Functional consequences of various leucine mutations in the M3/M4 loop of the Na+,K +-ATPase alpha-Subunit.
Leucines were mutated within the sequence L311 ILGYTWLE319 of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na+,K+-ATPase alpha-subunit. Replacement of Leu311 with Glu resulted in a considerable loss of Na+,K+-ATPase activity. Replacement of Leu313 with Glu shifted the equilibrium of E1P and E2P toward E1P and reduced the rate of the E1P to E2P 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 Leu313 mutant. Thus, Leu313 could be in the pathway of Na+ exit. Replacement of Leu318 with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K+, with an equilibrium shifted toward E2P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K+ (2)]E2 caused by inhibition of dephosphorylation through reduction of the K+ affinity of E2P. Thus, Leu318 may be a critical position in guiding external K+ to its binding site.
Eguchi
Hiroshi
H
Department of Cell Biology, School of Medicine, University of Occupational and Environmental Health, Kitakyushu 807-8555, Japan.
Morii
Magotoshi
M
Takahashi
Yuji
Y
Sakai
Hideki
H
Nakano
Masahiro
M
Ochiai
Hideo
H
Shirahata
Akira
A
Hara
Yukichi
Y
Kawamura
Masaru
M
Takeda
Kazuo
K
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D007930:Leucine / Q000235:genetics / Q000378:metabolism; D011188:Potassium / Q000276:immunology / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D012964:Sodium / Q000378:metabolism* / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014101:Torpedo; D014638:Vanadates / Q000378:metabolism / Q000494:pharmacology; D014982:Xenopus laevis
D014638:Vanadates; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D007930:Leucine; D011188:Potassium