major differences compared to WT NaK. First, hERG1-like NaK filter revealed four equivalent K+ binding sites. Second, the side chain of Phe66 residue establishes critical packing interactions with the adjacent aromatic residues from the pore helix. Third, hERG1-like NaK shows hydrogen bond interactions through a water molecule behind the selectivity filter, which is absent in WT NaK, but present in KcsA. We suggest that the hERG-NaK construct represents a unique tool to investigate the properties of the hERG1 channel selectivity filter at atomic level.