Millisecond association kinetics of K⁺ with triazacryptand-based K⁺ indicators measured by fluorescence correlation spectroscopy

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.