Scheiner-Bobis G, Schneider H
Institut für Biochemie und Endokrinologie, Fachbereich Veterinärmedizin, Justus-Liebig-Universität Giessen, Germany.
Eur J Biochem. 1997 Sep 15;248(3):717-23. doi: 10.1111/j.1432-1033.1997.00717.x.
It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+-ATPase. To investigate whether palytoxin-induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369-->Ala mutant of the alpha1 subunit of the sheep sodium pump was produced and coexpressed with beta subunits in the yeast Saccharomyces cerevisiae. This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump. Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild-type sodium pump, 60% of the total ATPase activity is ouabain-sensitive. Ouabain binds to yeast membranes containing either wild-type or mutant sodium pumps with similar Bmax (1.45+/-0.05 versus 1.37+/-0.02 pmol/mg) and Kd values (27.7+/-0.91 versus 29.57+/-0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme. In the presence of phosphate ouabain binds to microsomes containing the wild-type sodium pump with a Kd of 3.62+/-0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid. Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78+/-3 microM and 3.0+/-0.4 microM, respectively. Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation. Palytoxin causes K+ efflux from yeast cells expressing the wild-type or mutant sodium pumps with EC50 values of 3.5+/-0.4 nM and 6.2+/-0.9 nM, respectively. Palytoxin-induced efflux from cells expressing wild-type or mutant sodium pumps occurs with similar t1/2 values of 20.3+/-2.1 min and 22.2+/-3.1 min, respectively. Ouabain inhibits K+ efflux from both cell types with IC50 values of 28+/-2 microM and 210+/-15 microM, respectively. Cells expressing the Asp369-->Ala mutants have an IC50 7.5-fold higher than that obtained with cells expressing the wild-type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump. Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild-type enzyme by phosphate or ATP, palytoxin-induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump. Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features.
业已证明，短裸甲藻毒素可与钠钾ATP酶结合并在其中形成通道。为了研究短裸甲藻毒素在钠泵中诱导形成通道是否能够独立于该酶的ATP水解和磷酸化过程，制备了绵羊钠泵α1亚基的Asp369→Ala突变体，并与β亚基在酿酒酵母中共表达。这个天冬氨酸残基在离子转运过程中会从ATP接受磷酸化，对钠泵的功能至关重要。因此，正如预期的那样，从表达突变型钠泵的酵母中分离出的微粒体未表现出任何哇巴因敏感的ATP酶活性，而在表达野生型钠泵的酵母微粒体中，总ATP酶活性的60%是哇巴因敏感的。哇巴因与含有野生型或突变型钠泵的酵母膜结合，其Bmax值相似（分别为1.45±0.05和1.37±0.02 pmol/mg），Kd值也相似（分别为27.7±0.91和29.57±0.93 nM），这表明突变型钠泵在酵母中表达，且该突变并未对酶的构象产生显著影响。在有磷酸盐存在的情况下，哇巴因与含有野生型钠泵的微粒体结合，Kd为3.62±0.34 nM，这表明，虽然不是必需的，但磷酸化酶的形成会增强类固醇的结合。然而，磷酸盐或ATP会抑制哇巴因与含有突变型钠泵的微粒体的结合，IC50值分别为78±3 μM和3.0±0.4 μM。尽管突变型酶与哇巴因的相互作用发生了这些根本性变化，但与短裸甲藻毒素的相互作用并未受该突变影响。短裸甲藻毒素可使表达野生型或突变型钠泵的酵母细胞发生钾外流，EC50值分别为3.5±0.4 nM和6.2±0.9 nM。短裸甲藻毒素诱导表达野生型或突变型钠泵的细胞发生外流，其t1/2值相似，分别为20.3±2.1分钟和22.2±3.1分钟。哇巴因抑制两种细胞类型的钾外流，IC50值分别为28±2 μM和210±15 μM。表达Asp369→Ala突变体的细胞的IC50比表达野生型钠泵的细胞高7.5倍，这可能是因为酵母细胞胞质中存在的ATP或磷酸盐会影响并降低哇巴因与突变型钠泵的结合。因此，虽然磷酸盐或ATP使野生型酶磷酸化会促进哇巴因结合及相关的离子通量抑制，但短裸甲藻毒素诱导的通道形成独立于磷酸化过程，且可与钠泵的ATP酶功能分离。由于通过钠泵蛋白的离子通量不依赖于ATP水解，结果表明泵和离子通道的离子载体可能具有共同的结构特征。
