Mechanosensitive pore opening of a prokaryotic voltage-gated sodium channel

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Voltage-gated ion channels orchestrate electrical activities that drive mechanical functions in contractile tissues such as the heart and gut. In turn, contractions change membrane tension and impact ion channels. Voltage-gated ion channels are mechanosensitive, but the mechanisms of mechanosensitivity remain poorly understood. Here, we leverage the relative simplicity of NaChBac, a prokaryotic sodium channel from Bacillus halodurans , to investigate its mechanosensitivity. In whole-cell experiments on heterologously transfected HEK293 cells, shear stress reversibly altered the kinetic properties of NaChBac and increased its maximum current, comparably to the mechanosensitive eukaryotic sodium channel Na V 1.5. In single-channel experiments, patch suction reversibly increased the open probability of a NaChBac mutant with inactivation removed. A simple kinetic mechanism featuring a mechanosensitive pore opening transition explained the overall response to force, whereas an alternative model with mechanosensitive voltage sensor activation diverged from the data. Structural analysis of NaChBac identified a large displacement of the hinged intracellular gate, and mutagenesis at the hinge abolished NaChBac mechanosensitivity, further supporting the proposed mechanism. Overall, our results suggest that NaChBac responds to force because its pore is intrinsically mechanosensitive. This mechanism may apply to other voltage-gated ion channels, including Na V 1.5.

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  1. eLife Assessment:

    This manuscript presents a biophysical study of the nature of the mechanosensitivity of voltage-gated sodium channels. The identification of a voltage-independent mechanosensitive step is well founded, the proposal that this step is the intracellular gate is plausible speculation. It is expected to be of interest to scientists studying the physical basis of mechanosensitivity in electrophysiology.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

  2. Reviewer #1 (Public Review):

    This well-written manuscript presents a technically impressive and carefully controlled biophysical study of the nature of the mechanosensitivity of voltage-gated Na+ (Nav) channels. As a simplified model system, the study employs an inactivation-deficient bacterial Nav channel that appears to respond to mechanical stimulation in a similar fashion to mammalian Nav1.5. The results provide strong evidence that a step in the activation path which has little intrinsic voltage dependence is mechanosensitive. The manuscript proposes that the mechanosensitive step is pore opening. While this seems the most parsimonious explanation, it still seems possible that a conformational change other than the opening of the pore could be the voltage-independent mechanosensitive step. The swinging door model presented here seems apt and is conceptually valuable. However, it is not clear that the I228G hinge mutagenesis provides strong support for the swinging door model. Overall, the conclusion that a voltage-insensitive step of the bacterial channel is mechanosensitive is well-founded. The additional proposal that this mechanosensitive step is the opening of the intracellular S6 pore gate is best-considered speculation.

  3. Reviewer #2 (Public Review):

    Previous studies have shown that voltage-gated Na+ (Nav) channels are mechanosensitive, but the mechanistic basis had been difficult to study owing to fast kinetics, small currents, and the large size of eukaryotic Nav. Here, Strege et al. turn to the prokaryotic NaChBac as a simpler model. They combine whole cell and single channel recording, kinetic modelling, and structure-guided mutagenesis to convincingly demonstrates that NaChBac has intrinsic mechanosensitivity and that this sensitivity is mechanistically linked to changes in plane channel footprint associated with pore opening and closing.

    While the results and conclusion are overall analogous to earlier studies in other VGIC (in particular, voltage-gated K+ (Kv) channels [Schmidt & Mackinnon PNAS 2012]), this is an important contribution because it provides further support for the hypothesis that mechanosensitivity is widespread among ion channels. What remains unanswered is whether NaChBac's observed (and by induction Nav1.5's) mechanosensitivity occurs over a physiologically relevant regime; a quantitative relationship between applied pressure or shear (units of force/area) with membrane tension (units of force/distance) is not established. Much of the data are collected in patches that are known to be under non-zero tension before any application of additional pressure. This caveat should at least be explicitly considered and discussed. This consideration is also relevant when speculating about the selective evolutionary mechanism that conserved or tuned NaChBac mechanosensitivity. A more likely scenario is that mechanosensitivity is an intrinsic feature of membrane proteins that undergo conformation changes (e.g., pore opening) and deform the lipid bilayer (thickness deformation, change in area, midplane pending [Phillips & Sens Nature 2009]. Given the existence of specialized mechano-sensors, e.g., PIEZO, that is exquisitely sensitive to lateral membrane tension, it could be equally well argued that adaptive changes (e.g., those that would stabilize the close state of the pore) in other ion channels may have decreased this mechanosensitivity over evolutionary timescales time to improve the fidelity for sensing voltage and decrease 'gating noise' introduced by mechanical perturbation.

  4. Reviewer #3 (Public Review):

    Strege et al. addressed the mechanism underlying the well-known mechanosensitivity of voltage-gated sodium channels. They cleverly bypassed the complexities of working with the mammalian NaV channels using a non-inactivation version of the bacterial homologue NaChBac T220, validating its use as a model for studying mechanical modulation of voltage-gated Na channel gating.

    By performing a high-quality single channel recording the authors demonstrated that mechanosensitivity affected the channel Po nor single-channel conductance, and importantly, that the effects of pressure on channel gating were reversible. This is particularly appreciated due to the exceptionally low unitary conductance of the channel makes it exceedingly difficult to obtain this type of result.

    The authors performed kinetic modelling over the single channel recording at different voltages and pressures, using linear gating schemes that contrasted two extreme situations: mechanosensitivity was a property of the voltage sensing transitions - mechanosensitive activation (MSA) - or, alternatively, it was a feature of the voltage-independent step that opens the channel pore - mechanosensitive opening (MSO). The MSO performed much better than MSA model, suggesting that mechanosensitivity arises from conformational changes at channel regions different than the VSD during the pore opening. The latter gains further support with the experiments showing that pressure modulates the D93A - a mutation that stabilizes the VSD in its resting state - resembles wt channels, but is ineffective on the I228G channel, a channel with altered closure.

    Overall, the manuscript presents a nice experimental approach, making use of kinetic analysis in terms that are very accessible to the reader to shed light on a physiologically relevant question.