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: the 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 in Na+ and a modest increase in the 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.
Highlights
Voltage-gated sodium and calcium channels (NaVs and CaVs, respectively) are involved in a multitude of processes, including electrical signaling, secretion, and synaptic transmission [1].The malfunction or dysregulation of NaVs and CaVs leads to a wide range of neurological, cardiovascular, and muscular disorders, including periodic paralysis [2], arrhythmia [3], and epilepsy [4], which highlights the importance of these molecules.Eukaryotic NaVs and CaVs have similar structure and comprise a pore-forming α1 subunit of approximately 190–250 kDa, which co-assembles with a number of auxiliary subunits
The different behavior of the two species is reflected in the Potential of Mean Force (PMF) profile (Figure 3a,b), which is characterized by a single deep minimum centered at z = 4–5 Å for WT NavMs, and a minimum split into three sub-basins at z = 2.0 Å, z = 5.0 Å, and z = 8–9 Å, corresponding to three different binding regions, for the Qf = −5e mutant
The barriers between the sub-basins are in the order of 1–2 kcal/mol and can be overcome at the simulation temperature, yet the sodium ions linger in each binding site for longer than they would in case of a uniform probability distribution of occupancy
Summary
Voltage-gated sodium and calcium channels (NaVs and CaVs, respectively) are involved in a multitude of processes, including electrical signaling, secretion, and synaptic transmission [1].The malfunction or dysregulation of NaVs and CaVs leads to a wide range of neurological, cardiovascular, and muscular disorders, including periodic paralysis [2], arrhythmia [3], and epilepsy [4], which highlights the importance of these molecules.Eukaryotic NaVs and CaVs have similar structure and comprise a pore-forming α1 subunit of approximately 190–250 kDa, which co-assembles with a number of auxiliary subunits. Voltage-gated sodium and calcium channels (NaVs and CaVs, respectively) are involved in a multitude of processes, including electrical signaling, secretion, and synaptic transmission [1]. The malfunction or dysregulation of NaVs and CaVs leads to a wide range of neurological, cardiovascular, and muscular disorders, including periodic paralysis [2], arrhythmia [3], and epilepsy [4], which highlights the importance of these molecules. Eukaryotic NaVs and CaVs have similar structure and comprise a pore-forming α1 subunit of approximately 190–250 kDa, which co-assembles with a number of auxiliary subunits. The atomic level resolution of the structure of these molecules is essential to understand their structure-function relationships, but this task is challenging for eukaryotic NaV channels, due to them being
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
