title: Single-molecule patch-clamp FRET microscopy studies of NMDA receptor ion channel dynamics in living cells: revealing the multiple conformational states associated with a channel at its electrical off state.
abstract: Conformational dynamics plays a critical role in the activation, deactivation, and open-close activities of ion channels in living cells. Such conformational dynamics is often inhomogeneous and extremely difficult to be directly characterized by ensemble-averaged spectroscopic imaging or only by single channel patch-clamp electric recording methods. We have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy, to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging with single-channel electric current recording. Our approach is particularly capable of resolving ion channel conformational change rate process when the channel is at its electrically off states and before the ion channel is activated, the so-called "silent time" when the electric current signals are at zero or background. We have probed NMDA (N-methyl-D-aspartate) receptor ion channel in live HEK-293 cell, especially, the single ion channel open-close activity and its associated protein conformational changes simultaneously. Furthermore, we have revealed that the seemingly identical electrically off states are associated with multiple conformational states. On the basis of our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics.
authors.affiliation: Department of Chemistry and Center for Photochemical Sciences, Bowling Green State University , Bowling Green, Ohio 43403, United States.
authors.forename: Dibyendu Kumar
authors.initials: DK
authors.lastname: Sasmal
authors.forename: H Peter
authors.initials: HP
authors.lastname: Lu
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D016194:Receptors, N-Methyl-D-Aspartate / Q000032:analysis / Q000378:metabolism*
chemical_list: D016194:Receptors, N-Methyl-D-Aspartate