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A cyclic nucleotide modulated prokaryotic K+ channel.
Crina M Nimigean1, Tania Shane, Christopher Miller
1Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, 415 South St., Waltham, MA 02454, USA. cnimigea@brandeis.edu
The Journal of General Physiology
|September 1, 2004
Summary
Researchers discovered a bacterial protein, MloK1, that functions as a potassium channel. Cyclic nucleotides like cAMP and cGMP activate this channel, demonstrating a conserved mechanism across species.
Area of Science:
- Molecular Biology
- Biophysics
- Structural Biology
Background:
- Eukaryotic potassium (K+) channels of the S4 superfamily are crucial for cellular ion transport.
- Many of these channels possess a cyclic nucleotide-binding domain (CNBD) at their COOH terminus, regulating their activity.
- The structural requirements for signal transduction from CNBD to channel gating remain incompletely understood.
Purpose of the Study:
- To identify and characterize prokaryotic homologs of eukaryotic cyclic nucleotide-gated (CNG) K+ channels.
- To investigate the functional and structural properties of a newly identified bacterial K+ channel.
- To determine if the conserved eukaryotic 'C-linker' is essential for cyclic nucleotide-mediated channel gating.
Main Methods:
- Bioinformatic search for eukaryotic K+ channel homologs in prokaryotic genomes.
- Cloning and overexpression of the candidate gene (MloK1) from Mesorhizobium loti in Escherichia coli.
- Protein purification and size determination using gel filtration chromatography.
- Functional characterization using radioactive 86Rb+ flux assays to measure ionic transport.
- Assessment of cyclic nucleotide (cAMP, cGMP) effects on channel activity.
Main Results:
- A Mesorhizobium loti gene homologous to eukaryotic K+ channels with a CNBD was identified and cloned.
- The purified protein, MloK1, exists as a homogeneous homotetramer in the presence of cyclic nucleotides.
- MloK1 exhibits K+ selective ion flux, confirming its function as a potassium channel.
- Submicromolar concentrations of cAMP and cGMP significantly stimulate MloK1 ion transport.
- MloK1 lacks the eukaryotic 'C-linker' sequence, yet responds to cyclic nucleotides.
Conclusions:
- MloK1 represents a functional prokaryotic homolog of eukaryotic cyclic nucleotide-modulated K+ channels.
- Cyclic nucleotides directly regulate the activity of this bacterial potassium channel.
- The absence of the 'C-linker' in MloK1 demonstrates that this structural motif is not universally required for cyclic nucleotide-gated channel function.