The role of the PAS-cap in the function of hEAG1 and the role of hEAG1 in cancer cell progression

Human ether-a-go-go 1 (hEAG1) is a voltage gated K+ channel that is sensitive to inhibition by Ca2+-calmodulin. hEAG1 is located presynaptically in brain neurons and regulates Ca2+ influx and neurotransmitter release. It is also ectopically expressed in >70% of human tumours. hEAG1 has been shown to increase cell proliferation. The research here aims to understand the mechanism for Ca2+-calmodulin regulation of gating and whether specific intracellular domains are also important for effects on proliferation. The role of hEAG1 on cell migration is studied here for the first time. hEAG1 contains an N-terminal EAG domain consisting of a Per-Arnt-Sim (PAS) domain and a 26 amino acid PAS-cap region. On the C-terminus is a cyclic nucleotide binding homology domain (cNBHD). hEAG1 channels were mutated and channels characterised by expression in Xenopus laevis oocyte expression system and two electrode voltage clamp. At a membrane potential of +60mV, WT hEAG1 shows activation kinetics of 309.1 ± 16.4ms and does not inactivate in control conditions. WT hEAG1 is inhibited 85.5 ± 2.9% when Ca2+ is raised. Deleting the PAS-cap (Δ2-26) slows the activation kinetics to 801 ± 122ms and allows the non-inactivating channel to inactivate. When Ca2+ is raised, the Δ2-26 hEAG1 current increases by 1272.7 ± 197.1%. In this study we address, at the amino acid level, the interactions behind the PAS-cap’s role in regulating hEAG1. We also address whether the Ca2+ sensitivity and intracellular domains of hEAG1 influence its effect on proliferation by using a BrdU incorporation assay. The EAG domain and the E600 residue are important for this role as mutation of either resulted in a reduction in the number of proliferating cells. Overall the results suggest that PAS-cap, EAG and cNBH domain interactions regulate current responses to Ca2+-calmodulin and that the EAG domain and E600 are important for increased proliferation

Calmodulin regulates human ether à go-go 1 (hEAG1) potassium channels through interactions of the eag-domain with the cyclic nucleotide binding homology domain

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. E600 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