Function of extracellular M3/M4 loop of human non-gastric H⁺,K⁺-ATPase

Abstract

Human non-gastric H⁺,K⁺-ATPase, ATP1AL1, mediates Na⁺ or H⁺ efflux and K⁺ influx. It is reported that four amino acid residues of M3/M4 loop in Na⁺,K⁺-ATPase are essential for ouabain binding. To examine functions of the extracellular M3/M4 loop of ATP1AL1, the amino acid residues of ATP1AL1 were replaced with corresponding amino acid residues of Na⁺,K⁺-ATPase (Q334T, D337E, S338A, I339V). A tetra-mutant (TEAV) and four triple-mutants (EAV, TEA, TEV, TAV) were constructed, and they were transfected with HEK293 cells. Expression level of all ATP1AL1 mutants was not significantly different from that of wild-type, and the endogenous Na⁺,K⁺-ATPase level was unchanged by these transfections. In all of the mutant-expressing cells, 10 μM ouabain- sensitive K⁺-ATPase activity was not significantly different from that of wild-type. On the other hand, 3 mM ouabain-sensitive K⁺-ATPase activity of TEV, TEA and TEAV mutants was significantly decreased compared with that of wild-type. In the molecular dynamics simulation, we found that M3/M4 loop is indirectly involved in the closed ion gate formed from M1/M2 loop of ATP1AL1, and that the gate of TEV, TEA and TEAV mutants was unstable or disrupted. These results suggest that M3/M4 loop in ATP1AL1 is not essential for ouabain sensitivity, but that it may be important for stabilization of closed ion gate. [J Physiol Sci. 2008;58 Suppl:S54]