Asparagine 905 of the mammalian phospholipid flippase ATP8A2 is essential for lipid substrate-induced activation of ATP8A2 dephosphorylation.

The P-type ATPase protein family includes, in addition to ion pumps such as Ca2+-ATPase and Na+,K+-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.