Deletion of the Amino-Terminus Uncovers an Inactivated State in Eag1 Channels

Abstract

Ether-a-go-go (Eag) family channels, which include hErg1, are voltage-gated K+ channels that are important in cardiac and neural function and have a well-documented role in disease. Eag1 channels in particular are found throughout the central nervous system and are crucial regulators of cell cycle and tumor progression. Wild-type Eag1 channels exhibit voltage-gated activation and deactivation, but no apparent inactivation. Here we find that deletion of the entire intracellular amino-terminal domain uncovers an inactivated channel state at depolarizing potentials. We characterized this new inactivated state in inside-out patches excised from Xenopus oocytes expressing the mutant Eag1 channels. Intriguingly, we find that application of the membrane-impermeable cysteine modifying reagent MTSES to the intracellular side of the amino-terminal deletion mutant abolishes this inactivation. We have localized this cysteine-modifying effect to one or more of three cysteine residues in the intracellular carboxy-terminal domain. One of these residues resides in a domain that shares sequence similarity with the cyclic nucleotide binding domain (CNBD) of other channels and enzymes, and the other two reside in the C-linker which connects the CNBD-like region to the S6 transmembrane domain. As the CNBD-like region in does not bind to, and these channels are not regulated by cyclic nucleotides, the role of domain in channel gating is unknown. Our results suggest that the entire amino-terminus, or a fraction thereof, may act as a ligand that binds to the CNBD-like region to prevent inactivation in the wild-type channel.