In Vitro Single-Molecule Study of Nachbac using Planar Lipid Bilayer Device

Abstract

Voltage-gated sodium channels (Navs) are essential component for the generation and propagation of electric signals in excitable cells. The successes in the biochemical, biophysical and crystallographic studies on prokaryotic Nav in recent years has greatly promote the understanding of the molecular mechanism underlie these proteins and their eukaryotic counterparts. In this paper, we aim to investigate the conductance and ionic selectivity of the prokaryotic Nav NaChBac at single-molecule level. Purified NaChBac protein was first reconstituted into lipid vesicles to form proteoliposome, which is subsequently incorporated into lipid bilayer with known lipid composition by proteoliposome fusion and studied using a planar bilayer device. We were able to insert a single NaChBac into the bilayer by using a high lipid-to-protein ratio for the proteoliposome preparation. At single-molecule level, we observed three distinct conductance sub-states, 26 ± 6 pS (mean ± SEM, n=4), 92 ± 18 pS (n=3), and 268 ± 45 pS (n=2), respectively. The first two values are comparable to previous reported ones that were obtained by single-channel patch-clamp and multi-channel recording with planar bilayer device, respectively. But the third conductance sub-state is reported for the first time. In addition, our data at single-molecule recording shows similar permeability among Na+, K+ and Ca2+, indicating that the reconstituted NaChBac is non-selective in the artificial membrane environment. Study of NaChBac at single-molecule level in an artificial environment reveals new properties that were not observable in in vivo studies. Our results provide novel insight to understanding the biophysical properties of Navs.