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P4-ATPases, also known as flippases, translocate specific lipids from the exoplasmic leaflet to the cytoplasmic leaflet of biological membranes, thereby generating an asymmetric lipid distribution essential for numerous cellular functions. A debated issue is which pathway within the protein the lipid substrate follows during the translocation. Here we present a comprehensive mutational screening of all amino acid residues in the transmembrane segments M1, M2, M3, and M4 of the flippase ATP8A2, thus allowing the functionally important residues in these transmembrane segments to be highlighted on a background of less important residues. Kinetic analysis of ATPase activity of 130 new ATP8A2 mutants, providing V max values as well as apparent affinities of the mutants for the lipid substrate, support a translocation pathway between M2 and M4 ("M2-M4 path"), extending from the entry site, where the lipid substrate binds from the exoplasmic leaflet, to a putative exit site at the cytoplasmic surface, formed by the divergence of M2 and M4. The effects of mutations in the M2-M4 path on the function of the entry site, including loss of lipid specificity in some mutants, suggest that the M2-M4 path and the entry site are conformationally coupled. Many of the residues of the M2-M4 path possess side chains with a potential for interacting with each other in a zipper-like mode, as well as with the head group of the lipid substrate, by ionic/hydrogen bonds. Thus, the translocation of the lipid substrate toward the cytoplasmic bilayer leaflet is comparable to unzipping a zipper of salt bridges/hydrogen bonds. [ABSTRACT FROM AUTHOR]