Calmodulin Regulates Human Ether à Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain

Eva Lörinczi,Matthew Helliwell,Alina Finch,Phillip J Stansfeld,Noel W Davies,Martyn Mahaut-Smith,Frederick W Muskett,John S Mitcheson

doi:10.1074/jbc.m116.733576

Abstract

The ether à go-go family of voltage-gated potassium channels is structurally distinct. The N terminus contains an eag domain (eagD) that contains a Per-Arnt-Sim (PAS) domain that is preceded by a conserved sequence of 25–27 amino acids known as the PAS-cap. The C terminus contains a region with homology to cyclic nucleotide binding domains (cNBHD), which is directly linked to the channel pore. The human EAG1 (hEAG1) channel is remarkably sensitive to inhibition by intracellular calcium (Ca2+i) through binding of Ca2+-calmodulin to three sites adjacent to the eagD and cNBHD. Here, we show that the eagD and cNBHD interact to modulate Ca2+-calmodulin as well as voltage-dependent gating. Sustained elevation of Ca2+i resulted in an initial profound inhibition of hEAG1 currents, which was followed by a phase when current amplitudes partially recovered, but activation gating was slowed and shifted to depolarized potentials. Deletion of either the eagD or cNBHD abolished the inhibition by Ca2+i. However, deletion of just the PAS-cap resulted in a >15-fold potentiation in response to elevated Ca2+i. Mutations of residues at the interface between the eagD and cNBHD have been linked to human cancer. Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in hEAG1 currents that were profoundly potentiated by Ca2+i in a manner similar to the ΔPAS-cap mutant. These findings provide the first evidence that eagD and cNBHD interactions are regulating Ca2+-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding.

Highlights

The ether à go-go potassium channel family (KCNH)2 of voltage-gated potassium channels consists of three subgroups, ether à go-go (EAG), EAG-related gene (ERG), and EAG-like potassium (ELK) channels
Glu-600 on the cNBHD, when substituted with residues with a larger volume, resulted in human EAG1 (hEAG1) currents that were profoundly potentiated by Ca2؉i in a manner similar to the ⌬PAS-cap mutant. These findings provide the first evidence that eag domain (eagD) and cNBHD interactions are regulating Ca2؉-dependent gating and indicate that the binding of the PAS-cap with the cNBHD is required for the closure of the channels upon CaM binding
Wild-type hEAG1 Channels Undergo Two Distinct Phases of Inhibition in Response to Elevated Ca2ϩi—To characterize the response of wild-type hEAG1 currents to elevated Ca2ϩi, oocytes were voltage-clamped, and 2-s voltage steps to ϩ60 mV were applied repetitively at 10-s intervals from a holding potential of Ϫ90 mV. hEAG1 currents were characterized by relatively slow activation

Summary

Introduction

The ether à go-go potassium channel family (KCNH)2 of voltage-gated potassium channels consists of three subgroups, ether à go-go (EAG), EAG-related gene (ERG), and EAG-like potassium (ELK) channels. Elegant studies by Schonherr et al [12] and Ziechner et al [13] demonstrated that calmodulin (CaM) is the Ca2ϩi sensor and inhibits hEAG1 currents by binding to the channels in a Ca2ϩ-dependent manner.

Results

Conclusion