NCX_Mj - EC50 for Calcium and Magnesium
Short Description: Na+/sugar translocation is detected using a sugar concentration jump in presence of sodium. In absence of sodium, we found a pre steady-state signal, representing sugar binding. Under these conditions, no transport is observed.
Background and Assay Description:
We performed a double solution exchange to measure the transport activity of NCX_Mj. The GUVs containing NCX_Mj have been prepared in Na+-containing, but Ca2+-free solution. The first solution exchange provides a Na+-free solution (Na+ is replaced by K+) to establish an outward directed Na+ gradient; the second solution exchange provides the divalent cation, either Ca2+, Mg2+ or Sr2+ - to activate the ion exchanger. The resulting current traces show negative amplitude due to the stoichiometry of transport: NCX catalyzes efflux of 3 Na+ and influx of 1 divalent cation, reflecting the net charge movement of one positive charge out of the GUVs. By applying different concentrations of one divalent cation on the same sensor, we determined EC50 for Ca2+, Mg2+ and Sr2+.