High-speed AFM imaging of voltage gated sodium channel NaChBac and voltage application.

Abstract

High-speed atomic force microscopy (HS-AFM) has captured various biomolecular actions in physiological conditions. On membrane proteins, HS-AFM can reveal sub-nanometer conformational changes such as the movement of the E-F loop in bacteriorhodopsin upon the green light stimulus, and using HS-AFM line scanning (HS-AFM-LS) these molecular motions were characterized at millisecond temporal resolution. In addition, with the recent development of localization AFM (LAFM), Angstrom-range structural resolution can be extracted from HS-AFM data. With these recent progresses in HS-AFM, the study of the voltage sensor domain (VSD) movements in voltage-gated ion channels is within reach. The VSD is a well-conserved bundle of four transmembrane helices which responds to transmembrane voltage by moving along the electric field and induces pore opening of the voltage gated ion channels. Unfortunately, to date no direct experimental visualization of the VSD movements has been provided. Here, we develop novel high-speed AFM methodologies to apply voltage across supported lipid bilayers to study VSD movements upon voltage stimulus. We have applied this novel approach to the bacterial voltage gated sodium channels (NaChBac). NaChBac has no additional extra-membraneous domain, thus giving the HS-AFM tip access to probe the VSD and pore domains. Here we discuss our first results of high-resolution imaging of NaChBac and its response to voltage applications.