ChR2 - Stability of Retinal binding
We investigated the effects of retinal addition and removal on light-induced ChR2 activity using a prototypical SURFE2R N1 add-on for activation by light.
Background and Assay Description:
The membrane vesicles have been pre-loaded with Na+-containing solution before sensor preparation. After establishing an outward directed Na+ gradient by rinsing the sensor in Na+-free solution, we applied light pulses of 500 ms using LEDs with 470 nm wavelength and 20 mA to activate ChR2.
To increase the signal-to-noise ratio we incubated the sensors in solution containing 100 nM retinal and measured ChR2 activity after different time points. The signal was maximized after 10 minutes. Despite the covalent bond between ChR2 and Retinal, the signal again decays when rinsing the sensor with retinal-free solution, concluding that the stability of bound Retinal is low. We therefore decided to add retinal to all measurement solutions for enhancing the signal-to-noise ratio.
Author: Nanion Technologies and Axxam S.p.a.
Mode of transport: Light-gated Na+ flux
Organism: Chlamydomonas reinhardtii
Sample: Purified membrane vesicles from plasma membrane
Expression: Recombinant expression in HEK293 cells
Platform: SURFE²R N1