Geibel Sven, Zimmermann Dirk, Zifarelli Giovanni, Becker Anja, Koenderink Jan B, Hu Yi-Kang, Kaplan Jack H, Friedrich Thomas, Bamberg Ernst
Max-Planck-Institute of Biophysics, Kennedyallee 70, D-60596 Frankfurt/M., Germany.
Ann N Y Acad Sci. 2003 Apr;986:31-8. doi: 10.1111/j.1749-6632.2003.tb07136.x.
We used the method of site-directed fluorescence labeling in combination with voltage-clamp fluorometry for time-resolved recording of localized conformational transitions of the Na(+)/K(+)- and H(+)/K(+)-ATPase. Therefore, single cysteine mutations were introduced into the extracellular TM5-TM6 loop of the sheep Na(+)/K(+)-ATPase alpha(1)-subunit devoid of other extracellular cysteines. Upon expression in Xenopus oocytes and covalent attachment of tetramethylrhodamine-maleimide (TMRM) as a reporter fluorophore, Cys-mutant N790C showed large fluorescence changes of up to 5% in response to extracellular K(+) that were completely abolished by ouabain. When voltage jumps were applied under Na(+)/Na(+)-exchange conditions, we observed fluorescence changes that paralleled the transient currents originating from the E(1)P<-->E(2)P transition. These fluorescence changes were also completely inhibited by ouabain, as were the voltage jump-induced transient currents. Transient fluorescence changes could also be measured as a function of increasing K(+) concentrations, that is, under turnover conditions. As a result, the distribution between E(1) and E(2) states can be determined at any time and membrane potential. Very similar fluorescence signals were obtained for rat gastric H(+)/K(+)-ATPase upon expression in oocytes, when a single cysteine was introduced at a position homologous to N790 in Na(+)/K(+)-ATPase for attachment of the fluorophore. As to the high sequence similarity among P-type ATPases within the TM5 helix and the TM5-TM6 loop region, our results enable new means of kinetic investigation for these pumps under physiological conditions in living cells.
我们使用定点荧光标记法结合电压钳荧光测定法，对Na(+)/K(+)-ATP酶和H(+)/K(+)-ATP酶的局部构象转变进行时间分辨记录。因此，将单个半胱氨酸突变引入绵羊Na(+)/K(+)-ATP酶α(1)-亚基的细胞外TM5-TM6环中，该亚基不存在其他细胞外半胱氨酸。在非洲爪蟾卵母细胞中表达并共价连接四甲基罗丹明-马来酰亚胺（TMRM）作为报告荧光团后，半胱氨酸突变体N790C对细胞外K(+)的响应显示出高达5%的大荧光变化，而哇巴因可完全消除这种变化。当在Na(+)/Na(+)-交换条件下施加电压阶跃时，我们观察到荧光变化与源自E(1)P<-->E(2)P转变的瞬态电流平行。这些荧光变化也被哇巴因完全抑制，电压阶跃诱导的瞬态电流也是如此。瞬态荧光变化也可以作为K(+)浓度增加的函数进行测量，即在周转条件下。结果，可以在任何时间和膜电位下确定E(1)和E(2)状态之间的分布。当在卵母细胞中表达时，在与Na(+)/K(+)-ATP酶中N790同源的位置引入单个半胱氨酸以连接荧光团，大鼠胃H(+)/K(+)-ATP酶获得了非常相似的荧光信号。鉴于TM5螺旋和TM5-TM6环区域内P型ATP酶之间的高度序列相似性，我们的结果为在活细胞的生理条件下对这些泵进行动力学研究提供了新方法。
