Catalysis of Na+ permeation in the bacterial sodium channel NaVAb

Determination of a high-resolution 3D structure of voltage-gated sodium channel Na_VAb opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na⁺ through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of Na_VAb in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na⁺ through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na⁺ between the extracellular medium and the central cavity of the channel. Analysis of Na⁺ dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na⁺ ions, with a computed rate of translocation of (6 ± 1) × 10⁶ ions⋅s⁻¹ that is consistent with expectations from electrophysiological studies. The binding of Na⁺ is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na⁺ ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquid-like energy landscape propitious to Na⁺ diffusion. By stabilizing multiple ionic occupancy states while helping Na⁺ ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na⁺ permeation.The rapid passage of cations in and out of excitable cells through selective pathways underlies the generation and regulation of electrical signals in all living organisms (1–4). The metazoan cell membrane is exposed to a high-Na⁺, low-K⁺ concentration on the extracellular (EC) side, and to a low-Na⁺, high-K⁺ concentration on the intracellular (IC) side. Selective voltage-gated Na⁺ and K⁺ channels control the response of the cell to changes in the membrane potential. In particular, voltage-gated Na⁺ channels (Na_V) are responsible for the initiation and propagation of action potentials in cardiac and skeletal myocytes, neurons, and endocrine cells (1–4). Mutations in Na_V channel genes are responsible for a wide range of debilitating channelopathies, including congenital epilepsy, paramyotonia, erythromelalgia, familial hemiplegic migraine, paroxysmal extreme pain disorder, and periodic paralyses (5, 6), underlining the importance of deciphering the relationship between the structure and function of Na_V channels. Here, we use molecular simulations to study the binding and permeation of Na⁺ in bacterial sodium channel Na_VAb.Although several atomic structures of K⁺-selective channels have been solved over the past decade (7–12), the atomic structure of an Na⁺-selective channel from the bacterium Arcobacter butzleri, Na_VAb, was reported only recently (13). In the preopen state of Na_VAb (13), the pore is closed at the IC gate, but the selectivity filter (SF) appears to be in its open, functional state. The molecular structure of the SF of Na_VAb (TLESW) differs significantly from that of potassium channels such as KcsA (TVGYG), in that it is both wider and shorter. In KcsA, channel coordination of permeating cations consists almost entirely of direct interactions with backbone carbonyl oxygen atoms. In contrast, in Na_VAb, the SF is lined with amino acid side chains from S178 and E177 in addition to backbone carbonyl groups from T175 and L176 (7, 8, 10, 13). Due to the tetrameric domain arrangement of Na_VAb, the E177 site forms a ring of four glutamate side chains (EEEE) in the same sequence positions as the characteristic DEKA ring of eukaryotic sodium channels (14, 15). The presence of charged and titratable carboxylate groups in the SF of Na_v channels raises major questions about the catalytic mechanism for ionic permeation and the structural basis for ion selectivity.As a first step toward elucidating the structural basis of ionic permeation and selectivity, we examine the movement of Na⁺ ions in and out of the pore from equilibrium molecular dynamics (MD) simulations of Na_VAb in a hydrated lipid bilayer (Fig. S1). Forty-seven time trajectories totaling 21.6 μs were generated at 300 K in the presence of 150 mM NaCl to mimic the physiological environment of the periplasm. We analyzed Na⁺ diffusion at a potential of 0 mV, similar to the peak of macroscopic Na⁺ current during an action potential or a voltage clamp experiment in nerve or muscle cells. The analysis of hundreds of spontaneous events of Na⁺ diffusion through the SF provides detailed insight into a knock-on mechanism of Na⁺ permeation involving alternating ion-occupancy states and resulting in an estimated translocation rate of (6 ± 1) × 10⁶ ions⋅s⁻¹.