New Single-Molecule Patch-Clamp FRET Assay


The single channel patch clamp technique is a powerful tool that provides a direct measure of ion channel function in living cells. When the single channel technique is combined with fluorescence techniques (particularly FRET microscopy technology), it becomes a more powerful tool to study single molecules, and in which conformational domain (ie open-intermediate-close state) they bind. Conformational changes play a critical role in the activation, deactivation, and open–closed activities of ion channels and conformational dynamics is often extremely difficult to be directly characterized by spectroscopic imaging or by single channel patch-clamp alone.  In a recent publication in journal of American Chemistry Society (JACS) by Sasmal & Lu (2016), they have developed a new and combined technical approach, single-molecule patch-clamp FRET microscopy (FRET anisotropy microscopy), to probe ion channel conformational dynamics in living cell by simultaneous and correlated measurements of real-time single-molecule FRET spectroscopic imaging. They used the ligand gated ion channel NMDA to investigate how NMDA and glycine ligands bind to the binding domain. The method is capable of simultaneously recording single-molecule FRET efficiency and conformational changes of single NMDA receptor channels. This allows accurate characterisation of ligand binding events (binding and unbinding of ligands) in direct relation to changes in the receptor/channel conformation.

Schematic diagram showing the FRET microscopy technique – this is able to record single channel events and four channel fluorescence measurements simultaneously allowing correlation of: 1. electrically on and off state by patch clamp, 2. optically determined conformational close and open state by FRET and, 3. binding and unbinding state of glycine ligand by anisotropy measurement at the LBD of GluN1 subunit.

A                                                                                                     B.

a

image-a-b

 

J Am Chem Soc. 2016 Jul 20;138(28):8789-801.

The report provides new insight into some of the functional states of NMDA receptors, in this case recombinantly expressed in HEK-293 cells. Specifically the authors shows that seemingly identical electrically off states (ie channel is closed with no ions flowing) are associated with multiple conformational states. On the basis of experimental results, they have proposed a multistate clamshell model to interpret the NMDA receptor open–close dynamics (Diagram B). Thus single molecule FRET technique is a powerful and sensitive approach for probing the conformational intermediate state of protein and ion channel receptors.

C                                                                                              D

c

J Am Chem Soc. 2016 Jul 20;138(28):8789-801.

In conclusion, this study has demonstrated that binding kinetics as well as ion channel conformation dynamics and the method has the ability to detect the intermediate states in addition to electrical on and off states.

Reference:

 1) Sasmal DK1Yadav R1Lu HP1 (2016) J Am Chem Soc. 2016 Jul 20; 138(28):8789-801. doi: 10.1021/ jacs.6b03496. Epub 2016 Jul 12.