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

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.