Milanick Mark A, Arnett Krista L
Department of Physiology, School of Medicine, and Dalton Cardiovascular Research Center, University of Missouri, Columbia, MO 65211, USA.
J Gen Physiol. 2002 Oct;120(4):497-508. doi: 10.1085/jgp.20028573.
The effects of 0.3-10 nM extracellular protons (pH 9.5-8.0) on ouabain-sensitive rubidium influx were determined in 4,4'-diisocyanostilbene-2, 2'-disulfonate (DIDS)-treated human and rat erythrocytes. This treatment clamps the intracellular H. We found that rubidium binds much better to the protonated pump than the unprotonated pump; 13-fold better in rat and 34-fold better in human erythrocytes. This clearly shows that protons are not competing with rubidium in this proton concentration range. Bretylium and tetrapropylammonium also bind much better to the protonated pump than the unprotonated pump in human erythrocytes and in this sense they are potassium-like ions. In contrast, guanidinium and sodium bind about equally well to protonated and unprotonated pump in human red cells. In rat red cells, protons actually make sodium bind less well (about sevenfold). Thus, protons have substantially different effects on the binding of rubidium and sodium. The effect of protons on ouabain binding in rat red cells was intermediate between the effects of protons on rubidium binding and on sodium binding. Remarkably, all four cationic inhibitors (bretylium, guanidinium, sodium, and tetrapropylammonium) had similar apparent inhibitory constants for the unprotonated pump ( approximately 5-10 mM). The K(d) for proton binding to the human pump, with the empty transport site facing extracellularly is 13 nM, whereas the extracellular transport site loaded with sodium is 9.5 nM, and with rubidium is 0.38 nM. In rat red cells there is also a substantial difference in the K(d) for proton binding to the sodium-loaded pump (14.5 nM) and the rubidium-loaded pump (0.158 nM). These data suggest that important rearrangements occur at the extracellular pump surface as the pump moves between conformations in which the outward facing transport site has sodium bound, is empty, or has rubidium bound and that guanidinium is sodium-like and bretylium and tetrapropylammonium are rubidium-like.
在经4,4'-二异氰基芪-2,2'-二磺酸盐(DIDS)处理的人和大鼠红细胞中,测定了0.3 - 10 nM细胞外质子(pH 9.5 - 8.0)对哇巴因敏感的铷内流的影响。这种处理使细胞内的氢离子浓度保持稳定。我们发现,铷与质子化的泵结合比与未质子化的泵结合要好得多;在大鼠红细胞中好13倍,在人红细胞中好34倍。这清楚地表明,在这个质子浓度范围内,质子并不与铷竞争。在人红细胞中,溴苄铵和四丙基铵与质子化的泵结合也比与未质子化的泵结合要好得多,从这个意义上说,它们是类似钾的离子。相比之下,在人红细胞中,胍盐和钠与质子化和未质子化的泵结合的效果大致相同。在大鼠红细胞中,质子实际上使钠的结合变差(约7倍)。因此,质子对铷和钠结合的影响有很大不同。质子对大鼠红细胞中哇巴因结合的影响介于质子对铷结合和对钠结合的影响之间。值得注意的是,所有四种阳离子抑制剂(溴苄铵、胍盐、钠和四丙基铵)对未质子化的泵具有相似的表观抑制常数(约5 - 10 mM)。人泵的质子结合解离常数(K(d)),当空的转运位点面向细胞外时为13 nM,而当装载有钠的细胞外转运位点时为9.5 nM,装载有铷时为0.38 nM。在大鼠红细胞中,质子与装载有钠的泵(14.5 nM)和装载有铷的泵(0.158 nM)结合的K(d)也有很大差异。这些数据表明,当泵在构象之间移动时,细胞外泵表面会发生重要的重排,此时向外的转运位点分别结合有钠、为空或结合有铷,并且胍盐类似钠,溴苄铵和四丙基铵类似铷。