Revealing Function-Regulating Conformational Dynamics of an Ion Channel by Simultaneous Single-Molecule Fluorescence Imaging and Patch-Clamp Recording
reg Harms, Galya Orr, H. Peter Lu*
Pacific Northwest National Lab, US
Keywords: IOn Channel, Single-molecule spectroscopy and imaging, patch-clamp recording
By combining real-time single-molecule fluorescence imaging measurements with real-time single-channel current measurements in membranes of lipid bilayers or in living cells, we are now able to probe single ion-channel-protein conformational changes simultaneously correlated with single ion-channel current trajectories, providing an understanding at the molecular-level of the dynamics and mechanisms of ion-channel proteins. This technical innovation has been used to gain an understanding of how ion-channel activities are regulated by conformational change dynamics and assembly mechanisms of the dye-labeled gramicidin channels, which has revealed that the gramicidin channel activity is regulated by complex gramicidin dimer conformational changes. A new multiple-state model for gramicidin ion-channel dynamics, more complex than the classic two-state diffusion model, has been postulated based on our experimental results. The single-channel activity of recombinant NMDA receptors transiently expressed in a mammalian cell-line has been recorded in real-time, which begins to shed light on the molecular-level understanding of the ligand-receptor interaction dynamics in NMDA receptors in living cells.
NSTI Nanotech 2003 Conference Technical Program Abstract