Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na + ,K + -ATPase α-Subunit

Leucines were mutated within the sequence L 311 ILGYTWLE 319 of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na + ,K + -ATPase α-subunit. Replacement of Leu 311 with Glu resulted in a considerable loss of Na + ,K + -ATPase activity. Replacement of Leu 313 with Glu shifted the equilibrium of E 1 P and E 2 P toward E 1 P and reduced the rate of the E 1 P to E 2 P transition. The reduction of the transition rate and stronger inhibition of Na + ,K + -ATPase activity by Na + at higher concentrations together suggest that there is interference of Na + release on the extracellular side in the Leu 313 mutant. Thus, Leu 313 could be in the pathway of Na + exit. Replacement of Leu 318 with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K + , with an equilibrium shifted toward E 2 P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K + 2 ]E 2 caused by inhibition of dephosphorylation through reduction of the K + affinity of E 2 P. Thus, Leu 318 may be a critical position in guiding external K + to its binding site.