Solution NMR to Investigate Gating in the NaK Channel

Abstract

The NaK channel is small bacterial non-selective cation channel that has proved to be an excellent model system to study basic biochemical and biophysical question concerning ion channels. NaK has been crystalized in the closed state and as an open channel using a truncated construct (NaKΔ19) missing the M0 helix. NaK shares a homologous architecture to tradition K+ channel pores such as KcsA, but it is non selective. However, small mutation in the selectivity filter can change NaK into a K+ selective channel. Solution NMR spectra of NaK solubilized in isotropic bicelles show superior spectral resolution, allowing for detailed solution NMR studies of NaK structure and dynamics in bicelles. Regulation of NaK is currently unknown, however the location of the amphipathic M0 helix on the membrane surface packed against two adjacent monomers makes it a likely contributor to channel gating. Preliminary work suggests that the full length NaK channel exists in at least two states in slow exchange at 45oC. Mutations to the M0 helix shift the equilibrium to a single population, supporting a role for the M0 helix in determining the structural and functional state of the channel. NaKΔ19 NMR spectra are also sensitive to addition of amino acids just before the M1 helix. This suggests that this region connecting the M0 helix to the M1 helix is important in determining NaK dynamics. Comparison of our results with functional studies highlights specific interactions between the M0 helix and the pore domain that determine the structural and functional state of the channel.