A Family of HCN Channel Homologs in Bacteria

Abstract

HCN channels belong to the superfamily of six transmembrane domain voltage-gated ion channels. Functionally, these channels activate upon membrane hyperpolarization and carry an inward current that is weakly selective between potassium and sodium and can be modulated by cyclic nucleotides. At the structural level, insight into these channels is limited to a crystal structure of the intracellular C-linker and cyclic nucleotide binding domain, which assembles into a tetramer. To advance our structural understanding of HCN channels, we investigated bacterial homologs of HCN channels as candidates for large-scale expression and future structural studies. Using a BLAST search of the bacterial genome database we identified different homologs, which have a sequence identity of 24-32% with the human HCN2 channel. Using a C-terminal fusion with green fluorescent protein (GFP) we investigated whether the bacterial HCN homologs could be expressed in E. coli. We identified homologs that could be expressed at a whole-cell fluorescence level of ∼40% compared to KcsA-GFP as a positive control. Next, we conducted detergent screening using fluorescence size exclusion chromatography (FSEC). We found that dodecylmaltoside or undecylmaltoside are suitable detergents to solubilize bacterial HCN channels in a tetrameric monodisperse state.In parallel, we investigated the functional properties of the bacterial HCN channel homologs using expression of C-terminal GFP fusions in Xenopus oocytes. Using cell-attached patch recordings we observed in a minority of patches a current that may closely resemble the kinetics of the invertebrate spHCN channel as clear current inactivation is observed at most hyperpolarizing potentials. Confocal microscopy demonstrates marked fluorescence just below the oocyte membrane, indicating that trafficking to the oocyte cell membrane may be compromised and limits our success in obtaining patches for detailed functional characterization.Together, our biochemical characterization paves the way for large-scale production and crystallization screening.