Single-Molecule Patch-Clamp FRET Imaging Microscopy in Living Cells

Bowling Green State University, Bowling Green OH

Investigators

Linked publications & trials

Abstract

DESCRIPTION (provided by applicant): We propose to use single-molecule spectroscopy imaging simultaneously with single-channel current recording to interrogate the dynamics in the conformational motions of a single ion-channel, to better understand the mechanism of channel function at the cell membranes. To meet a major methodology and technical need and challenge in the medical life sciences, the primary goal of our proposal is to solve a critical problem that holds understanding ion channel protein biological function and dynamics over the last four decades ever since the patch-clamp electrophysiological technique demonstrated. Specifically, we propose to conduct a systematic technical development and demonstration. Our project consists of three primary aims: (1) Achieve an experimental understanding of the sub-unit conformational changes that control the open-close activity of the NMDA receptor; (2) Resolve the conformational changes of the NMDA receptor in desensitization and inactivation; and (3) Demonstrate high time resolution for single-molecule ion channel dynamics studies. Our proposed technical approach, patch-clamp confocal single-molecule fluorescence imaging microscopy, will be applicable for a broad range of ion channel proteins and receptors in various cells, including all of the cellular systems that have been traditionally studied by conventional patch-clamp electric current measurements and proteins that can be probed by fluorescence. We will use the N-methyl-D-aspartate (NMDA) receptor in living cells as a model system to demonstrate our innovative physical technique and its unprecedented applications in life sciences and medical researchers. The dynamics of NMDA receptor mediated calcium currents is crucial to the normal function of the brain. The temporal behavior of NMDA receptor activity is regulated by the dynamic conformational changes of the protein in response to agonist binding and dissociation. Identifying the conformational motions of the receptor is critical for understanding the mechanisms of receptor function. Inhomogeneous conformational motions of the receptor control the activation, inactivation, desensitization and deactivation of the channel that in turn shape the calcium transients.