Functional Consequences of Alterations to Ile279, Ile283, Glu284, His285, Phe286, and His288 in the NH2-terminal Part of Transmembrane Helix M3 of the Na+,K+-ATPase

Abstract

Mutations3 Ala displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of E 2 and E 2 P, respectively, and showed a unique enhancement of the E 1 P 3 E 2 P transition rate. These effects suggest that M3 undergoes significant rearrangements in relation to E 1 -E 2 and E 1 P-E 2 P conformational changes. Because the E 1 -E 2 and E 1 P-E 2 P conformational equilibria were differentially affected by some of the mutations, the phosphorylated conformations seem to differ significantly from the dephospho forms in the M3 region. Mutation of His 285 furthermore increased the Na ؉ -activated ATPase activity in the absence of K ؉

Expression of Mutant α1 Na/K-ATPase Defective in Conformational Transition Attenuates Src-mediated Signal Transduction*

“…1F). In general, the expressed I279A and F286A mutants exhibited enzymatic properties similar to those reported previously (17). Although I279A had much lower vanadate sensitivity, F286A mutant was more sensitive to vanadate than the wild-type ␣1.…”

“…As reported (17), vanadate sensitivity of cell lysates from I3 cells was significantly reduced in comparison to that of AAC-19 cells, whereas a large increase was noted in F19 cells (data not shown).…”

“…Moreover, these two mutants are from the same transmembrane domain (17). Finally, these two mutants produce more pronounced kinetic changes than most of the other mutants (17). Functional studies of these mutant-expressing cell lines demonstrate that inhibition of conformation transition is sufficient to attenuate the capability of Na/K-ATPase to regulate cellular Src and consequently ligand-induced activation of protein kinase cascades in cultured cells.…”

“…Prior studies showed that I279A mutation accumulated the pump at the E1 state, whereas F286A mutation increased the E2 state Na/K-ATPase. Moreover, these two mutants are from the same transmembrane domain (17). Finally, these two mutants produce more pronounced kinetic changes than most of the other mutants (17).…”

“…Prior studies have identified many ␣1 mutants that inhibit the E1/E2 conformational transition by favoring the pump at either E1-or E2-like state (15)(16)(17)(18). These mutants exhibit altered pumping properties.…”