G Protein-Coupled Receptors (GPCRs) are a very large, diverse family of transmembrane receptors in eukaryotes. These receptors detect molecules outside the cell and activate internal signaling pathways by coupling with G proteins. Once a GPCR is activated, β-arrestins translocate to the cell membrane and bind to the occupied receptor, uncoupling it from G proteins and promoting its internalization.
Reporter tags are useful for studying the dynamics of GPCRs and associated proteins, but large tags can disrupt the receptors’ native functioning, and often overexpression of the tagged protein is required to obtain sufficient signal. Here is one example of how researchers have used the small, bright NanoLuc® luciferase to overcome these common challenges and answer questions about GPCRs. Continue reading “Lighting Up GPCR Research with Bioluminescent Tagging”
I confess that I struggled through biophysics, and my Bertil Hille textbook Ion Channels of Excitable Membranes lies neglected somewhere in a box in my basement (I have not tossed it into the recycle bin—I can’t bear too, I spent too much time bonding with that book in graduate school).
My struggles in that graduate class and my attendance at the seminars of my grad school colleagues who were conducting electrophysiological studies left me with a sincere awe and appreciation of both the genius and the artistry required to produce nice electrophysiology data. The people who are good at these experiments are artists—they have the golden touch when it comes to generating that megaohm seal between a piece of cell membrane and a finely pulled glass pipette. And, they are brilliant scientists, they really understand the physics, the chemistry and the biology of the cells they study from a perspective that very few scientists ever develop.
Electrophysiology data, which often demonstrate the gating of a single channel protein in response to a single stimulus in real time–ions crossing a membrane through a single protein–are amazing for their ability, unlike virtually any other experimental data for the story they can tell about what is going on in a cell in real time under physiological conditions.
When constructs were ectopically expressed in HEK 293T/17 cells, we obtained very similar kinetics for the GPCR-driven responses between NanoBRET™ biosensors and the patch clamp recordings.
Indeed, the activation rates that we observed were very similar to those of GPCR-stimulated GIRKs [G protein-coupled, inwardly rectifying K+ channel] in native cells, suggesting that the conditions of this assay closely match the in vivo setting. This finding further demonstrates the ability of the system to resolve the fast, physiological relevant kinetics of GPCR signaling.