In this report the Catterall laboratory succeeded in solving the high resolution structure of a voltage-gated Na+-channel (Nav) in its resting state (1). Why is this difficult and why is this important? It is difficult because Navs exist in the resting state only at very negative voltages but not at a zero membrane potential required for structural analysis by X-ray crystallography or cryo-EM. Accordingly, all high resolution structures of Navs, whether pro- or eukaryotic, have so far reported channels with the voltage-sensing domains in the depolarized state, i.e. the positively charges S4 helices of the voltage sensors moved “up” towards the extracellular side. Therefore it is not known how the activation gate of the ion pore (formed by the four S6 helices) is kept closed by the voltage sensor in its resting position, i.e. with the S4-helices “down”. Some predictions about how this might work was inferred from structural work on related voltage-gated ion channels (e.g in a TPC1 channel, 2) or from a study in which a chimeric Nav construct was trapped in a closed (“deactivated”) state by a toxin (3). The elegant work presented here by Wideschaisri and colleagues (1) directly addressed this important question by generating suitable mutants of the bacterial Nav, NavAb (4). One mutant (KAV mutation) was engineered to shift its activation threshold to much higher voltages thus holding the channel in the resting state also at 0 mV for structural studies. Moreover, they introduced disulfide crosslinks locking the voltage-sensor in the desired resting (S4 “down”) or activated (S4 “up”) state. The stabilization of these states in these mutants were verified in functional studies to ensure that the structural data have clear functional correlates. Analysis of the X-ray structures (and cryo-EM structure for the KAV mutant) provided important novel insight into the structural rearrangements associated with the transition from the activated/open to the resting/closed state. This includes changes of the helical structure of S4 associated with its striking inward movement of about 11.5 A compatible with a “sliding helix” model. Rearrangements of the four S4-S5 linkers were found to tighten the “collar” around the S5 and S6 segments thus keeping the pore closed.
All this was possible because they used the bacterial NavAb, for their experiments. This comes with the advantage that this channel exists as a tetramer of identical subunits and therefore the mutations are present in all four voltage-sensors. This also facilitated disulfide cross-linking, because these channels lack endogenous cysteines. The disadvantage of NavAb is that it does not reflect the more complex structure of eukaryotic Nav and voltage-gated Ca2+ channels (Cavs) in which all four voltage-sensing- and pore forming elements are different and are tethered together in a single molecule. Nevertheless, there is no doubt that this “sliding helix” model of electromechanical coupling will also apply to eukarytic Navs and Cavs. Understanding all the conformational rearrangements occurring between resting and activated channel states will provide new opportunities for the discovery of state-dependent and subtype-selective Nav- and Cav- channel blocking drugs.
Comments by Jörg Striessnig, University of Innsbruck
(1) Wisedchaisri, G., Tonggu, L., McCord, E., Gamal El-Din, T.M., Wang, L., Zheng, N., Catterall, W.A., 2019. Resting-State Structure and Gating Mechanism of a Voltage-Gated Sodium Channel. Cell 178, 993-1003. doi: 10.1016/j.cell.2019.06.031. [PMID: 31353218]
(2) Kintzer, A.F., Green, E.M., Dominik, P.K., Bridges, M., Armache, J.-P., Deneka, D., Kim, S.S., Hubbell, W., Kossiakoff, A.A., Cheng, Y., Stroud, R.M., 2018. Structural basis for activation of voltage sensor domains in an ion channel TPC1. Proc. Natl. Acad. Sci. U.S.A. 115, E9095–E9104. doi: 10.1073/pnas.1805651115. [PMID: 30190435]
(3) Xu, H., Li, T., Rohou, A., Arthur, C.P., Tzakoniati, F., Wong, E., Estevez, A., Kugel, C., Franke, Y., Chen, J., Ciferri, C., Hackos, D.H., Koth, C.M., Payandeh, J., 2019. Structural Basis of Nav1.7 Inhibition by a Gating-Modifier Spider Toxin. Cell 176, 702-715.e14. doi: 10.1016/j.cell.2018.12.018. [PMID: 30661758]
(4) Payandeh, J., Scheuer, T., Zheng, N., Catterall, W.A., 2011. The crystal structure of a voltage-gated sodium channel. Nature 475, 353–358. doi: 10.1038/nature10238. [PMID: 21743477]