Functional significance of E2 state stabilization by specific alpha/beta-subunit interactions of Na,K- and H,K-ATPase.
The beta-subunits of Na,K-ATPase and H,K-ATPase have important functions in maturation and plasma membrane targeting of the catalytic alpha-subunit but also modulate the transport activity of the holoenzymes. In this study, we show that tryptophan replacement of two highly conserved tyrosines in the transmembrane domain of both Na,K- and gastric H,K-ATPase beta-subunits resulted in considerable shifts of the voltage-dependent E1P/E2P distributions toward the E1P state as inferred from presteady-state current and voltage clamp fluorometric measurements of tetramethylrhodamine-6-maleimide-labeled ATPases. The shifts in conformational equilibria were accompanied by significant decreases in the apparent affinities for extracellular K+ that were moderate for the Na,K-ATPase beta-(Y39W,Y43W) mutation but much more pronounced for the corresponding H,K-ATPase beta-(Y44W,Y48W) variant. Moreover in the Na,K-ATPase beta-(Y39W,Y43W) mutant, the apparent rate constant for reverse binding of extracellular Na+ and the subsequent E2P-E1P conversion, as determined from transient current kinetics, was significantly accelerated, resulting in enhanced Na+ competition for extracellular K+ binding especially at extremely negative potentials. Analogously the reverse binding of extracellular protons and subsequent E2P-E1P conversion was accelerated by the H,K-ATPase beta-(Y44W,Y48W) mutation, and H+ secretion was strongly impaired. Remarkably tryptophan replacements of residues in the M7 segment of Na,K- and H,K-ATPase alpha-subunits, which are at interacting distance to the beta-tyrosines, resulted in similar E1 shifts, indicating their participation in stabilization of E2. Thus, interactions between selected residues within the transmembrane regions of alpha- and beta-subunits of P2C-type ATPases exert an E2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions.
Dürr
Katharina L
KL
Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany. Katharina.Duerr@TU-Berlin.DE
Tavraz
Neslihan N
NN
Dempski
Robert E
RE
Bamberg
Ernst
E
Friedrich
Thomas
T
D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain / Q000502:physiology*; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D007700:Kinetics; D020125:Mutation, Missense; D011188:Potassium / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D012756:Sheep; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
D006859:Hydrogen; D012964:Sodium; D017506:H(+)-K(+)-Exchanging ATPase; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium