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, 825827). 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.
ASJC Scopus subject areas
- Physical and Theoretical Chemistry
- Surfaces, Coatings and Films
- Materials Chemistry