Striking structural differences between voltage-gated sodium (Nav) channels from prokaryotes (homotetramers) and eukaryotes (asymmetric, four-domain proteins) suggest the likelihood of different molecular mechanisms for common functions. For these two channel families, our data show similar selectivity sequences among alkali cations (relative permeability, Pion/PNa) and asymmetric, bi-ionic reversal potentials when the Na/K gradient is reversed. We performed coordinated experimental and computational studies, respectively, on the prokaryotic Nav channels NaChBac and NavAb. NaChBac shows an “anomalous,” nonmonotonic mole-fraction dependence in the presence of certain sodium–potassium mixtures; to our knowledge, no comparable observation has been reported for eukaryotic Nav channels. NaChBac’s preferential selectivity for sodium is reduced either by partial titration of its highly charged selectivity filter, when extracellular pH is lowered from 7.4 to 5.8, or by perturbation—likely steric—associated with a nominally electro-neutral substitution in the selectivity filter (E191D). Although no single molecular feature or energetic parameter appears to dominate, our atomistic simulations, based on the published NavAb crystal structure, revealed factors that may contribute to the normally observed selectivity for Na over K. These include: (a) a thermodynamic penalty to exchange one K+ for one Na+ in the wild-type (WT) channel, increasing the relative likelihood of Na+ occupying the binding site; (b) a small tendency toward weaker ion binding to the selectivity filter in Na–K mixtures, consistent with the higher conductance observed with both sodium and potassium present; and (c) integrated 1-D potentials of mean force for sodium or potassium movement that show less separation for the less selective E/D mutant than for WT. Overall, tight binding of a single favored ion to the selectivity filter, together with crucial inter-ion interactions within the pore, suggests that prokaryotic Nav channels use a selective strategy more akin to those of eukaryotic calcium and potassium channels than that of eukaryotic Nav channels.

Significance: Voltage-gated proton (Hv1) channels play important roles in various physiological processes, such as the innate immune response. However, the mechanism by which this channel closes and opens its proton permeation pathways is unknown, due to the lack of structural information about the closed and open states of the channel. This study uses both simulation and experimental approaches to develop models of the closed and open states of the Hv1 channel. These models suggest a mechanism for how the channel closes and opens. The models also suggest a mechanism explaining why a blocker only binds to the open state of the channel. These structural models will be essential for future investigations of this channel and the development of new pharmacological blockers.

The differential modulation of agonist and antagonist binding to opioid receptors (ORs) by sodium (Na⁺) has been known for decades. To shed light on the molecular determinants, thermodynamics, and kinetics of Na⁺ translocation through the μ-OR (MOR), we used a multi-ensemble Markov model framework combining equilibrium and non-equilibrium atomistic molecular dynamics simulations of Na⁺ binding to MOR active or inactive crystal structures embedded in an explicit lipid bilayer. We identify an energetically favorable, continuous ion pathway through the MOR active conformation only, and provide, for the first time: i) estimates of the energy differences and required timescales of Na⁺ translocation in inactive and active MORs, ii) estimates of Na⁺-induced changes to agonist binding validated by radioligand measurements, and iii) testable hypotheses of molecular determinants and correlated motions involved in this translocation, which are likely to play a key role in MOR signaling. Notwithstanding years of research supporting the notion that μ-opioid receptor (MOR) function can be modulated by sodium ions (Na⁺), a complete understanding of Na⁺ translocation through the receptor and its effect on ligand binding at MOR requires additional information. Here, we use computer simulations to elucidate the energetics involved in sodium binding at inactive and active MOR, the timescales of sodium translocation through these receptor conformations, and the molecular determinants involved in this process.