Catalysis of Na ⁺ permeation in the bacterial sodium channel Na _V Ab

Abstract

Determination of a high-resolution 3D structure of voltage-gated sodium channel Na V Ab 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 V Ab 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) × 10 6 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, liquidlike 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.T he 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, voltagegated 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 V Ab.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 V Ab, wa...

Ion conduction and conformational flexibility of a bacterial voltage-gated sodium channel

“…With its wide pore and Glulined SF, subject to the rotational isomerizations, the Na v Ab pore displays more flexibility than that familiar in K + channels. We have observed large movements of Glu side chains, which are closely correlated with ion translocation through the SF (Movie S1 and Movie S2), as also described in a recent study (23). Fig.…”

“…1B) is in a conducting state, allowing for studies of multiple ion movements. We examine the effects of EEEE protonation and reveal extensive protein fluctuations, unseen in previous simulations on shorter timescales (11,(20)(21)(22), but suggested to play a role in a recent study (23). We will demonstrate that protein fluctuations away from the published crystal structure are critical for describing ion conduction mechanisms.…”

A structural, functional, and computational analysis suggests pore flexibility as the base for the poor selectivity of CNG channels

“…The rotameric change in conformation of the Glu66 side chains herein observed is reminiscent of the mobility recently reported for Na + bacterial channels, where the conformational isomerization of a ring of four glutamate side chains lining the selectivity filter is coupled to ionic coordination (24,25,48). We propose that this flexibility, together with the number of equivalent and contiguous ion binding sites in the filter (26,34,49), underscores the poor ionic selectivity of CNG channels and reveals a conduction mode that differs substantially from that of classical K + channels, which are highly selective and have a fairly rigid molecular structure.…”

“…We demonstrate that the extracellular entrance of the selectivity filter and the filter itself exhibit a dynamic structure capable of structural rearrangements, which can be partially captured by X-ray crystallography and are best visualized and understood through MD simulations. Our results indicate that the pore of CNG channels is highly flexible, with a liquidlike energy landscape (24,25). This flexibility underlies the poor selectivity of CNG channels and their strong coupling between gating and permeation.…”

Molecular basis of ion permeability in a voltage‐gated sodium channel

“…The resolution of the first such structure (3.5 Å), however, was too low to determine the details of the sites of the ions in the SF, whilst the latter structures contained channel blocker ligands which interfered with ion binding. The sodium ion binding site locations and mechanisms for ion selectivity have also been predicted by a number of molecular dynamics (MD) studies, although they differ considerably in their details (Amaral et al , 2012; Chakrabarti et al , 2013; Stock et al , 2013; Ulmschneider et al , 2013; Xia et al , 2013; Zhang et al , 2013; Boiteux et al , 2014; Furini et al , 2014). …”