The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca²⁺ dissipates the H⁺ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca²⁺ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg²⁺ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca²⁺ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.

The gastric H+,K+‐ATPase is an ATP‐driven proton pump responsible for generating a million‐fold proton gradient across the gastric membrane. We present the structure of gastric H+,K+‐ATPase at 6.5 Å resolution as determined by electron crystallography of two‐dimensional crystals. The structure shows the catalytic α‐subunit and the non‐catalytic β‐subunit in a pseudo‐E2P conformation. Different from Na+,K+‐ATPase, the N‐terminal tail of the β‐subunit is in direct contact with the phosphorylation domain of the α‐subunit. This interaction may hold the phosphorylation domain in place, thus stabilizing the enzyme conformation and preventing the reverse reaction of the transport cycle. Indeed, truncation of the β‐subunit N‐terminus allowed the reverse reaction to occur. These results suggest that the β‐subunit N‐terminus prevents the reverse reaction from E2P to E1P, which is likely to be relevant for the generation of a large H+ gradient in vivo situation. There is a [Have you seen ...?][1] (June 2009) associated with this Article. [1]: http://dx.doi.org/10.1038/emboj.2009.137

Since the discovery of Na/K-ATPase, evidence has accumulated to suggest that 1 mol of ATP hydrolysis occurs via the Na⁺-occluded ADP-sensitive phosphoenzyme, the K⁺-sensitive phosphoenzyme and the K⁺-occluded enzyme accompanying active transport of 3Na⁺ and 2K⁺ according the Post-Albers scheme. However, some controversial issues have arisen concerning whether the functional unit of the enzyme is an αβ-protomer or a much higher oligomer, which would be related to the mechanism of transport, either sequential or simultaneous. Detailed studies of oligomer interaction and the reactivity of the enzyme and a comparison of the extent of phosphorylation with ligand-binding capacities in the presence or absence of ATP hydrolysis and others strongly suggest that the functional unit of the enzyme in the membrane is a tetraprotomer, (αβ)₄. They also suggest that each reaction intermediate of the Post-Albers scheme, respectively, reflects half of the site property of the intermediate and that another half binds ATP. These data may be useful not only to answer the long-standing question of whether the mechanism functions in the presence of both Na⁺ and K⁺ but also contribute to a better understanding of the mechanism of P-type pump ATPase in general.

ATP11C, a plasma membrane phospholipid flippase, maintains the asymmetric distribution of phosphatidylserine accumulated in the inner leaflet. Caspase-dependent inactivation of ATP11C is essential for an apoptotic "eat me" signal, phosphatidylserine exposure, which prompts phagocytes to engulf cells. We show six cryo-EM structures of ATP11C at 3.0-4.0 Å resolution in five different states of the transport cycle. A structural comparison reveals phosphorylation-driven domain movements coupled with phospholipid binding. Three structures of phospholipid-bound states visualize phospholipid translocation accompanied by the rearrangement of transmembrane helices and an unwound portion at the occlusion site, and thus they detail the basis for head group recognition and the locality of the protein-bound acyl chains in transmembrane grooves. Invariant Lys880 and the surrounding hydrogen-bond network serve as a pivot point for helix bending and precise P domain inclination, which is crucial for dephosphorylation. The structures detail key features of phospholipid translocation by ATP11C, and a common basic mechanism for flippases is emerging.