K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Nav Channels

Abstract

Crystal structures of several bacterial Nav channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Nav channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial NavAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Nav channels. The non-equilibrium simulations indicate that two or three extracellular K+ ions can block the entrance to the selectivity filter of NavAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be ‘locked’ in place by modest applied forces. In contrast to K+, three Na+ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na+ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K+ block is equivalent to large applied potentials experimentally measured for two bacterial Nav channels to induce inward currents of K+ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.

Bases of Bacterial Sodium Channel Selectivity Among Organic Cations

“…It is important to stress that virtually all the studied ions show binding wells in calculated 1D PMF near or at the locations marked by E/D-191 (WT and E/D, respectively). This finding is in excellent agreement with the results of equilibrium simulations and previous studies of Na + and K + binding to Nav SF 17,19 . 1D PMF decomposition from ion-protein interactions (Figs S1A and S2A) shows marked differences in cation interactions near the main SF binding site between WT and D191 mutants.…”

“…The analysis of equilibrium occupancies of NH 4 + shows almost no in-plane binding mode for this cation. The Hz + binding pocket organization near E191 is reminiscent of what is found for single K + binding to the selectivity filter of NavAb 17,30 . The binding site for Hz + was notably shifted compared to the one observed for Na + and is reminiscent to the binding pocket mapped for K + in previously reported 1D PMF calculations 19,30 .…”

“…In the Cryo-EM structure of the eukaryotic (putative) Nav channel (NavPaS) 12 from the American cockroach ( Periplaneta americana ), the DEKA selectivity filter is a few Å more constricted than its prokaryotic counterparts, nevertheless it is larger than Hille’s estimates for the frog node Nav SF. Despite of a relatively large selectivity filter, the sidechains of the acidic and lysine residues can stretch out 4–6 Å away from the backbone and appear to be sufficiently dynamic to reach out to either the opposite or proximal sidechains, effectively decreasing the cross-sectional area of the filter 17,18 , thus dynamically lowering ionic flux. In particular, movements of the glutamate sidechains appear to be able to create an efficient blockage by potassium ions 17,19,20 directly supporting mechanisms proposed by French and collaborators in 1994 21 .…”

Ion-triggered selectivity in bacterial sodium channels

“…The transitions between metastable states observed in trajectories with K + might be the result of a direct effect of the positive membrane potential on the glutamate residues or of ion movements in the outward direction. The second hypothesis is supported by steered MD simulations, which showed that the selectivity filter of bacterial Na + channels behaves asymmetrically for inward and outward fluxes, even at null membrane potential (23). The quantitative comparison with experimental data requires a throughout understanding of the individual contributions of the direction of ion fluxes and of the membrane potential to selectivity, and this is not possible with the simulation protocol employed in the present study.…”

Molecular Dynamics of Ion Conduction through the Selectivity Filter of the NaVAb Sodium Channel

“…This situation allows an ion to pass-by another, an action previously observed in several simulation studies. 42–44, 82 This is often described as a mechanism by which one ion can pass-by another to exit the selectivity filter first. However, because these events are energetically equivalent, repeated ion-ion passing are unproductive, or “redundant”, in contrast with the progressive movements of ions through the pore according to a single file knock-on mechanism.…”