Ion-dependent structure, dynamics, and allosteric coupling in a non-selective cation channel

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.

Reassessing the helix bundle crossing model for gating in a non-selective ion channel

“…We did not observe all of the expected possible inter-methyl cross-peaks in the NaK crystal structure in this distance range, but 110 of these "missing" peaks are expected to be weak (79 of these unobserved cross-peaks report on atoms separated by more that 8Å) or would overlap with the diagonal peaks in our 3D experiments (31 crosspeaks had too small separation of their diagonal 13 C frequencies). The absence of peaks originating from spin diffusion was further verified by comparing the spectra of ILV labeled NaK recorded in both D2O, and H2O 46 , and confirms that the NOESY cross-peaks correspond directly to the distance between methyl groups.…”

“…There is broadening of both the amide and methyl resonances of G89 and L90, along with the L98 methyl resonance (Figure 2, Figure SI2), but resonances downstream of the hinge region, such as the Cγ1/2 methyls of V100 and V102, do not appear to be broadened. As we have previously reported 46 , the flexibility of the M2 helix in the region V102-L108 has increased ps-ns timescale dynamics, which could explain why this region is not observable in the solid-state data. The data also suggest that HBC has enhanced dynamics in different lipid environments (DMPC/DHPC bicelles here and Lewis et al 46 , asolectin bilayers in Shi et al 43 ).…”

“…As we have previously reported 46 , the flexibility of the M2 helix in the region V102-L108 has increased ps-ns timescale dynamics, which could explain why this region is not observable in the solid-state data. The data also suggest that HBC has enhanced dynamics in different lipid environments (DMPC/DHPC bicelles here and Lewis et al 46 , asolectin bilayers in Shi et al 43 ). The ability to observe the terminal parts of M1 and M2 helices using solution NMR gives us a unique opportunity to probe the structure of the inner gate of a K + -like channel more directly by acquiring NMR distance restraints.…”

“…We have thus tried to detect any minor conformations which could potentially correspond to the closed HBC conformation of NaK. However, we could not detect such evidence in the vicinity of the HBC on the millisecond time scale (a timescale at which conformational changes of such magnitude would be expected) in neither NaKΔ18 46 nor in NaK FL (Figure SI 9). We do observe somewhat enhanced millisecond dynamics for I88 Cδ1 methyl group, which is in line with the broadening we observe for G89 and L90, indicating some conformational dynamics in this region.…”

“…NaK has 32 methyl bearing Ile, Leu, and Val residues (out of 110 total residues) for 53 probes per monomer in total, which are dispersed rather uniformly throughout the structure. The methyl groups were assigned previously 46 . We then compared the methyl chemical shifts for U-[ 15 N 12 C 2 H] Ile (δ1), Leu/Val labelled NaK∆18 and NaK FL.…”