Sodium Binding Sites and Permeation Mechanism in the NaChBac Channel: A Molecular Dynamics Study

Abstract

NaChBac was the first discovered bacterial sodium voltage-dependent channel, yet computational studies are still limited due to the lack of a crystal structure. In this work, a pore-only construct built using the NavMs template was investigated using unbiased molecular dynamics and metadynamics. The potential of mean force (PMF) from the unbiased run features four minima, three of which correspond to sites IN, CEN, and HFS discovered in NavAb. During the run, the selectivity filter (SF) is spontaneously occupied by two ions, and frequent access of a third one is often observed. In the innermost sites IN and CEN, Na is fully hydrated by six water molecules and occupies an on-axis position. In site HFS sodium interacts with a glutamate and a serine from the same subunit and is forced to adopt an off-axis placement. Metadynamics simulations biasing one and two ions show an energy barrier in the SF that prevents single-ion permeation. An analysis of the permeation mechanism was performed both computing minimum energy paths in the axial-axial PMF and through a combination of Markov state modeling and transition path theory. Both approaches reveal a knock-on mechanism involving at least two but possibly three ions. The currents predicted from the unbiased simulation using linear response theory are in excellent agreement with single-channel patch-clamp recordings.

On the selectivity of the NaChBac channel: an integrated computational and experimental analysis of sodium and calcium permeation

“…In order to gain a molecular level insight into these differences, we performed long timescale MD equilibrium simulations of the NaChBac system bathed in a CaCl 2 solution following the same protocol employed to study the channel in the presence of NaCl. 22 Similar to that reported for the simulation in the NaCl solution, water molecules permeate spontaneously in the channel accessing from both the lower and the upper bath. The number of water molecules in the cavity of the channel in the axial range À20 o z o 10 Å increases during the first 20 ns of the simulation and thereafter oscillates around a plateau level of about 80 water molecules.…”

“…The details of the model building as well as of the simulations in 0.5 M NaCl are provided elsewhere. 22 The protocol for the simulations in 0.5 M CaCl 2 was as follows. The NaChBac homology model was embedded in a bilayer comprising 248 molecules of 1-palmitoyl-2-oleoylsn-glycero-3-phosphocholine (POPC) and solvated by 15 712 water molecules.…”

“…Further details on the equilibration protocol are provided in ref. 22 and the values of the force constants used in the six stages can be found in Table S1 of the ESI. ‡ The production run was carried out in the isothermal isobaric (NPT) ensemble for 105 ns.…”

Ionic Coulomb blockade and anomalous mole fraction effect in the NaChBac bacterial ion channel and its charge-varied mutants

“…We take both the water and the protein to be homogeneous continua describable by relative permittivities e w ¼ 80 and e p = 2, respectively, together with an implicit model of ion hydration whose validity is discussed elsewhere [11]. These model parameters are assumed to be appropriate for the NaChBac channel, both for the wild type and for its mutants [23]. Of course, our reduced model represents a significant simplification of the actual electrostatics and dynamics of ions and water molecules within the narrow selectivity filter due to, for example: the application of continuum electrostatics; the use of the implicit solvent model; and the assumption of 1D (i.e., single-file) movement of ions inside the selectivity filter.…”

Effect of local binding on stochastic transport in ion channels

“…The effect of local binding on conduction and occupancy (analytic result (9)) is found to agree with Brownian dynamics simulations, thus putatively accounting for how the position of the resonant point M 0 is influenced by the radius of the Glutamate/Aspartate ring in NaChBac channels and their mutants [18], [23], [24].…”