Phosphatidylserine flipping by the P4-ATPase ATP8A2 is electrogenic

Abstract

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.

Keywords: CAMRQ syndrome; SSM method; charge displacement; flippase; phospholipid transport.

Fig. 1.

Simplified scheme of the ATP8A2 reaction cycle and schematic diagram of the SSM method. (A) The scheme is based on functional similarities to the ion-transporting P2-ATPases (5, 6). E₁, E₁P, E₂P, and E₂ are different enzyme conformational states, “P” indicates covalently bound phosphate. The phospholipid substrate, PL (PS or PE), enters the cycle from the exoplasmic leaflet of the lipid bilayer by binding to the E₂P phosphoenzyme, thereby stimulating the dephosphorylation and release of the lipid substrate toward the cytoplasmic leaflet as consequence of the E₂ to E₁ conformational change. (B) Reconstituted proteoliposome containing ATP8A2 adsorbed on the SSM surface and subjected to an ATP concentration jump (not drawn to scale). If the ATP concentration jump induces net charge displacement across the ATPase, a current signal is detected along the external circuit (the blue spheres represent electrons) to keep constant the potential difference (ΔV) applied across the whole system. RE, reference electrode.

Fig. 2.

ATP8A2-related current transients in the presence of PS. (A) Current transients observed upon 100 µM ATP concentration jumps on 90PC:10PS proteoliposomes containing ATP8A2 in the absence (black line) or in the presence (red line) of 50 μM orthovanadate in 150 mM NaCl, 1 mM DTT, 5 mM MgCl₂, 50 mM Hepes-Tris (“basic medium”), pH 7.5. The average displaced charge, obtained by numerical integration of the ATP-induced current transient in the absence of orthovanadate (Fig. 3B) is 41 ± 2 pC (n = 7); n, number of independent measurements; ±, SEM. Inset shows the average current signal induced by a 100 µM ATP concentration jump on native SR vesicles containing Ca²⁺-ATPase (present at a concentration of 0.5 mg/mL similar to ATP8A2 during adsorption on the SSM surface) in 100 mM KCl, 1 mM MgCl₂, 25 mM Mops (pH 7.0), 0.2 mM DTT, 0.25 mM EGTA, and 0.25 mM CaCl₂ (10 µM free Ca²⁺). The average displaced charge is in this case 84 ± 5 pC (n = 9). (B) Current traces recorded with 90PC:10PS liposomes devoid of ATP8A2 (control liposomes) upon a 100 µM ATP concentration jump (black line) or upon exchange with basic medium without ATP added (red line).

Fig. 3.

Effect of different phospholipids on the ATP8A2-related current transients. (A) Current transients observed upon 100 µM ATP concentration jumps on 90PC:10PS (black line), 90PC:10PE (red line), 50PC:50PE (blue line), and 100PC (green line) proteoliposomes containing ATP8A2 in basic medium, pH 7.5. (B) Average displaced charges obtained by numerical integration of the ATP-induced current transients observed for 90PC:10PS (PS, 41 ± 2 pC, n = 7), 50PC:50PE (50PE, 13 ± 1 pC, n = 5), 90PC:10PE (10PE, 9 ± 1 pC, n = 5), and 100PC (PC, 3.0 ± 0.7 pC, n = 3) proteoliposomes containing ATP8A2; n, number of independent measurements; error bars and ±, SEM. Individual data points are shown by open circles.

Fig. 4.

Effects of pH and specific mutations on the ATP8A2-related current transients. (A and B) Current transients induced by 100 µM ATP concentration jumps on 90PC:10PS (A) and 50PC:50PE (B) proteoliposomes containing ATP8A2 in basic medium, pH 7.5 (black lines) or 6.7 (red lines). (C and D) Current transients observed following 100 µM ATP concentration jumps on 90PC:10PS proteoliposomes containing ATP8A2 wild-type enzyme (C and D, black lines), mutant E198Q (C, green line), or mutant I364M (D, green line) in basic medium, pH 7.5.