Millisecond Association Kinetics of K+ with Triazacryptand-Based K+ Indicators Measured by Fluorescence Correlation Spectroscopy

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Abstract
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.