Functional Significance of E2 State Stabilization by Specific α/β-Subunit Interactions of Na,K- and H,K-ATPase

被引:22
作者
Duerr, Katharina L. [1 ]
Tavraz, Neslihan N. [1 ]
Dempski, Robert E. [2 ]
Bamberg, Ernst [2 ,3 ]
Friedrich, Thomas [1 ]
机构
[1] Tech Univ Berlin, Inst Chem, D-10623 Berlin, Germany
[2] Max Planck Inst Biophys, Dept Biophys Chem, D-60438 Frankfurt, Germany
[3] Goethe Univ Frankfurt, Chem & Pharmaceut Sci Dept, D-60439 Frankfurt, Germany
关键词
VOLTAGE-CLAMP FLUOROMETRY; NA; K-ATPASE BETA-SUBUNIT; ATP-ADP EXCHANGE; P-TYPE ATPASES; NA+/K+-ATPASE; INTRACELLULAR-TRANSPORT; GASTRIC H+; K+-ATPASE; XENOPUS OOCYTES; ACID-SECRETION; SODIUM-IONS;
D O I
10.1074/jbc.M808101200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 E-1 shifts, indicating their participation in stabilization of E-2. Thus, interactions between selected residues within the transmembrane regions of alpha- and beta-subunits of P-2C-type ATPases exert an E-2-stabilizing effect, which is of particular importance for efficient H+ pumping by H,K-ATPase under in vivo conditions.
引用
收藏
页码:3842 / 3854
页数:13
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