Exploring the Mechanism of Cyclic Nucleotide Activation in the MLOTIK1 Potassium Channel

Abstract

Cyclic nucleotides are essential elements in the cellular responses to hormones, light and smell. These molecules bind to their receptors through a well conserved cyclic nucleotide binding (CNB) domain, propagating a conformational change to an effector domain. We seek to understand the molecular details of the cyclic nucleotide activation mechanism. Our model protein is the CNB domain of a cyclic nucleotide regulated potassium channel from Mesorhizobium loti, a soil bacterium. This CNB domain is formed by a β-roll and three α-helices, named αA, αB and αC. A major difference between the bound and unbound states is the relative position of αC helix, which raises the hypothesis that αC helix motion could be the primary event in cyclic nucleotide binding.To address the hypothesis, we have monitored the conformational change in the isolated CNB domain. We tested two mutants having a single cysteine in αB or αC helix using a cysteine-reacting probe. Reaction kinetics were quantified by determining rate constants, which reflect the relative exposure of the cysteine. The cyclic nucleotides tested were not all equivalent in their rate constants. Moreover, the ratios between rate constants determined in the same conditions were different for αB and αC helix mutants. This indicates that ligand binding does not have the same effect on the two connected helices or, in other words, that the conformational change in αB helix is not totally dependent on that in αC helix. Therefore, the primary event in ligand binding is not the αC helix motion. Accordingly, point mutations in a functionally relevant residue in αC helix affected activity of the full-length channel only partially, suggesting that residues outside this helix must be involved in the activation mechanism.