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A cyclic nucleotide modulated prokaryotic K+ channel.

Nimigean CM, Shane T, Miller C - J. Gen. Physiol. (2004)

Bottom Line: A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus.Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations.Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, 415 South St., Waltham, MA 02454, USA. cnimigea@brandeis.edu

ABSTRACT
A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive (86)Rb(+) flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K(+) channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

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Cyclic nucleotide dependence of 86Rb+ uptake. Flux time courses into liposomes reconstituted with 5 μg MloK1/mg lipid in the presence of cAMP (A) and cGMP (B), at 0 (white), 0.1 (light gray), 1 (dark gray), and 10 (black) μM concentration. (C) Cyclic nucleotide dose dependence of 86Rb+ uptake. Uptake values at 15 min (squares) and 45 min (circles) are normalized to both maximum (saturating concentration of cNMP) and minimum (no cNMP) values. Smooth curves are drawn according to single-site binding functions, with K1/2 = 60 nM for cAMP (black) and 600 nM for cGMP (gray). Each symbol represents mean ± SEM for three different experiments.
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fig7: Cyclic nucleotide dependence of 86Rb+ uptake. Flux time courses into liposomes reconstituted with 5 μg MloK1/mg lipid in the presence of cAMP (A) and cGMP (B), at 0 (white), 0.1 (light gray), 1 (dark gray), and 10 (black) μM concentration. (C) Cyclic nucleotide dose dependence of 86Rb+ uptake. Uptake values at 15 min (squares) and 45 min (circles) are normalized to both maximum (saturating concentration of cNMP) and minimum (no cNMP) values. Smooth curves are drawn according to single-site binding functions, with K1/2 = 60 nM for cAMP (black) and 600 nM for cGMP (gray). Each symbol represents mean ± SEM for three different experiments.

Mentions: Cyclic nucleotides bias the open–closed equilibrium of eukaryotic CNG and HCN channels toward open conformations. This behavior is mirrored in MloK1, although the maximum stimulatory effect of cAMP is only twofold (Fig. 4). To examine the ligand dependence of this effect, we reconstituted the channel into liposomes and used the uptake assay to determine dose–response curves for cAMP and cGMP. 86Rb+ flux time courses in the presence of varying cyclic nucleotide concentrations are shown in Fig. 7 (A and B). Both cAMP and cGMP stimulate the uptake, and both give similar approximately twofold maximal stimulation. Dose–response curves were derived from these time courses by normalizing the uptake values measured at 15 or 45 min to the corresponding isochronal uptake at maximal ligand concentration. These follow a simple Langmuir-type concentration dependence (Hill coefficient of unity), with cAMP being ∼10-fold more effective than cGMP (K1/2 = 60 and 600 nM, respectively).


A cyclic nucleotide modulated prokaryotic K+ channel.

Nimigean CM, Shane T, Miller C - J. Gen. Physiol. (2004)

Cyclic nucleotide dependence of 86Rb+ uptake. Flux time courses into liposomes reconstituted with 5 μg MloK1/mg lipid in the presence of cAMP (A) and cGMP (B), at 0 (white), 0.1 (light gray), 1 (dark gray), and 10 (black) μM concentration. (C) Cyclic nucleotide dose dependence of 86Rb+ uptake. Uptake values at 15 min (squares) and 45 min (circles) are normalized to both maximum (saturating concentration of cNMP) and minimum (no cNMP) values. Smooth curves are drawn according to single-site binding functions, with K1/2 = 60 nM for cAMP (black) and 600 nM for cGMP (gray). Each symbol represents mean ± SEM for three different experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233883&req=5

fig7: Cyclic nucleotide dependence of 86Rb+ uptake. Flux time courses into liposomes reconstituted with 5 μg MloK1/mg lipid in the presence of cAMP (A) and cGMP (B), at 0 (white), 0.1 (light gray), 1 (dark gray), and 10 (black) μM concentration. (C) Cyclic nucleotide dose dependence of 86Rb+ uptake. Uptake values at 15 min (squares) and 45 min (circles) are normalized to both maximum (saturating concentration of cNMP) and minimum (no cNMP) values. Smooth curves are drawn according to single-site binding functions, with K1/2 = 60 nM for cAMP (black) and 600 nM for cGMP (gray). Each symbol represents mean ± SEM for three different experiments.
Mentions: Cyclic nucleotides bias the open–closed equilibrium of eukaryotic CNG and HCN channels toward open conformations. This behavior is mirrored in MloK1, although the maximum stimulatory effect of cAMP is only twofold (Fig. 4). To examine the ligand dependence of this effect, we reconstituted the channel into liposomes and used the uptake assay to determine dose–response curves for cAMP and cGMP. 86Rb+ flux time courses in the presence of varying cyclic nucleotide concentrations are shown in Fig. 7 (A and B). Both cAMP and cGMP stimulate the uptake, and both give similar approximately twofold maximal stimulation. Dose–response curves were derived from these time courses by normalizing the uptake values measured at 15 or 45 min to the corresponding isochronal uptake at maximal ligand concentration. These follow a simple Langmuir-type concentration dependence (Hill coefficient of unity), with cAMP being ∼10-fold more effective than cGMP (K1/2 = 60 and 600 nM, respectively).

Bottom Line: A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus.Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations.Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, 415 South St., Waltham, MA 02454, USA. cnimigea@brandeis.edu

ABSTRACT
A search of prokaryotic genomes uncovered a gene from Mesorhizobium loti homologous to eukaryotic K(+) channels of the S4 superfamily that also carry a cyclic nucleotide binding domain at the COOH terminus. The gene was cloned from genomic DNA, and the protein, denoted MloK1, was overexpressed in Escherichia coli and purified. Gel filtration analysis revealed a heterogeneous distribution of protein sizes which, upon inclusion of cyclic nucleotide, coalesces into a homogeneous population, eluting at the size expected for a homotetramer. As followed by a radioactive (86)Rb(+) flux assay, the putative channel protein catalyzes ionic flux with a selectivity expected for a K(+) channel. Ion transport is stimulated by cAMP and cGMP at submicromolar concentrations. Since this bacterial homologue does not have the "C-linker" sequence found in all eukaryotic S4-type cyclic nucleotide-modulated ion channels, these results show that this four-helix structure is not a general requirement for transducing the cyclic nucleotide-binding signal to channel opening.

Show MeSH
Related in: MedlinePlus