Although x-ray crystal structures exist for several voltage-gated ion channel (VGIC) proteins, the mechanism of coupling voltage dependent conformational changes in the voltage sensor domains to opening and closing the pore domain remains controversial.  This situation persists because the usual physical techniques for protein structure determination cannot access the dependence of their structures on a transmembrane voltage.  As a result, less direct experimental approaches and sophisticated computational techniques have been utilized to develop consensus models of the voltage-dependent conformations responsible for electromechanical coupling in VGIC proteins.  Alternatively, we have developed two methods for vectorially-orienting VGIC proteins within single phospholipid bilayer membranes at the solid-liquid interface within electrochemical cells designed for the investigation of the scattering-length density (SLD) profile structures of these membranes by x-ray and neutron reflectivity.  The spatial resolution, or sensitivity to detail within the profile structure, can be dramatically enhanced using interferometric techniques.  We have employed time-resolved “pump-probe” techniques to investigate changes in both the x-ray and neutron SLD profile structures of the isolated voltage-sensor domain of a voltage-gated potassium channel as a function of the applied transmembrane voltage.  The changes in each of these two independent SLD profile structures were found to be consistent with molecular dynamics simulations of the voltage sensor domain within a fully-hydrated phospholipid bilayer investigated as a function of the transmembrane voltage.  This agreement allowed us to relate the voltage-dependent changes in the SLD profile structure to the conformational changes in the 3-D atomic level structure of the voltage sensor.  These approaches are now being applied to the complete homotetrameric VGIC proteins wherein each subunit contains a voltage sensor and ¼ of the pore domain.  This is more complicated because unlike their isolated voltage sensor domains, the complete VGIC proteins typically exhibit inactivation upon prolonged exposure to depolarizing voltages, with respect to the resting transmembrane voltage, which requires the cyclic application of polarizing and depolarizing voltages of much shorter duration on the order of milliseconds.  Our goal is to understand how anesthetics interfere with electromechanical coupling.

Investigating the mechanism of electromechanical coupling in voltage-gated ion channels by time-resolved x-ray & neutron interferometry

251st National Meeting (2016)

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