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Final published version
Licence: CC BY: Creative Commons Attribution 4.0 International License
Research output: Contribution to Journal/Magazine › Journal article › peer-review
Research output: Contribution to Journal/Magazine › Journal article › peer-review
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TY - JOUR
T1 - Changes in ion selectivity following asymmetrical addition of charge to the selectivity filter of bacterial sodium channels
AU - Fedorenko, Olena
AU - Khovanov, Igor
AU - Roberts, Stephen
AU - Guardiani, Carlo
PY - 2020/12/31
Y1 - 2020/12/31
N2 - Abstract: Voltage-gated sodium channels (NaVs) play fundamental roles in eukaryotes but their exceptional size hinders their structural resolution. Bacterial NaVs are simplified homologues of their eukaryotic counterparts but their use as models of eukaryotic Na+ channels is limited by their homotetrameric structure at odds with the asymmetric Selectivity Filter (SF) of eukaryotic NaVs. This work aims at mimicking the SF of eukaryotic NaVs by engineering radial asymmetry into the SF of bacterial channels. This goal was pursued with two approaches: co-expression of different monomers of the NaChBac bacterial channel to induce the random assembly of heterotetramers, and the concatenation of four bacterial monomers to form a concatemer that can be targeted by site-specific mutagenesis. Patch-clamp measurements and Molecular Dynamics simulations showed that an additional gating charge in the SF leads to a significant increase of Na+ and a modest increase in Ca2+ conductance in the NavMs concatemer in agreement with the behavior of the population of random heterotetramers with the highest proportion of channels with charge −5e. We thus showed that charge, despite being important, is not the only determinant of conduction and selectivity and we created new tools extending the use of bacterial channels as models of eukaryotic counterparts.
AB - Abstract: Voltage-gated sodium channels (NaVs) play fundamental roles in eukaryotes but their exceptional size hinders their structural resolution. Bacterial NaVs are simplified homologues of their eukaryotic counterparts but their use as models of eukaryotic Na+ channels is limited by their homotetrameric structure at odds with the asymmetric Selectivity Filter (SF) of eukaryotic NaVs. This work aims at mimicking the SF of eukaryotic NaVs by engineering radial asymmetry into the SF of bacterial channels. This goal was pursued with two approaches: co-expression of different monomers of the NaChBac bacterial channel to induce the random assembly of heterotetramers, and the concatenation of four bacterial monomers to form a concatemer that can be targeted by site-specific mutagenesis. Patch-clamp measurements and Molecular Dynamics simulations showed that an additional gating charge in the SF leads to a significant increase of Na+ and a modest increase in Ca2+ conductance in the NavMs concatemer in agreement with the behavior of the population of random heterotetramers with the highest proportion of channels with charge −5e. We thus showed that charge, despite being important, is not the only determinant of conduction and selectivity and we created new tools extending the use of bacterial channels as models of eukaryotic counterparts.
U2 - 10.3390/e22121390
DO - 10.3390/e22121390
M3 - Journal article
VL - 22
JO - Entropy
JF - Entropy
SN - 1099-4300
IS - 12
M1 - 998416
ER -