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Address correspondence to Crina M. Nimigean, Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, 415 South St., Waltham, MA 02454. Fax: (781) 736-2365; email: cnimigea{at}brandeis.edu
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 86Rb+ 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.
Key Words: transport • reconstitution • flux • HCN • CNG
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