title: HCN2 and HCN4 isoforms self-assemble and co-assemble with equal preference to form functional pacemaker channels.
abstract: Hyperpolarization-activated cyclic nucleotide-modulated (HCN) "pacemaker" channel subunits are integral membrane proteins that assemble as tetramers to form channels in cardiac conduction tissue and nerve cells. Previous studies have suggested that the HCN2 and HCN4 channel isoforms physically interact when overexpressed in mammalian cells, but whether they are able to co-assemble and form functional channels remains unclear. The extent to which co-assembly occurs over self-assembly and whether HCN2-HCN4 heteromeric channels are formed in native tissue are not known. In this study, we show co-assembly of HCN2 and HCN4 in live Chinese hamster ovary cells using bioluminescence resonance energy transfer (BRET(2)), a novel approach for studying tetramerization of ion channel subunits. Together with results from electrophysiological and imaging approaches, the BRET(2) data show that HCN2 and HCN4 subunits self-assemble and co-assemble with equal preference. We also demonstrate colocalization of HCN2 and HCN4 and a positive correlation of their intensities in the embryonic mouse heart using immunohistochemistry, as well as physical interactions between these isoforms in the rat thalamus by coimmunoprecipitation. Together, these data support the formation of HCN2-HCN4 heteromeric channels in native tissue.
authors.affiliation: Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Gina M
authors.initials: GM
authors.lastname: Whitaker
authors.forename: Damiano
authors.initials: D
authors.lastname: Angoli
authors.forename: Hamed
authors.initials: H
authors.lastname: Nazzari
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Shigemoto
authors.forename: Eric A
authors.initials: EA
authors.lastname: Accili
mesh_terms: D000818:Animals; D001683:Biological Clocks; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D054815:Cyclic Nucleotide-Gated Cation Channels; D004594:Electrophysiology; D006321:Heart / Q000196:embryology; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007150:Immunohistochemistry; D007473:Ion Channels / Q000737:chemistry / Q000502:physiology*; D051379:Mice; D008954:Models, Biological; D015221:Potassium Channels; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats
chemical_list: D054815:Cyclic Nucleotide-Gated Cation Channels; C578711:Hcn2 protein, mouse; C578712:Hcn2 protein, rat; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D007473:Ion Channels; D015221:Potassium Channels; D020033:Protein Isoforms