ChR2 - Symmetry of transport
Depending on the direction of the Na+ gradient, we observed positive or negative transient currents after ChR2 activation by light. A negative control without ChR2 shows no current signal upon light activation.
Background and Assay Description:
We have compared the current signals from three different sensors. The light blue trace was recorded from membrane vesicles which have been pre-loaded with Na+-free solution before sensor preparation. Before ChR2 activation by a 470 nm light pulse, we established an inward directed Na+ gradient by rinsing the sensor with a Na+ containing solution. Inward transport of sodium leads to a positive current signal.
The red trace was recorded from membrane vesicles which have been pre-loaded with Na+-containing solution before sensor preparation. Before ChR2 activation by a 470 nm light pulse, we established an outward directed Na+ gradient by rinsing the sensor with sodium free solution. Outward transport of sodium leads to a negative current signal.
In addition, we have used membranes from cells lacking ChR2 expression. Here, the same workflows yield no current signals.
Author: Nanion Technologies and Axxam S.p.a.
Target: T159C ChR2
Family / Type: Ion-translocating Microbial Rhodopsin Family (MRF, TCDB: 3.E.1)
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