X-ray and Neutron Interferometry Studies of Anesthetic Interactions with Voltage

University Of Pennsylvania, Philadelphia PA

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Abstract

In Project 5, we will investigate how anesthetic binding perturbs the mechanism of electromechanical coupling in anesthetic-sensitive, prototypical voltage-gated sodium and potassium channels. Aim 1 concerns the prokaryotic Nav-channels NaChBac and NavAb possessing a dominant transmembrane domain. Aim 2 concerns the eul<aryotic Shaw2 Kv-channel expressed to contain only its transmembrane domain, as well as full-length Shaw 2 and its chimeras with Kv1.2, in collaboration with Project 2 (Covarrubias). Synchrotron xray & neutron interferometry techniques will be applied to single, reconstituted phospholipid membranes containing the vectorially-oriented Nav-channel or Kv-channel protein within an electrochemical cell to probe the profile structure ofthe channel as a function ofthe applied transmembrane electric potential (voltage). The x-ray interferometry experiments will be time-resolved with serial time-frames of less than -1ms each, providing 10-30 frames over the channel's kinetic response to the depolarizing step-wise change in the potential, and thereby sensitive to changes in the profile structure ofthe channel on the physiologically relevant time-scales of channel activation separated from subsequent pore opening. The neutron interferometry experiments will be either steady-state at each potential, or partially time-resolved with serial time-frames of 16ms each, sampling only the first and second halves ofthe channel's response to the depolarizing step-wise change in the potential. The x-ray interferometry experiments will also determine the localization of anesthetic binding sites and their occupancy within the channel's profile structure, utilizng the heavy halogen atoms of several prototypical volatile anesthetics. The spatial resolution ofthe structural studies will be substantially enhanced through site-directed labeling, with heavy resonant atoms in the x-ray case and deuterium in the neutron case, to achieve a the positional accuracy of better than 1 A. These studies should provide an experimental structural basis for understanding how anesthetic binding alters the mechanism of electromechanical coupling, for comparison with the theoretical predictions from the molecular dynamics computer simulations of Project 4.