Dürr Katharina L, Tavraz Neslihan N, Dempski Robert E, Bamberg Ernst, Friedrich Thomas
Technical University of Berlin, Institute of Chemistry, D-10623 Berlin, Germany.
J Biol Chem. 2009 Feb 6;284(6):3842-54. doi: 10.1074/jbc.M808101200. Epub 2008 Dec 8.
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.
钠钾ATP酶和氢钾ATP酶的β亚基在催化性α亚基的成熟和质膜靶向中具有重要功能，同时也调节全酶的转运活性。在本研究中，我们发现，用色氨酸取代钠钾ATP酶和胃氢钾ATP酶β亚基跨膜结构域中两个高度保守的酪氨酸，导致电压依赖性E1P/E2P分布显著向E1P状态偏移，这是通过对四甲基罗丹明-6-马来酰亚胺标记的ATP酶进行稳态前电流和电压钳荧光测量推断得出的。构象平衡的偏移伴随着细胞外K⁺表观亲和力的显著降低，钠钾ATP酶β-(Y39W,Y43W)突变的这种降低较为适度，而相应的氢钾ATP酶β-(Y44W,Y48W)变体的降低则更为明显。此外，在钠钾ATP酶β-(Y39W,Y43W)突变体中，由瞬态电流动力学测定的细胞外Na⁺反向结合及随后E2P-E1P转化的表观速率常数显著加快，导致Na⁺对细胞外K⁺结合的竞争增强，尤其是在极负电位时。类似地，氢钾ATP酶β-(Y44W,Y48W)突变加速了细胞外质子的反向结合及随后的E2P-E1P转化，并且H⁺分泌受到严重损害。值得注意的是，用色氨酸取代钠钾ATP酶和氢钾ATP酶α亚基M7片段中与β酪氨酸相互作用距离内的残基，导致类似的E1偏移，表明它们参与了E2的稳定。因此，P2C型ATP酶α亚基和β亚基跨膜区域内选定残基之间的相互作用发挥了E2稳定作用，这对于体内条件下氢钾ATP酶高效泵出H⁺尤为重要。
