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

Carlo Guardiani,Olena A Fedorenko,Stephen K Roberts,Igor A Khovanov

doi:10.1039/c7cp05928k

Abstract

Ion channel selectivity is essential for their function, yet the molecular basis of a channel's ability to select between ions is still rather controversial. In this work, using a combination of molecular dynamics simulations and electrophysiological current measurements we analyze the ability of the NaChBac channel to discriminate between calcium and sodium. Our simulations show that a single calcium ion can access the Selectivity Filter (SF) interacting so strongly with the glutamate ring so as to remain blocked inside. This is consistent with the tiny calcium currents recorded in our patch-clamp experiments. Two reasons explain this scenario. The first is the higher free energy of ion/SF binding of Ca2+ with respect to Na+. The second is the strong electrostatic repulsion exerted by the resident ion that turns back a second potentially incoming Ca2+, preventing the knock-on permeation mechanism. Finally, we analyzed the possibility of the Anomalous Mole Fraction Effect (AMFE), i.e. the ability of micromolar Ca2+ concentrations to block Na+ currents. Current measurements in Na+/Ca2+ mixed solutions excluded the AMFE, in agreement with metadynamics simulations showing the ability of a sodium ion to by-pass and partially displace the resident calcium. Our work supports a new scenario for Na+/Ca2+ selectivity in the bacterial sodium channel, challenging the traditional notion of an exclusion mechanism strictly confining Ca2+ ions outside the channel.

Highlights

Voltage gated sodium channels (Navs) play a key role in the onset and propagation of electric signals in excitable cells due to their involvement in the rising branch of the action potential.[1]
In NaChBac, the first discovered bacterial sodium channel identified in Bacillus halodurans,[8] as well as in other prokaryotic Navs, a further link with calcium channels is at the level of the Selectivity Filter (SF) sequence that conforms to the pattern FxxxTxExW typical of eukaryotic calcium channels
Current–voltage data were collected by recording responses to a consecutive series of step pulses from a holding potential of À100 mV at intervals of À15 mV beginning at +95 mV in SBSNa followed by perfusion with a bath solution in which Na+ was completely replaced with 100 mM Ca2+ (SBS-Ca) or Cs+ (SBS-Cs)

Summary

Introduction

Voltage gated sodium channels (Navs) play a key role in the onset and propagation of electric signals in excitable cells due to their involvement in the rising branch of the action potential.[1] Their physiological importance makes them potential targets for a wide range of drugs including anticonvulsants, antiarrhythmics and local anaesthetics.[2,3]. Complexity, so far it has not been possible to attain high resolution crystal structures of eukaryotic Navs. This is why the smaller prokaryotic Navs (Fig. 1) represent amenable model systems for simulation and experimental studies. Mutagenesis experiments on NaChBac showed that serine to aspartate mutations switch the channel from sodium selective to calcium selective.[9]

Methods

Results

Conclusion