Principal Supervisor: Dr John Mitcheson - Department of Molecular and Cell Biology
Co-supervisors: Prof Emma Raven and Dr Ralf Schmid
PhD project title: The role of heme in regulating potassium channel function
University of Registration: Leicester
Heme-containing proteins are found in all species and carry out diverse and vital functions such as electron transfer (the cytochromes), and catalysis (the P450s, peroxidases). Heme commonly acts as a protein cofactor and will often confer gas sensitivity (O2, N0, CO). Recently, a novel role for heme as a potent modulator of a small subset of channels has been discovered. Indeed, earlier this year we published an article that demonstrated heme activation of cardiac ATP-sensitive K+ (KATP) channels and identified the heme-binding location .
We now want to investigate the structural basis of heme modulation of the ether-a-go-go (EAG, Kv10-12) family of K+ channels, which are structurally very different from KATP channels and regulated by voltage, phosphoinositides and in the case of EAG1 by calcium-calmodulin. The EAG channel family are regulators of neuronal and cardiac cell action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia and cardiac sudden death. These channels are also ectopically expressed in many cancers and contribute to progression of this disease . We know that human EAG1 (hEAG1) channels are inhibited by low nanomolar concentrations of intracellular heme (extracellular application has no effect). Available evidence indicates that heme forms a stable and direct interaction with hEAG1, but the location and mechanism of binding are unknown.
Our project is timely because in August a cryo-EM structure of the entire rat EAG1 channel bound to calmodulin was published in Science . This structure provides an ideal opportunity for making progress on solving the mechanism of heme inhibition of hEAG1. Our project brings together experts in ion channels (Dr Mitcheson), heme protein structures (Prof Emma Raven) and molecular modelling and bioinformatics (Dr Ralf Schmid). The overall objective is to establish the mechanism of heme regulation of the EAG family of voltage-gated potassium channels, focussing initially on hEAG1.
The specific objectives are:
- The generation of molecular models of hEAG in different conformations based on the rat EAG cryo-EM structure of the channel. Bioinformatic and modelling approaches will be applied to identify putative heme binding sites to test in functional studies (objective 2).
- To investigate the heme regulation of hEAG1 channel function using two-electrode voltage-clamp and inside-out patch-clamp techniques. hEAG1 channels will be expressed in xenopus oocytes. Intracellular domains will be systematically deleted to determine regions required for heme regulation. We have already made these constructs and shown that they are functional . Putative heme binding motifs will be tested by site directed mutagenesis and electrophysiological characterisation.
- Quantification of heme binding to hEAG1 channels by uv-visible and EPR spectroscopy. We will also assess binding of O2, CO and NO to the heme ligand and if the results looking promising do follow up functional studies (objective 2).
- The above studies will be complemented by crystallography and/or NMR spectroscopy studies to gain structural insights at atomic resolution of heme in complex with channel binding domains. This multidisciplinary approach will build a framework for understanding K+ channel regulation by heme.
- Burton, M.J., et al., A heme-binding domain controls regulation of ATP-dependent potassium channels. PNAS, 2016. 113(14): p. 3785-90.
- Pardo, L.A. and W. Stuhmer, The roles of K+ channels in cancer. Nat Rev Cancer, 2014. 14(1): p. 39-48.
- Whicher, J.R. and R. MacKinnon, Structure of the voltage-gated K+ channel Eag1 reveals an alternative voltage sensing mechanism. Science, 2016. 353(6300): p. 664-9.
- Lorinczi, E., et al., Calmodulin Regulates Human Ether a Go-Go 1 (hEAG1) Potassium Channels through Interactions of the Eag Domain with the Cyclic Nucleotide Binding Homology Domain. JBC, 2016. 291(34): p. 17907-18. 3,960 characters
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Bioinformatics and in-silico molecular modelling
- Excised inside-out macropatch recording
- Two-electrode voltage-clamp of Xenopus oocytes
- Molecular biology – including site-directed mutagenesis, DNA and RNA purification
- Protein over-expression and purification
- Protein characterization using biophysical techniques (circular dichroism, biolayer interferometry)
- Structural analysis using crystallography, high-resolution nuclear magnetic resonance (NMR) spectroscopy and small-angle X-ray scattering (SAXS)
Contact: Dr John Mitcheson, University of Leicester