ATP8A2 (Flippase) - Inhibition with vanadate and negative control
We measured ATP8A2 mediated and ATP-dependent flips of phosphatidylserine. When the ATPase activity is inhibited or when liposomes without ATP8A2 are used, no charge translocation is measured.
Background and Assay Description:
We performed 100 μM ATP concentration jumps on Proteoliposomes containing ATP8A2, a mammalian PS-flippase, both in absence (dark blue trace) and presence (light blue trace) of the ATPase inhibitor orthovanadate. As a negative control we performed the same assay on empty liposomes (red trace). Orthovanadate caused a strong suppression of the current transient, thus confirming that the measured current is actually generated by ATP8A2 activity. We observed that the sign of the ATP8A2-related current is positive. The displacement of positive charge in one direction is electrically equivalent to the movement of negative charge in the opposite direction. PS possesses a negatively charged head group and is a substrate of ATP8A2, whereas PC, also present in the proteoliposomes, is not a substrate and possesses an electrically neutral head group. Thus, the ATP8A2 current signal can be attributed to displacement of negatively charged PS toward the outside of the proteoliposomes (the ATP8A2 cytoplasmic side facing the external aqueous solution) in connection with ATP utilization.
Author: Francesco Tadini-Buoninsegni, University of Florence
Family/Type: Phospholipid-Flippase, P4-type-ATPase
Mode of transport: Phospholipid translocation between membrane leaflets
Sample: Proteoliposomes from purified protein (90% PC, 10% PS), liposomes without ATP8A2 protein (negative control)
Expression: Recombinant expression in HEK293 cells
Platform: SURFE²R N1