Zouzoulas Athina, Therien Alex G, Scanzano Rosemarie, Deber Charles M, Blostein Rhoda
Department of Biochemistry, McGill University, Montreal, Quebec H3G 1A4.
J Biol Chem. 2003 Oct 17;278(42):40437-41. doi: 10.1074/jbc.M308610200. Epub 2003 Aug 7.
The enzymatic activity of the Na,K-ATPase, or sodium pump, is modulated by members of the so-called FXYD family of transmembrane proteins. The best characterized member, FXYD2, also referred to as the gamma subunit, has been shown to decrease the apparent Na+ affinity and increase the apparent ATP affinity of the pump. The effect on ATP affinity had been ascribed to the cytoplasmic C-terminal end of the protein, whereas recent observations suggest that the transmembrane (TM) segment of gamma mediates the Na+ affinity effect. Here we use a novel approach involving synthetic transmembrane mimetic peptides to demonstrate unequivocally that the TM domain of gamma effects the shift in apparent Na+ affinity. Specifically, we show that incubation of these peptides with membranes containing alphabeta pumps modulates Na+ affinity in a manner similar to transfected full-length gamma subunit. Using mutated gamma peptides and transfected proteins, we also show that a specific glycine residue, Gly-41, which is associated with a form of familial renal hypomagnesemia when mutated to Arg, is important for this kinetic effect, whereas Gly-35, located on an alternate face of the transmembrane helix, is not. The peptide approach allows for the analysis of mutants that fail to be expressed in a transfected system.
钠钾ATP酶（即钠泵）的酶活性受所谓跨膜蛋白FXYD家族成员的调节。研究最充分的成员FXYD2，也被称为γ亚基，已被证明可降低泵对钠离子的表观亲和力，并增加其对ATP的表观亲和力。对ATP亲和力的影响归因于该蛋白的胞质C末端，而最近的观察结果表明，γ亚基的跨膜（TM）区段介导了对钠离子亲和力的影响。在此，我们采用一种涉及合成跨膜模拟肽的新方法，明确证明γ亚基的TM结构域影响了表观钠离子亲和力的变化。具体而言，我们发现，将这些肽与含有αβ泵的膜一起孵育，会以类似于转染全长γ亚基的方式调节钠离子亲和力。利用突变的γ肽和转染蛋白，我们还表明，一个特定的甘氨酸残基Gly-41对这种动力学效应很重要，该残基突变为精氨酸时与一种家族性肾性低镁血症有关，而位于跨膜螺旋另一面的Gly-35则无关。肽法允许对在转染系统中无法表达的突变体进行分析。
