Virkki Leila V, Murer Heini, Forster Ian C
Institute for Physiology and the Center for Integrative Human Physiology, University of Zurich, Switzerland.
J Gen Physiol. 2006 May;127(5):539-55. doi: 10.1085/jgp.200609496.
Voltage clamp fluorometry (VCF) combines conventional two-electrode voltage clamp with fluorescence measurements to detect protein conformational changes, as sensed by a fluorophore covalently attached to the protein. We have applied VCF to a type IIb Na+-coupled phosphate cotransporter (NaPi-IIb), in which a novel cysteine was introduced in the putative third extracellular loop and expressed in Xenopus oocytes. Labeling this cysteine (S448C) with methanethiosulfonate (MTS) reagents blocked cotransport function, however previous electrophysiological studies (Lambert G., I.C. Forster, G. Stange, J. Biber, and H. Murer. 1999. J. Gen. Physiol. 114:637-651) suggest that substrate interactions with the protein can still occur, thus permitting study of a limited subset of states. After labeling S448C with the fluorophore tetramethylrhodamine MTS, we detected voltage- and substrate-dependent changes in fluorescence (DeltaF), which suggested that this site lies in an environment that is affected by conformational change in the protein. DeltaF was substrate dependent (no DeltaF was detectable in 0 mM Na+) and showed little correlation with presteady-state charge movements, indicating that the two signals provide insight into different underlying physical processes. Interpretation of ion substitution experiments indicated that the substrate binding order differs from our previous model (Forster, I., N. Hernando, J. Biber, and H. Murer. 1998. J. Gen. Physiol. 112:1-18). In the new model, two (rather than one) Na+ ions precede Pi binding, and only the second Na+ binding transition is voltage dependent. Moreover, we show that Li+, which does not drive cotransport, interacts with the first Na+ binding transition. The results were incorporated in a new model of the transport cycle of type II Na+/Pi cotransporters, the validity of which is supported by simulations that successfully predict the voltage and substrate dependency of the experimentally determined fluorescence changes.
电压钳荧光测定法（VCF）将传统的双电极电压钳与荧光测量相结合，以检测蛋白质构象变化，这种变化由共价连接到蛋白质上的荧光团感知。我们已将VCF应用于IIb型Na⁺偶联磷酸盐共转运体（NaPi-IIb），其中在假定的第三个细胞外环中引入了一个新的半胱氨酸，并在非洲爪蟾卵母细胞中表达。用甲硫基磺酸盐（MTS）试剂标记这个半胱氨酸（S448C）会阻断共转运功能，然而先前的电生理研究（兰伯特G.、I.C.福斯特、G.施坦格、J.比伯和H.穆勒。1999年。《普通生理学杂志》114:637 - 651）表明底物与蛋白质之间仍可发生相互作用，从而允许研究有限的一部分状态。在用荧光团四甲基罗丹明MTS标记S448C后，我们检测到了荧光的电压和底物依赖性变化（ΔF），这表明该位点所处的环境会受到蛋白质构象变化的影响。ΔF是底物依赖性的（在0 mM Na⁺中未检测到ΔF），并且与稳态前电荷移动几乎没有相关性，这表明这两个信号揭示了不同的潜在物理过程。离子替代实验的解释表明，底物结合顺序与我们之前的模型（福斯特I.、N.埃尔南多、J.比伯和H.穆勒。1998年。《普通生理学杂志》112:1 - 18）不同。在新模型中，两个（而非一个）Na⁺离子先于Pi结合，并且只有第二个Na⁺结合转变是电压依赖性的。此外，我们表明不驱动共转运的Li⁺与第一个Na⁺结合转变相互作用。这些结果被纳入了II型Na⁺/Pi共转运体转运循环的新模型中，该模型的有效性得到了模拟的支持，这些模拟成功预测了实验测定的荧光变化的电压和底物依赖性。
