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

Igor A Khovanov,Stephen K Roberts,Olena A Fedorenko,Carlo Guardiani,P Mark Rodger

doi:10.1021/acs.jctc.6b01035

Igor A Khovanov, Stephen K Roberts + Show 3 more

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https://doi.org/10.1021/acs.jctc.6b01035

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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.

Highlights

Multicellular organisms rely on voltage-dependent cation channels for the onset and rapid propagation of electrical signals triggering key cellular events such as muscle contraction or neurosecretion
We investigated ion transport across the NaChBac channel by means of molecular dynamics simulations
The potential of mean force computed from the unbiased run showed the existence of four main binding sites, three of which corresponded to sites IN, CEN, and HFS that were predicted by Catterall and co-workers[8] based on their analysis of the NavAb crystal structure

Summary

Introduction

Multicellular organisms rely on voltage-dependent cation channels for the onset and rapid propagation of electrical signals triggering key cellular events such as muscle contraction or neurosecretion. Eukaryotic voltage-gated sodium and calcium channels are large monomeric proteins consisting of four homologous domains each containing a voltage-sensor subdomain encompassing helices S1−S4 and a pore subdomain comprizing helices S5−S6 and the intervening.[1,2]. The importance of these channels is testified by the wide range of channelopathies arising from function impairing mutations or autoimmune reactions. The first sodium channel to be identified, NaChBac (Figure 1) from Bacillus halodurans,[5] is a homotetrameric protein whereby each subunit shows the same structural organization of the domains of eukaryotic Navs. Site-directed mutation experiments[6] showed that serine to aspartate mutations in the TLESWAS sequence make the channel calcium selective

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