Zillikens S, Gisselmann G, Glitsch H G
Arbeitsgruppe Muskelphysiologie, Ruhr-Universität Bochum, Germany.
Biochim Biophys Acta. 2000 Dec 20;1509(1-2):155-66. doi: 10.1016/s0005-2736(00)00289-3.
The current (I(p)) generated by the wild-type or the glutamate (E) 779 alanine (A) mutant of the rat Na(+)/K(+) pump alpha1-subunit expressed in HEK 293 cells was studied at 35 degrees C by means of whole-cell recording in Na(+)-free and Na(+)-containing solution. Glutamate 779 is located in the fifth transmembrane domain of the alpha-subunit of the Na(+)/K(+)-ATPase. Compared with the wild-type, the E779A mutant exhibited an apparent K(+)(o)-affinity decreased by a factor of 3-4 both in Na(+)-free and in Na(+)-containing media. The competition of Na(+)(o) and K(+)(o) for cation binding sites of the pump remained unchanged. Similarly, in Na(+)-free solution the shape of the I(p)-V curves for various external K(+)-concentrations (K(+)) was essentially the same. However, in Na(+)-containing solutions the shape of I(p)-V curves from cells expressing the mutant of the rat alpha1-subunit clearly differed from the shape observed in cells expressing the wild-type, but voltage dependence of the pump current persisted. A prominent Na(+)(o)-activated, electrogenic Na(+)-transport mediated by the pump, displaying little voltage dependence in the potential range tested (-80 to +60 mV), was present in the cells expressing the E779A mutant pump. The data suggest that exchanging E779 for A in the rat Na(+)/K(+) pump alpha1-subunit causes a modest decrease in the apparent K(+)(o) affinity and a profound, Na(+)(o)-dependent alteration in the electrogenicity of the mutant pump expressed in HEK 293 cells.
在35℃下，通过在无钠和含钠溶液中进行全细胞记录，研究了在HEK 293细胞中表达的大鼠Na(+)/K(+)泵α1亚基的野生型或谷氨酸(E)779丙氨酸(A)突变体产生的电流(I(p))。谷氨酸779位于Na(+)/K(+)-ATPaseα亚基的第五个跨膜结构域。与野生型相比，E779A突变体在无钠和含钠培养基中表现出明显的K(+)(o)亲和力下降3-4倍。Na(+)(o)和K(+)(o)对泵阳离子结合位点的竞争保持不变。同样，在无钠溶液中，各种外部K(+)浓度(K(+))下的I(p)-V曲线形状基本相同。然而，在含钠溶液中，表达大鼠α1亚基突变体的细胞的I(p)-V曲线形状与表达野生型的细胞中观察到的形状明显不同，但泵电流的电压依赖性仍然存在。在表达E779A突变体泵的细胞中，存在由泵介导的显著的Na(+)(o)激活的电致Na(+)转运，在测试的电位范围内(-80至+60 mV)几乎没有电压依赖性。数据表明，在大鼠Na(+)/K(+)泵α1亚基中用A替换E779会导致明显的K(+)(o)亲和力适度下降，并使在HEK 293细胞中表达的突变体泵的电致性发生深刻的、Na(+)(o)依赖性改变。
