2018FINCHAPhD.pdf (7.91 MB)

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

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posted on 14.02.2019, 16:02 by Alina Finch
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.
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Mitcheson, John; Muskett, Fred

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Department of Molecular and Cell Biology

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University of Leicester

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