Ligand Binding and Activation in a Prokaryotic Cyclic Nucleotide-modulated Channel

Nimigean, Crina M.; Pagel, Matthew D.

doi:10.1016/j.jmb.2007.06.030

Cited by 20 publications

(19 citation statements)

References 38 publications

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“…Mutating this arginine to glutamate decreased the affinity for cNMP dramatically in both eukaryotic CNG and HCN channels (45,46). A similar effect was observed when neutralizing this residue in the prokaryotic MloK1 (42). Interestingly, this conserved arginine is also present in the sequences of all prokaryotic channels described in our study (Fig.…”

Section: Discussionsupporting

confidence: 83%

“…Bacterial CNG channels could only be successfully studied as reconstituted proteins in liposomes (13,42) or in giant E. coli spheroplasts (22). In the present study, fluorescence imaging and electrophysiological patch-clamp recording demonstrated that SthK-GFP can be expressed as a functional ion channel protein in the plasma membrane of Xenopus oocytes.…”

Section: Discussionmentioning

confidence: 54%

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Family of prokaryote cyclic nucleotide-modulated ion channels

Brams

Kusch

Spurný

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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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.cyclic AMP | cyclic GMP | electrophysiology | protein purification H yperpolarization-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 Clinker 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-regula...

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Section: Discussionsupporting

confidence: 83%

Section: Discussionmentioning

confidence: 54%

Family of prokaryote cyclic nucleotide-modulated ion channels

Brams

Kusch

Spurný

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…ity) than the K1 ⁄ 2 previously reported for MloK1 (25). The Hill coefficient of ϳ1 is consistent with what was previously found for MloK1 (34,37).…”

Section: Construction Of the Kcsa/mlok1supporting

confidence: 91%

“…1A) and a variant with an E71A mutation, which removes inactivation at the KcsA selectivity filter (35). Following gel filtration, protein was reconstituted into liposomes as reported previously (36,37) by mixing a ratio of 5-10 g of protein per mg of lipid with a 3:1 molar ratio of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol solubilized in reconstitution buffer (400 mM KCl, 10 mM HEPES, 5 mM N-methyl-D-glucamine, 200 M cAMP) with 34 mM CHAPS, which was subsequently applied over a 20-ml G-50 Sephadex column to remove the detergent. Liposome aliquots in reconstitution buffer were frozen in liquid nitrogen and stored for future use.…”

Section: Methodsmentioning

confidence: 99%

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

McCoy

Rusinova

Kim

et al. 2014

Journal of Biological Chemistry

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Background:The mechanism of ligand gating in physiologically important cyclic nucleotide-modulated channels is unknown. Results: We constructed and purified a chimeric ion channel with activity modulated by cAMP and used it to measure ligand-binding energetics. Conclusion: cAMP binds with high lipid-dependent affinity to the chimeric channel. Significance: The availability of a good protein preparation enables assays that shed light on ligand gating.

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“…-channel, designated MloK1, has been identified in Mesorhizobium loti (Nimigean et al 2004;Nimigean and Pagel 2007). The channel consists of four identical subunits and each subunit encompasses six transmembrane domains, a 'GYG' signature sequence for K ?…”

mentioning

confidence: 99%

Resonance assignments of the nucleotide-free wildtype MloK1 cyclic nucleotide-binding domain

et al. 2010

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Cyclic nucleotide-sensitive ion channels, known as HCN and CNG channels play crucial roles in neuronal excitability and signal transduction of sensory cells. These channels are activated by binding of cyclic nucleotides to their intracellular cyclic nucleotide-binding domain (CNBD). A comparison of the structures of wildtype ligand-free and ligand-bound CNBD is essential to elucidate the mechanism underlying nucleotide-dependent activation of CNBDs. We recently reported the solution structure of the Mesorhizobium loti K1 (MloK1) channel CNBD in complex with cAMP. We have now extended these studies and achieved nearly complete assignments of (1)H, (13)C and (15)N resonances of the nucleotide-free CNBD. A completely new assignment of the nucleotide-free wildtype CNBD was necessary due to the sizable chemical shift differences as compared to the cAMP bound CNBD and the slow exchange behaviour between both forms. Scattering of these chemical shift differences over the complete CNBD suggests that nucleotide binding induces significant overall conformational changes.

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Ligand Binding and Activation in a Prokaryotic Cyclic Nucleotide-modulated Channel

Cited by 20 publications

References 38 publications

Family of prokaryote cyclic nucleotide-modulated ion channels

Family of prokaryote cyclic nucleotide-modulated ion channels

A KcsA/MloK1 Chimeric Ion Channel Has Lipid-dependent Ligand-binding Energetics

Resonance assignments of the nucleotide-free wildtype MloK1 cyclic nucleotide-binding domain

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