Klymchenko Andrey S, Stoeckel Herrade, Takeda Kenneth, Mély Yves
UMR 7175 CNRS, Département Pharmacologie et Physicochimie, Université Louis Pasteur (Strasbourg 1), Faculté de Pharmacie, 74 route du Rhin, 67401 Illkirch, France.
J Phys Chem B. 2006 Jul 13;110(27):13624-32. doi: 10.1021/jp062385z.
Development of fast-response potentiometric probes for measuring the transmembrane potential Vm in cell plasma membranes remains a challenge. To overcome the limitations of the classical charge-shift potentiometric probes, we selected a 3-hydroxychromone fluorophore undergoing an excited-state intramolecular proton transfer (ESIPT) reaction that generates a dual emission highly sensitive to electric fields. To achieve the highest sensitivity to the electric field associated to Vm, we modified the fluorophore by adding two rigid legs containing terminal polar sulfonate groups to allow a deep vertical insertion of the fluorophore into the membrane. Fluorescence spectra of the new dye in lipid vesicles and cell membranes confirm the fluorophore location in the hydrophobic region of the membranes. Variation of Vm in lipid vesicles and cell plasma membranes results in a change of the intensity ratio of the two emission bands of the probe. The ratiometric response of the dye in cells is approximately 15% per 100 mV, and is thus quite large in comparison with most single-fluorophore, fast-response probes reported to date. Combined patch-clamp/fluorescence data further show that the ratiometric response of the dye in cells is faster than 1 ms. Analysis of the excitation and emission shifts further suggests that the probe responds to changes in Vm by a mechanism based on electrochromic modulation of its ESIPT reaction. Thus, for the first time, the ESIPT reaction has been successfully applied as a sensing principle for detection of transmembrane potential, allowing to couple classical electrochromic band shifts with changes in the relative intensities of the two well-separated emission bands. The fast two-band ratiometric response as well as the relatively high sensitivity of the new probe are the key features that make it useful for rapid detection of Vm changes in cell suspensions and single cells. Moreover, the new design principles proposed in the present work should allow further improvement of the probe sensitivity.
开发用于测量细胞质膜跨膜电位Vm的快速响应电位探针仍然是一项挑战。为了克服传统电荷转移电位探针的局限性，我们选择了一种3-羟基色酮荧光团，它经历激发态分子内质子转移（ESIPT）反应，产生对电场高度敏感的双重发射。为了实现对与Vm相关的电场的最高灵敏度，我们通过添加两个含有末端极性磺酸根基团的刚性支链来修饰荧光团，以使荧光团能够深入垂直插入膜中。新染料在脂质体和细胞膜中的荧光光谱证实了荧光团位于膜的疏水区域。脂质体和细胞质膜中Vm的变化导致探针两个发射带强度比的变化。该染料在细胞中的比率响应约为每100 mV 15%，因此与迄今为止报道的大多数单荧光团快速响应探针相比相当大。膜片钳/荧光联合数据进一步表明，该染料在细胞中的比率响应快于1 ms。对激发和发射位移的分析进一步表明，该探针通过基于其ESIPT反应的电致变色调制机制对Vm的变化作出响应。因此，ESIPT反应首次成功地用作检测跨膜电位的传感原理，从而能够将经典的电致变色带位移与两个分离良好的发射带的相对强度变化相结合。新探针的快速双波段比率响应以及相对较高的灵敏度是使其可用于快速检测细胞悬浮液和单细胞中Vm变化的关键特性。此外，本工作中提出的新设计原则应能进一步提高探针的灵敏度。
