Millisecond Association Kinetics of K + with Triazacryptand-Based K + Indicators Measured by Fluorescence Correlation Spectroscopy Mazin Magzoub, † Prashant Padmawar, † James A. Dix, ‡ and A. S. Verkman* ,† Departments of Medicine and Physiology, UniVersity of California at San Francisco, San Francisco, California 94143-0521, and Department of Chemistry, State UniVersity of New York at Binghamton, Binghamton, New York 13902-6000 ReceiVed: May 30, 2006; In Final Form: August 25, 2006 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(τ), 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 ∼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(τ) 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. Introduction Ion-sensing fluorescent indicators have applications in the measurement of ion content and transport in cellular and extracellular aqueous compartments. The response kinetics of indicator fluorescence to changes in ion concentration are particularly important in applications involving rapid neural signal transduction and ion channel gating. Available pH and Cl - indicators, with sensing mechanisms that involve protona- tion/deprotonation and collisional quenching, respectively, have sub-microsecond response kinetics. 1,2 However, sensors that involve cation binding to ionophores, such as crown ethers and cryptands, are predicted to have substantially slower response kinetics. 3-6 We recently introduced a water-soluble K + -sensing fluores- cent indicator, TAC-Red, consisting of a K + -binding triaza- cryptand ionophore coupled to a xanthylium chromophore. 7 TAC-Red fluorescence is low in the absence of K + because of charge-transfer quenching, increasing by >20-fold upon K + / triazacryptand association. TAC-Red was applied to visualize K + waves in the brain cortex in living mice in a spreading depression model of neuroexcitability in which brain extracel- lular space K + concentration increases locally from ∼5 to >40 mM over 5-10 s, producing a K + wave propagating over the cortical surface at a velocity of 3-4 mm/min. The purpose of this study was to determine the K + response kinetics of TAC-Red fluorescence as well as that of a new, longer-wavelength K + sensor introduced here, TAC-Crimson. Because the response kinetics of indicator fluorescence to changes in K + were faster than those resolvable by stopped- flow analysis, we applied fluorescence correlation spectroscopy (FCS), exploiting indicator fluorescence enhancement upon K + binding. In FCS, fluorescent molecules are excited by a sharply focused laser beam, and the fluorescence in a small observation volume is recorded over time. Temporal autocorrelation of fluorescence intensity fluctuations, which are produced either by fluorescent molecules diffusing into and out of the volume element or by transitions between chromophore states of differing intrinsic fluorescence, provide quantitative information about chromophore diffusion and kinetics. 8-10 Methods Synthesis of Triazacryptand-Based K + Indicators. TAC- Red (Figure 1A) was synthesized as described previously. 7 For synthesis of TAC-Crimson (Figure 1B), intermediate 2 was synthesized by stirring aldehyde (1, 72 mg, 0.10 mmol) and 9-hydroxyjulolidine (41 mg, 0.22 mmol) in 1 mL of propionic acid with catalytic amount of p-toluenesulfonic acid (PTSA) for 20 h at 60-70 °C. After being cooled, 2 was precipitated with 3 M sodium acetate, and the precipitated solid was collected by centrifugation, washed with water, and dried, giving 0.106 g of a brown-rose solid, which was used immediately for subsequent reaction. TAC-Crimson (3) was synthesized by stirring 2 (106 mg, 0.1 mmol) and tetrachloro-1,4-benzoquinone (49 mg, 0.2 mmol) in methanol/chloroform (1:1) at ambient temperature for 16-20 h. Excess tetrachloro-1,4-benzoquinone was removed by filtration, and the reaction mixture was concentrated under reduced pressure. The residue was purified twice by chromatography on silica gel using CHCl 3 /MeOH/ AcOH (9:1:0.1) as eluent, yielding 7 mg of TAC-Crimson as a crimson-dark violet semisolid (overall yield ∼5%). 1 H NMR (400 MHz, CDCl 3 , ppm): δ 7.9-6.4 (m, 11H), 4.8-2.5 (m, * Author to whom correspondence should be addressed. Phone: (415) 476-8530. Fax: (415) 665-3847. E-mail: verkman@itsa.ucsf.edu. † University of California at San Francisco. ‡ State University of New York at Binghamton. 21216 J. Phys. Chem. B 2006, 110, 21216-21221 10.1021/jp0633392 CCC: $33.50 © 2006 American Chemical Society Published on Web 09/27/2006