Ligand binding and activation in a prokaryotic cyclic nucleotide-modulated channel.

TitleLigand binding and activation in a prokaryotic cyclic nucleotide-modulated channel.
Publication TypeJournal Article
Year of Publication2007
AuthorsNimigean CM, Pagel MD
JournalJ Mol Biol
Date Published2007 Aug 31
KeywordsAmino Acid Sequence, Cyclic AMP, Cyclic GMP, Dose-Response Relationship, Drug, Escherichia coli, Escherichia coli Proteins, Ion Channels, Kinetics, Ligands, Liposomes, Molecular Conformation, Molecular Sequence Data, Mutation, Protein Binding, Sensitivity and Specificity, Sequence Homology, Amino Acid

We designed a technique that directly determines binding of cyclic nucleotides to the prokaryotic cyclic nucleotide modulated ion channel MloK1. The ability to purify large quantities of MloK1 facilitated equilibrium binding assays, which avoided the inherent problem of relatively low affinity binding which hindered the use of eukaryotic channels. We found that MloK1 specifically binds cAMP and cGMP with affinity values in the range of those observed for activity assays for eukaryotic channels. Notably, the concentration of ligand that elicited 50% of maximum response in (86)Rb flux assays (K1/2), also referred to as ligand sensitivity, was smaller than the corresponding value obtained from binding assays (Kd) potentially indicating significant channel activity in partially liganded states. To gain further insight into the mechanism of binding and activation of these channels, we mutated several amino acids in the ligand-binding pocket of MloK1, known from electrophysiological studies of homologous eukaryotic channels to affect ligand selectivity and binding efficacy. The S308V MloK1 mutant (a mutation which decreases cGMP selectivity in eukaryotic channels) decreased both the observed cGMP binding affinity and the sensitivity to cGMP relative to the wild-type (WT) channel, leaving those for cAMP unchanged. Conversely, the A352D MloK1 mutant (a mutation which increases cGMP selectivity in eukaryotic channels) increased both the affinity and the sensitivity for cGMP relative to the WT channel, again leaving those for cAMP unchanged. Mutations at R307 in MloK1, the most conserved residue in the binding pocket of cyclic nucleotide-binding proteins, were not tolerated as these mutants do not form functional channels. Furthermore, for each mutation, changes in binding affinities were mirrored by equivalent changes in ligand sensitivity. These data, together with the evidence that partially liganded channels open significantly, suggested strong coupling between cyclic nucleotide binding and MloK1 channel opening.

Alternate JournalJ. Mol. Biol.
PubMed ID17619023