Family of prokaryote cyclic nucleotide-modulated ion channels

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.Hyperpolarization-activated cyclic nucleotide-modulated (HCN) and cyclic nucleotide-gated (CNG) channels belong to the superfamily of voltage-gated K⁺ channels. Both types of channels share a similar domain topology with six transmembrane domains, a C-linker region, and a cyclic nucleotide binding domain (CNBD). The S5–S6 segment forms the channel pore, including the selectivity filter for cations. The S4 segment contains several positively charged amino acids, suggesting that it acts as voltage sensor. Despite these similarities in sequence, the function of HCN and CNG channels is noticeably different: HCN channels activate upon membrane hyperpolarization and can be modulated by cyclic nucleotides. They are weakly selective for K⁺ over Na⁺ ions (for reviews, see refs. 1–3). In contrast, CNG channels are activated by the binding of cyclic nucleotides solely and their activity depends only weakly on voltage. The ionic current is carried by both monovalent and divalent cations (for reviews, see refs. 4 and 5).Insight into the structure of HCN channels has been gained only from crystal structures of the isolated intracellular C-linker and CNBD of mammalian HCN1, HCN2, HCN4, and invertebrate spHCN1. These parts of the channel assemble into tetramers (6–9). Further structural information comes from prokaryote ion channels that are homologous to HCN and CNG channels, such as the bacterial cyclic nucleotide-regulated K⁺ channel MloK1 (10–13). MloK1 lacks a C-linker region, but has a CNBD with an overall structure that is remarkably similar to the CNBD of eukaryote HCN channels (10). Based on the dimer assembly of the MloK1 CNBD in the crystal structure, a gating mechanism has been proposed in which the pore opening in the tetrameric channel arises from the action of the four CNBDs as a dimer of dimers (10). The crystal structure of the MloK1 transmembrane domain (11) reveals a domain topology that resembles that of the voltage-gated K⁺ channel Kv1.2 (14), but with important differences. The MloK1 structure suggests that the S1–S4 domain and its associated linker in MloK1 can serve as a clamp to constrain the gate and possibly function in concert with the CNBD to regulate channel opening (11). Additionally, crystal structures have also been determined for the C-linker and cyclic nucleotide binding homology domain (CNBHD) of related ion channels, including the zebrafish EAG-like (ELK) K⁺ channel (15), the mosquito ERG K⁺ channel (16), and the mouse EAG1 K⁺ channel (17). Structural insight into the mechanism of ion permeation has been derived from a prokaryote ion channel NaK (18), which was mutated to mimic the CNG channel pore region (19). Collectively, these structural data have brought valuable information about the determinants of ion permeation, domain assembly, ligand recognition, channel gating and regulation, as well as effects of disease-causing mutations (20).Despite this tremendous progress, crystal structures for whole-eukaryote HCN and CNG channels are still not available at present, and structural insight into fundamental aspects of ion channel function is still lacking, such as the inverse voltage sensitivity in HCN channels and the coupling between cyclic nucleotide binding and channel opening by the C-linker domain, which is, as mentioned, absent in the MloK1 channel (10). In contrast, a putative voltage-gated K⁺ channel containing a C-linker region and CNBD similar to eukaryote channels was identified in the genome of the cyanobacterium Trichodesmium erythraeum (21), here termed TerK, and it was suggested to possibly represent an ancestral HCN or CNG channel (21). However, neither structure nor function of this prokaryote homolog is known. In this study, we report the characterization of TerK and four additional prokaryote ion channels, which all contain six putative transmembrane domains, a C-linker region, and a CNBD, and apparently form a family of prokaryote ion channels with close similarity to eukaryote HCN and CNG channels. We describe the expression in Escherichia coli, detergent screening and biochemical purification of these different homologs. Moreover, we identified two homologs, SthK (from Spirochaeta thermophila) and AmaK (from Arthrospira maxima), which can be stably extracted with detergents and purified in sufficiently high amounts for biochemical and structural studies. Using confocal fluorescence microscopy and electrophysiological recordings, we describe essential functional properties of one homolog, SthK. We find that SthK has electrophysiological properties that closely resemble those of eukaryote CNG channels as it is gated by intracellular cAMP and produces large unitary currents, whereas its activity is relatively insensitive to voltage. However, unlike CNG channels, SthK contains the selectivity filter sequence -TIGYGD-, which is more similar to HCN channels and other K⁺ selective channels. We could experimentally demonstrate that SthK channels are highly selective for K⁺ over Na⁺ ions. Importantly, SthK has several sequence features that closely resemble eukaryote cyclic nucleotide-modulated channels, including a C-linker region, which is missing in previously studied prokaryote homologs, such as MloK1 (10, 12, 13) and MmaK (22). Together, these data make the SthK channel a promising candidate for future structural analysis to learn more about how mammalian CNG and HCN channels work.