Phospholipid flippases (P4-ATPases) utilize
ATP to translocate specific
phospholipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thus generating and maintaining transmembrane
lipid asymmetry essential for a variety of cellular processes. P4-ATPases belong to the
P-type ATPase protein family, which also encompasses the ion transporting P2-
ATPases: Ca2+-
ATPase, Na+,K+-
ATPase, and H+,K+-
ATPase. In comparison with the P2-ATPases, understanding of P4-ATPases is still very limited. The electrogenicity of P4-ATPases has not been explored, and it is not known whether
lipid transfer between membrane bilayer leaflets can lead to displacement of charge across the membrane. A related question is whether P4-ATPases countertransport
ions or other substrates in the opposite direction, similar to the P2-ATPases. Using an electrophysiological method based on solid supported membranes, we observed the generation of a transient electrical current by the mammalian P4-ATPase ATP8A2 in the presence of
ATP and the negatively charged
lipid substrate
phosphatidylserine, whereas only a diminutive current was generated with the
lipid substrate
phosphatidylethanolamine, which carries no or little charge under the conditions of the measurement. The current transient seen with
phosphatidylserine was abolished by the mutation E198Q, which blocks dephosphorylation. Likewise, mutation I364M, which causes the
neurological disorder cerebellar ataxia,
mental retardation, and disequilibrium (CAMRQ) syndrome, strongly interfered with the electrogenic
lipid translocation. It is concluded that the electrogenicity is associated with a step in the
ATPase reaction cycle directly involved in translocation of the
lipid. These measurements also showed that no charged substrate is being countertransported, thereby distinguishing the P4-ATPase from P2-ATPases.