title: Family of prokaryote cyclic nucleotide-modulated ion channels.
abstract: Cyclic nucleotide-modulated ion channels are molecular pores that mediate the passage of ions across the cell membrane in response to cAMP or GMP. Structural insight into this class of ion channels currently comes from a related homolog, MloK1, that contains six transmembrane domains and a cytoplasmic cyclic nucleotide binding domain. However, unlike eukaryote hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels, MloK1 lacks a C-linker region, which critically contributes to the molecular coupling between ligand binding and channel opening. In this study, we report the identification and characterization of five previously unidentified prokaryote homologs with high sequence similarity (24-32%) to eukaryote HCN and CNG channels and that contain a C-linker region. Biochemical characterization shows that two homologs, termed AmaK and SthK, can be expressed and purified as detergent-solubilized protein from Escherichia coli membranes. Expression of SthK channels in Xenopus laevis oocytes and functional characterization using the patch-clamp technique revealed that the channels are gated by cAMP, but not cGMP, are highly selective for K(+) ions over Na(+) ions, generate a large unitary conductance, and are only weakly voltage dependent. These properties resemble essential properties of various eukaryote HCN or CNG channels. Our results contribute to an understanding of the evolutionary origin of cyclic nucleotide-modulated ion channels and pave the way for future structural and functional studies.
authors.affiliation: Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and.
authors.forename: Marijke
authors.initials: M
authors.lastname: Brams
authors.affiliation: Institute of Physiologie II, University Hospital Jena, 07743 Jena, Germany.
authors.forename: Jana
authors.initials: J
authors.lastname: Kusch
authors.affiliation: Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and.
authors.forename: Radovan
authors.initials: R
authors.lastname: Spurny
authors.affiliation: Institute of Physiologie II, University Hospital Jena, 07743 Jena, Germany.
authors.forename: Klaus
authors.initials: K
authors.lastname: Benndorf
authors.affiliation: Laboratory of Structural Neurobiology, Department of Cellular and Molecular Medicine, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium; and chris.ulens@med.kuleuven.be.
authors.forename: Chris
authors.initials: C
authors.lastname: Ulens
mesh_terms: D000818:Animals; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D019143:Evolution, Molecular; D018613:Microscopy, Confocal; D005810:Multigene Family / Q000235:genetics*; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000378:metabolism; D017385:Sequence Homology; D014981:Xenopus
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; D000242:Cyclic AMP; D011188:Potassium
keywords: cyclic AMP; cyclic GMP; electrophysiology; protein purification