Concurrent measurements of the free cytosolic concentrations of H⁺ and Na⁺ ions with fluorescent indicators

Abstract

We report a method for the concurrent measurement of intracellular [Na⁺] ([Na⁺]_i) and pH (pH_i) in cells co-loaded with SBFI, a Na⁺-sensitive fluorophore, and either carboxy SNARF-1 or SNARF-5F, H⁺-sensitive fluorophores. With the optical filters specified, fluorescence emissions from SBFI and either SNARF derivative were sufficiently distinct to allow the accurate measurement of [Na⁺]_i and pH_i in rat hippocampal neurons. Neither the Na⁺ sensitivity of SBFI nor the pH sensitivities of carboxy SNARF-1 or SNARF-5F was affected by the presence of a SNARF derivative or SBFI, respectively. In addition, the calibration parameters obtained in neurons single-loaded with SBFI, carboxy SNARF-1 or SNARF-5F were not significantly influenced by the presence of a second fluorophore. In contrast to the established weak sensitivity of SBFI for protons, both SNARF derivatives appeared essentially insensitive to changes in [Na⁺]_i. The utility of the technique was demonstrated in neurons co-loaded with SBFI and SNARF-5F, which was found to have a lower pK_a in situ than carboxy SNARF-1. There were no significant differences in the changes in [Na⁺]_i and pH_i observed in response either to intracellular acid loads imposed by the NH₄⁺ prepulse technique or to transient periods of anoxia in neurons single-loaded with SBFI or SNARF-5F or co-loaded with both probes. The findings support the feasibility of using SBFI in conjunction with either carboxy SNARF-1 or SNARF-5F to concurrently and accurately measure [Na⁺]_i and pH_i.