Abstract
The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. NaK is a non-selective cation channel that conducts Na+ and K+ with equal efficiency. Crystal structures of NaK suggested a rigid SF structure, but later solid-state NMR and MD simulations questioned this interpretation. Here, we use solution NMR to characterize how bound Na+ vs. K+ affects NaK SF structure and dynamics. We find that the extracellular end of the SF is flexible on the ps-ns timescale regardless of bound ion. On a slower timescale, we observe a structural change between the Na+ and K+-bound states, accompanied by increased structural heterogeneity in Na+. We also show direct evidence that the SF structure is communicated to the pore via I88 on the M2 helix. These results support a dynamic SF with multiple conformations involved in non-selective conduction. Our data also demonstrate allosteric coupling between the SF and pore-lining helices in a non-selective cation channel that is analogous to the allosteric coupling previously demonstrated for K+-selective channels, supporting the generality of this model.
Highlights
The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores
To understand the impact of binding of Na+ and K+ on the structure and dynamics of the NaK channel, 1H–15N TROSY-HSQC spectra of U-15N,2H labeled NaK were collected in 100 mM K+ and 600 mM Na+
Spectra of refolded NaK solubilized in isotropic bicelles overlay very well with spectra of NaK that did not undergo the refolding protocol, but the peak intensity is increased for many weak peaks in the K+ sample and many more peaks are visible in the Na+ sample (Supplementary Fig. 1)
Summary
The selectivity filter (SF) determines which ions are efficiently conducted through ion channel pores. We employ NaK as a model non-selective channel to ask how the structure and dynamics of the SF are affected by ion identity and to what extent ion-dependent conformational changes in the SF are propagated throughout the pore. Allosteric coupling between M2, the SF, and pore helix was observed via ssNMR to mediate the anionic lipid-induced opening of KirBac1.126 These data suggest that coupling between the SF and M2 may be an important, general regulator of ion channel conductance properties. Our group has shown that NaK and NaK2K, a K+-selective mutant of NaK with a different SF structure[27], have distinct NMR chemical shifts for residues far from the sites of mutation in the SF28 This demonstrates that in principle the state of the putative inner gate can be influenced by the structure of the SF. In ssNMR spectra of NaK, peaks for residues below V91 on M2 were not visible[13], making it impossible to assess how or if the SF structure is communicated throughout the pore in the wild-type channel
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