title: Chimeric hERG channels containing a tetramerization domain are functional and stable.
abstract: Biochemical and detailed structural information of human ether-a-go-go-related gene (hERG) potassium channels are scarce but are a prerequisite to understand the unwanted interactions of hERG with drugs and the effect of mutations that lead to long QT syndrome. Despite the huge interest in hERG, to our knowledge, procedures that provide a purified, functional, and tetrameric hERG channel are not available. Here, we describe hybrid hERG molecules, termed chimeric hERG channels, in which the N-terminal Per-Arnt-Sim (PAS) domain is deleted and the C-terminal C-linker as well as the cyclic nucleotide binding domain (CNBD) portion is replaced by an artificial tetramerization domain. These chimeric hERG channels can be overexpressed in HEK cells, solubilized in detergent, and purified as tetramers. When expressed in Xenopus laevis oocytes, the chimeric channels exhibit efficient trafficking to the cell surface, whereas a hERG construct lacking the PAS and C-linker/CNBD domains is retained in the cytoplasm. The chimeric hERG channels retain essential hERG functions such as voltage-dependent gating and inhibition by astemizole and the scorpion toxin BeKm-1. The chimeric channels are thus powerful tools for helping to understand the contribution of the cytoplasmic hERG domains to the gating process and are suitable for in vitro biochemical and structural studies.
authors: From the Department of Biochemistry, University of Zürich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
authors: Georg J
authors: GJ
authors: Hausammann
authors: Markus G
authors: MG
authors: Grütter
mesh_terms: D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008564:Membrane Potentials; D009712:Nucleotides, Cyclic / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D055503:Protein Multimerization; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012995:Solubility; D014982:Xenopus laevis
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D009712:Nucleotides, Cyclic; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C116037:PAS domain kinases; D017346:Protein Serine-Threonine Kinases