ChR2 - Membrane potential as driving force
We investigated the effect of a membrane potential - generated by Na+/Ca2+ exchange activity of NCX1 - on the signal amplitude of light induced Na+ flux by ChR2.
Background and Assay Description:
The aim of this experiment was to test the influence of a membrane potential as additional driving force for the sodium flux mediated by ChR2.
To apply a membrane potential, we co-expressed the Na+/Ca2+ exchanger NCX1 with ChR2 and activated NCX1 by a Ca2+ concentration jump before activating ChR2. 3Na+/Ca2+ exchange by NCX1 rapidly increases the membrane potential within 500 ms. By timing the light pulse for ChR2 activation at different time points within these 500 ms, we observe that ChR2 activity increases with the membrane potential built up by NCX1.
The NCX1 equilibrium potential finally leads to an acceleration of ChR2 mediated Na+ flux by a factor of 10.
Author: Nanion Technologies and Axxam S.p.a.
Target: T159C ChR2 and NCX1
Family / Type: Ion-translocating Microbial Rhodopsin Family (MRF, TCDB: 3.E.1) (ChR2) and Ca2+:Cation Antiporter Family (CaCA. TCDB: 2.A.19) (NCX1)
Mode of transport: Light-gated Na+ flux (ChR2) and 3Na+/Ca2+ exchange (NCX1)
Organism: Chlamydomonas reinhardtii (ChR2), human (NCX1)
Sample: Purified membrane vesicles from plasma membrane
Expression: Recombinant co-expression in HEK293 cells
Platform: SURFE²R N1