Simplified Bacterial “Pore” Channel Provides Insight into the Assembly, Stability, and Structure of Sodium Channels

Abstract

Eukaryotic sodium channels are important membrane proteins involved in ion permeation, homeostasis, and electrical signaling. They are long, multidomain proteins that do not express well in heterologous systems, and hence, structure/function and biochemical studies on purified sodium channel proteins have been limited. Bacteria produce smaller, homologous tetrameric single domain channels specific for the conductance of sodium ions. They consist of N-terminal voltage sensor and C-terminal pore subdomains. We designed a functional pore-only channel consisting of the final two transmembrane helices, the intervening P-region, and the C-terminal extramembranous region of the sodium channel from the marine bacterium Silicibacter pomeroyi. This sodium “pore” channel forms a tetrameric, folded structure that is capable of supporting sodium flux in phospholipid vesicles. The pore-only channel is more thermally stable than its full-length counterpart, suggesting that the voltage sensor subdomain may destabilize the full-length channel. The pore subdomains can assemble, fold, and function independently from the voltage sensor and exhibit similar ligand-blocking characteristics as the intact channel. The availability of this simple pore-only construct should enable high-level expression for the testing of potential new ligands and enhance our understanding of the structural features that govern sodium selectivity and permeability.

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

“…Functional studies on wild‐type and mutant prokaryotic channels (Ren et al , 2001; Yue et al , 2002; McCusker et al , 2011; Shaya et al , 2011; Tang et al , 2014) have suggested which regions and residues may be important for ion binding, selectivity and translocation within the SF. However, the detailed locations and environments surrounding the sodium ions within the channels have not been discernable from the crystal structures published to date.…”

Bacterial Voltage-Gated Sodium Channels (BacNaVs) from the Soil, Sea, and Salt Lakes Enlighten Molecular Mechanisms of Electrical Signaling and Pharmacology in the Brain and Heart

“…Rather than having the twenty-four transmembrane segment architecture of eukaryotic Na V s, BacNa V s are built from a six transmembrane segment architecture comprising a VSD and PD (Fig. 1b) that assembles into homotetramers 34; 35; 36; 37; 38; 39 in a manner similar to many voltage-gated potassium channels 1 . Initial studies demonstrated that BacNa V s had an ion selectivity profile that was similar to Na V s 29; 40 , even though the actual selectivity BacNa V selectivity filter sequence has more in common with those from Ca V s than Na V s 29; 33; 41 (Fig.…”

Structure of the C-terminal domain of the prokaryotic sodium channel orthologue NsvBa

“…This aberrant running of full length constructs has also been observed for other orthologues (e.g. NaChBac, NavSp) (Nurani et al, 2008;McCusker et al, 2011).…”