Structures of closed and open states of a voltage-gated sodium channel

Abstract

Bacterial voltage-gated sodium channels (BacNavs) serve as models of their vertebrate counterparts. BacNavs contain conserved voltage-sensing and pore-forming domains, but they are homotetramers of four identical subunits, rather than pseudotetramers of four homologous domains. Here, we present structures of two NaAb mutants that capture tightly closed and open states at a resolution of 2.8-3.2 Å. Introduction of two humanizing mutations in the S6 segment (NaAb/FY: T206F and V213Y) generates a persistently closed form of the activation gate in which the intracellular ends of the four S6 segments are drawn tightly together to block ion permeation completely. This construct also revealed the complete structure of the four-helix bundle that forms the C-terminal domain. In contrast, truncation of the C-terminal 40 residues in NaAb/1-226 captures the activation gate in an open conformation, revealing the open state of a BacNav with intact voltage sensors. Comparing these structures illustrates the full range of motion of the activation gate, from closed with its orifice fully occluded to open with an orifice of ∼10 Å. Molecular dynamics and free-energy simulations confirm designation of NaAb/1-226 as an open state that allows permeation of hydrated Na, and these results also support a hydrophobic gating mechanism for control of ion permeation. These two structures allow completion of a closed-open-inactivated conformational cycle in a single voltage-gated sodium channel and give insight into the structural basis for state-dependent binding of sodium channel-blocking drugs.

Optimized expression and purification of NavAb provide the structural insight into the voltage dependence

“…The similar density was observed in NavAb I217C structure [12]. CHAPSO was also observed in the closed structure of NavAb mutant (pdb code: 5VB2) [17], but the positions of CHAPSO in each structure were different (Fig. S1D), was observed between the cytoplasmic end of the poredomain S5 helix and the adjacent-subunit voltage sensor domain (VSD) (Fig.…”

Conservation and variability of the pore-lining helices in P-loop channels

“…The deviations increase above 10 A at the C-terminal halves of the inner helices, which contain residues contributing to the activation gate; positions of these residues obviously differ between the open-and closed-state structures. Alpha carbon deviations of the open-state sodium channels NavAb 6 and NavMs 4 from the open-state Kv1.2 are rather small all along the S6 helices (Fig. 2B).…”

“…In the open-state structure of NavAb, 6 asparagines N Xi20 donate H-bonds to the sidechains of aspartates D (X-1)i28 , which are the last C-terminal S6 residues resolved in the X-ray structure. Importantly, the C-ends of the inner helices adopt the 3 10 helix conformations, which are not seen in the X-ray structures of NavMs or closed-state NavAb.…”

“…[1][2][3][4][5][6][7] and cryoelectron microscopy, e.g. 8,9 Voltagegated channels (4 £ 6TM) have four voltage-sensing domains and a pore domain.…”

“…When the first X-ray structures of potassium channels become available, 2,11 various sequence alignments were proposed between these channels and medically important sodium and calcium channels. 3,[12][13][14] More recent 3D structures of sodium, 4,6,7,10 calcium, 9 TRPV1 15 and two-pore 16 channels opened a possibility to obtain 3D alignment of various P-loop channels, compare sequence-based and 3D-based alignments and analyze similarity and differences of these channels. Results of this comparison reveal some problems in sequence alignments that previously seemed unambiguous.…”