--- - |2+ Key points In the human ether‐a‐go‐go related gene (hERG) channel, both the ether‐a‐go‐go (EAG) domain in the N‐terminal and the cyclic nucleotide (CN) binding homology (CNBH) domain in the C‐terminal cytoplasmic region are known to contribute to the characteristic slow deactivation. Mutations of Phe860 in the CNBH domain, reported to fill the CN binding pocket, accelerate the deactivation and decrease the fluorescence resonance energy transfer (FRET) efficiencies between the EAG and CNBH domains. An electrostatic interaction between Arg696 and Asp727 in the C‐linker domain, critical for HCN and CNG channels, is not formed in the hERG channel. Mutations of newly identified electrostatically interacting pair, Asp727 in the C‐linker and Arg752 in the CNBH domains, accelerate the deactivation and decrease FRET efficiency. Voltage‐dependent changes in FRET efficiency were not detected. These results suggest that the acceleration of the deactivation by mutations of C‐terminal domains is a result of the lack of interaction between the EAG and CNBH domains. Abstract The human ether‐a‐go‐go related gene (hERG) channel shows characteristic slow deactivation, and the contribution of both of the N‐terminal cytoplasmic ether‐a‐go‐go (EAG) domain and the C‐terminal cytoplasmic cyclic nucleotide (CN) binding homology (CNBH) domain is well known. The interaction between these domains is known to be critical for slow deactivation. We analysed the effects of mutations in the CNBH domain and its upstream C‐linker domain on slow deactivation and the interaction between the EAG and CNBH domains by electrophysiological and fluorescence resonance energy transfer (FRET) analyses using Xenopus oocyte and HEK293T cell expression systems. We first observed that mutations of Phe860 in the CNBH domain, which is reported to fill the CN binding pocket as an intrinsic ligand, accelerate deactivation and eliminate the inter‐domain interaction. Next, we observed that the salt bridge between Arg696 and Asp727 in the C‐linker domain, which is reported to be critical for the function of CN‐regulated channels, is not formed. We newly identified an electrostatically interacting pair critical for slow deactivation: Asp727 and Arg752 in the CNBH domain. Their mutations also impaired the inter‐domain interaction. Taking these results together, both mutations of the intrinsic ligand (Phe860) and a newly identified salt bridge pair (Asp727 and Arg752) in the hERG channel accelerated deactivation and also decreased the interaction between the EAG and CNBH domains. Voltage‐dependent changes in FRET efficiency between the two domains were not detected. The results suggest that the CNBH domain contributes to slow deactivation of the hERG channel by a mechanism involving the EAG domain. - The Journal of Physiology, Volume 596, Issue 19, Page 4629-4650, 1 October 2018.