Structural Characterization of the Voltage Sensor Domain of the KvAP Channel Vectorially-Oriented within a Phospholipid Bilayer Membrane

Voltage-gated cation (Na+, K+) channels are responsible for the generation and propagation of action potentials in neurological signal transmission. Kv-channels are transmembrane proteins consisting of a homo-tetramer of 4 subunits that assemble about a 4-fold axis normal to the membrane plane to form the K+ ion-selective pore. Each of the four subunits is comprised of six transmembrane helices, the S1-S4 helices forming the voltage-sensor domain (VSD) and the S5-S6 helices contributing to form the pore domain (PD). Despite several advances in the field, a complete understanding of the mechanism of electromechanical coupling interconverting the closed-to-open states is yet to be achieved. Positively charged arginine residues predominately in the S4 helix of the VSD are responsible for voltage sensing and the VSD's are arranged around the periphery of the PD in extensive contact with the lipid bilayer. This prompted us to focus initially on the structure of VSD itself within a phospholipid bilayer environment for the present study. A hydrated, phospholipid bilayer membrane environment has been reconstituted for the VSD of KvAP, vectorially oriented on the surface of inorganic multilayer substrates. This has been established by X-ray and neutron reflectivity (enhanced by interferometry), the latter employing a specifically deuterated phospholipid and water contrast variation, for the reconstituted membrane at both the solid-vapor and solid-liquid interfaces. This accomplishment now allows an investigation of the profile structure of the VSD within the lipid bilayer as a function of the applied transmembrane electric potential via x-ray reflectivity with millisecond time-resolution, employing high energy x-rays (> 20KeV) & pixel array detectors, and neutron reflectivity, employing selectively deuterium-labeled VSD proteins achieved via semi-synthesis. The same approach can be extended to the intact KvAP channel.