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.
Highlights
Voltage-gated Na+- selective (Nav) and K+- selective (Kv) ion channels provide the molecular pathways for most ion current flow across cell membranes during electrical activity in biological systems [1]
The bacterial Nav channels are composed of four polypeptide subunits, similar to Kv channels, whereas mammalian Nav channels are composed of a single polypeptide
Molecular dynamics simulations of ion permeation through bacterial Nav channels have predicted unperturbed-free-energy barrier differences for permeation of Na+ and K+ of 1–3 kcal/mol that are consistent with estimates for the PNa/PK permeability ratio of 20–40 obtained from experimental values for reversal potentials obtained for eukaryotic Nav channels under bi-ionic conditions[11,12,13,14,15,16]
Summary
Voltage-gated Na+- selective (Nav) and K+- selective (Kv) ion channels provide the molecular pathways for most ion current flow across cell membranes during electrical activity in biological systems [1]. Finol-Urdaneta et al, for example, have observed that in the presence of 140 mM intracellular Na+ and 140 mM extracellular K+, the NaChBac channel is strongly outwardly rectifying for K+, with a reversal potential for K+ current obtained by extrapolation from the linear portion of the I-V plot approximately 40 mV less negative than the actual value of the reversal potential obtained from instantaneous I-V data [17] These investigators observed that the PK/PNa permeability ratio differed by a factor of 10 with oppositely directed Na+/K+ gradients, which they characterized as a functional asymmetry of the channel. These experimental results show that potassium ions are prevented from entering the selectivity filter from the extracellular side of the membrane, and that transmembrane voltages of 78–128 mV are needed to overcome the block of potassium ion current
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