Magzoub Mazin, Padmawar Prashant, Dix James A, Verkman A S
Department of Medicine, University of California at San Francisco, 94143-0521, USA.
J Phys Chem B. 2006 Oct 26;110(42):21216-21. doi: 10.1021/jp0633392.
We recently introduced a water-soluble, long-wavelength K(+)-sensing indicator, TAC-Red, consisting of a triazacryptand K(+)-selective ionophore coupled to a xanthylium chromophore (Nat. Methods 2005, 2, 825-827). Stopped-flow kinetic analysis indicated that in response to changes in K(+) concentration TAC-Red fluorescence enhancement occurs in milliseconds or less. Here, we use fluorescence correlation spectroscopy to quantify the binding kinetics of K(+) with TAC-Red and a new, longer-wavelength sensor, TAC-Crimson. Autocorrelation functions, G(tau), were similar at 0 and high (150 mM) K(+) concentrations, with the appearance of a prominent kinetic process with a correlation time in the millisecond range for K(+) concentrations between approximately 20 and 60 mM. Control experiments with increased illumination volume and solution viscosity indicated that the millisecond component represented K(+)/TAC-Red association. K(+)-dependent G(tau) data, modeled using a global regression to a binding/diffusion model, gave association and dissociation rate constants of 0.0020 +/- 0.0003 mM(-1) ms(-1) and 0.12 +/- 0.02 ms(-1), respectively, for TAC-Red. Similar results were obtained for TAC-Crimson. The rapid K(+) binding kinetics with triazacryptand-based sensors support their utility for measuring changes in K(+) concentrations during rapid neural signaling and ion channel gating.
我们最近推出了一种水溶性长波长钾离子传感指示剂TAC-Red，它由与呫吨鎓发色团偶联的三氮杂环穴状化合物钾离子选择性离子载体组成（《自然方法》，2005年，第2卷，825 - 827页）。停流动力学分析表明，响应钾离子浓度变化时，TAC-Red荧光增强在数毫秒或更短时间内发生。在此，我们使用荧光相关光谱法定量钾离子与TAC-Red以及一种新的、波长更长的传感器TAC-Crimson的结合动力学。在0和高（150 mM）钾离子浓度下，自相关函数G(τ)相似，当钾离子浓度在约20至60 mM之间时，出现一个相关时间在毫秒范围内的显著动力学过程。增加光照体积和溶液粘度的对照实验表明，毫秒级分量代表钾离子/TAC-Red缔合。使用全局回归到结合/扩散模型对依赖钾离子的G(τ)数据进行建模，得到TAC-Red的缔合和解离速率常数分别为0.0020 ± 0.0003 mM⁻¹ ms⁻¹和0.12 ± 0.02 ms⁻¹。TAC-Crimson也得到了类似结果。基于三氮杂环穴状化合物的传感器快速的钾离子结合动力学支持了它们在测量快速神经信号传导和离子通道门控期间钾离子浓度变化方面的实用性。
