Patti Monica, Forster Ian C
Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland.
Biophys J. 2014 Apr 15;106(8):1618-29. doi: 10.1016/j.bpj.2014.02.028.
To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na(+)-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na(+), providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.
为深入了解电生性次级主动协同转运蛋白在其转运循环过程中结构重排的稳态和动态特征，研究了两种构象变化的测量方法（稳态前电流弛豫和报告荧光团发出的荧光强度）作为膜电位和外部底物的函数。在IIb型Na(+)-偶联无机磷酸协同转运蛋白（SLC34A2比目鱼同工型）中三个被认为是新的位点进行了半胱氨酸取代，这些位点预计参与构象变化。在一个位点进行标记导致转运活性大幅抑制，而对于其他位点，功能与野生型相当。对于这些突变体，每个突变体的稳态前电荷弛豫特性相似，而荧光强度变化则有显著差异。荧光变化可以通过使用五态模型的模拟来解释，根据标记位点为每个状态分配一组独特的表观荧光强度。从一个位点报告的荧光与内向和外向构象相关，而对于其他位点，包括四个先前确定的位点，发射主要与转运蛋白的一种或另一种取向相关。相同的膜电位变化在某些位点诱导荧光的互补变化，这表明各个荧光团的微环境经历了极性的伴随变化。响应电压的阶跃变化，稳态前电流弛豫和荧光强度变化的时间进程由单指数描述。对于一个突变体，有无外部Na(+)时的时间常数匹配良好，提供了直接证据表明该标记报告了伴随内在电荷移动和阳离子相互作用的构象变化。
