Discovery of a heme-binding domain in a neuronal voltage-gated potassium channel

Mark J Burton,Joel Cresser-Brown,Morgan Thomas,Nicola Portolano,Jaswir Basran,Samuel L Freeman,Hanna Kwon,Andrew R Bottrill,Manuel J Llansola-Portoles,Andrew A Pascal,Rebekah Jukes-Jones,Tatyana Chernova,Ralf Schmid,Noel W Davies,Nina M Storey,Pierre Dorlet,Peter C.E Moody,John S Mitcheson,Emma L Raven

doi:10.1074/jbc.ra120.014150

Abstract

The EAG (ether-à-go-go) family of voltage-gated K+ channels are important regulators of neuronal and cardiac action potential firing (excitability) and have major roles in human diseases such as epilepsy, schizophrenia, cancer, and sudden cardiac death. A defining feature of EAG (Kv10-12) channels is a highly conserved domain on the N terminus, known as the eag domain, consisting of a Per-ARNT-Sim (PAS) domain capped by a short sequence containing an amphipathic helix (Cap domain). The PAS and Cap domains are both vital for the normal function of EAG channels. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity. In addition, we expressed the Cap and PAS domain of hERG3 in Escherichia coli and, using spectroscopy and kinetics, identified the PAS domain as the location for heme binding. The results identify heme as a regulator of hERG3 channel activity. These observations are discussed in the context of the emerging role for heme as a regulator of ion channel activity in cells.

Highlights

Per–ARNT–Sim (PAS) domains, first identified by sequence homology in the Drosophila proteins period and single-minded, and the vertebrate aryl hydrocarbon receptor nuclear transporter (ARNT), are well-known sensory modules present in a variety of signaling proteins in organisms ranging from bacteria to humans [1,2,3]
We identified a number of proteins known to bind heme, as well as proteins with unknown heme affinity; this included hERG3 (Fig. S1)
We have identified heme-dependent regulation of hERG3 channel activity

Summary

Introduction

Per–ARNT–Sim (PAS) domains, first identified by sequence homology in the Drosophila proteins period and single-minded, and the vertebrate aryl hydrocarbon receptor nuclear transporter (ARNT), are well-known sensory modules present in a variety of signaling proteins in organisms ranging from bacteria to humans [1,2,3]. Using heme-affinity pulldown assays and proteomics of lysates from primary cortical neurons, we identified that an EAG channel, hERG3 (Kv11.3), binds to heme. In whole-cell electrophysiology experiments, we identified that heme inhibits hERG3 channel activity.

Results

Conclusion