An open state of a voltage-gated sodium channel involving a π-helix and conserved pore-facing Asparagine

Abstract

Voltage-gated sodium (Nav) channels play critical roles in propagating action potentials and otherwise manipulating ionic gradients in excitable cells. These channels open in response to membrane depolarization, selectively permeating sodium ions until rapidly inactivating. Structural characterization of the gating cycle in this channel family has proved challenging, particularly due to the transient nature of the open state. A structure from the bacterium Magnetococcus marinus Nav (NavMs) was initially proposed to be open, based on its pore diameter and voltage-sensor conformation. However, the functional annotation of this model, and the structural details of the open state, remain disputed. In this work, we used molecular modeling and simulations to test possible open-state models of NavMs. The full-length experimental structure, termed here the α-model, was consistently dehy-drated at the activation gate, indicating an inability to conduct ions. Based on a spontaneous transition observed in extended simulations, and sequence/structure comparison to other Nav channels, we built an alternative π-model featuring a helix transition and the rotation of a conserved asparagine residue into the activation gate. Pore hydration, ion permeation and state-dependent drug binding in this model were consistent with an open functional state. This work thus offers both a functional annotation of the full-length NavMS structure, and a detailed model for a stable Nav open state, with potential conservation in diverse ion-channel families.

An α-π transition in S6 shapes the conformational cycle of the bacterial sodium channel NavAb

“…Alternative models of NavAb with contracted or expanded pores were built by introducing a π-helix in a region of the S6 helix of 5VB2 and 5VB8, respectively. This was done using MODELLER 9.22 (34) following a protocol implemented in previous work on NavMs (29). Briefly, this consisted of shifting the structural alignment in S6 of the π-versus template α-models in order to introduce an unpaired backbone carbonyl.…”

“…Alternative models of NavAb with contracted or expanded pores were built by introducing a πhelix in a region of the S6 helix of 5VB2 and 5VB8, respectively. This was done using MODELLER 9.22 (34) following a protocol implemented in previous work on NavMs (29).…”

“…This resulted in rotation of the pore helix following the π helix around T1206. As a result of this, residues facing the pore in the α-model rotate away from it in the π-model, while a different set of residues-including N1211-now face the pore [29]. Since all the residues in this region except N1211 are hydrophobic, the protonation states of the residues are not affected by the rotation.…”

“…In our recent study of the related bacterial channel NavMs (29), we found that a structure initially thought to represent an open state, with an expanded pore with entirely α-helical S6 helices, was dehydrated and non-conductive to sodium ions. However, when the S6 helices transitioned to containing a short π-helical stretch resulting in a rotation of the S6 C-terminus, the pore was hydrated, allowing sodium ion permeation.…”

“…Reverting the I1217C mutation dehydrated the pore, leading to a model that was impermeable to sodium ions. Given our previous model of NavMs pore opening (29), we then constructed an alternate model of the NavAb pore, in which introducing a π-helix in S6 results in a hydrated and conductive pore. This open-pore model was validated by sodium ion and local-anesthetic permeation free energy calculations.…”