NMDA Receptor Ion Channel Dynamics in Living Cells by a Novel Single-Molecule Patch-Clamp FRET Microscopy: Revealing the Multiple Conformational States Associated with a Channel at its Electrical Off State

Abstract

Stochastic and inhomogeneous conformational changes regulate the function and dynamics of ion channels. The 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 combined approaches of using single ion channel patch-clamp electrical recording and single-molecule fluorescence imaging for probing ion channel conformational changes simultaneously with the electrical single channel recording. We were able to probe single ion-channel-protein conformational changes simultaneously with the electric on-off signals, and thus providing an understanding the dynamics and mechanism of ion-channel proteins at the molecular level.(1,2) 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. Based on our experimental results, we have proposed a multistate clamshell model to interpret the NMDA receptor open-close dynamics. Our results shed light on new perspectives of the intrinsic interplay of lipid membrane dynamics, solvation dynamics, and the ion channel functions.Reference:1. Dibyendu Kumar Sasmal, H. Peter Lu, “ 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,” J. Am. Chem. Soc., 136, 12998-13005 (2014).2. Suneth P. Rajapaksha, Xuefei Wang, H. Peter Lu, “Suspended Lipid Bilayer for Optical and Electrical measurements of Single Ion Channel Proteins,” Anal. Chem., 85, 8951-8955 (2013).