All solutions were degassed to avoid CO2 contamination. The titrant (0.1 M NaOH,) was calibrated selleck compound with analytically pure, crystalline potassium hydrogen phthalate (KHP). The titration experiments were run at 25°C controlled by a circulating thermostatted bath. The ionic strength was fixed with

100 mM KCl. Data analysis and calculation of association constants were performed with HYPERQUAD software. All kinetic measurements were performed in pH 7 buffered solutions containing 50 mM of PIPES and 100 mM KCl. Millipore purified water was used to prepare all aqueous solutions. A glass electrode (Orion, Boston), calibrated before each use, was employed to determine solution pH. The kinetics of fluorescence quenching experiment was performed on a Photon Technology International (Lawrenceville, NJ) Quanta Master 4 L-format scanning spectrofluorimeter equipped with an LPS-220B 75-W xenon lamp and power supply, an A-1010B lamp housing with integrated igniter, a switchable

814 photon-counting/analog photomultiplier detection unit, and a MD-5020 motor driver. Samples were held in 1 × 1 cm quartz cuvettes (3.5 ml volume, Starna, Atascadero, CA). The kinetic traces www.selleckchem.com/products/s-gsk1349572.html were obtained by following fluorescence emission at 515 nm (λex = 494 nm); the fluorescence was recorded every one second for a total of 600 s. Double-mixing stopped-flow kinetics studies were performed with a Hi-Tech SF-61 DX2 apparatus equipped with fluorescence detection. Excitation was provided at 494 nm. A GG455 glass cutoff filter (<455 nm) was placed over the exit to the photomultiplier tube, and emission was monitored from 455 to 700 nm. The observed rate constants obtained from all sets of experiments were calculated by employing the Kinet-Assyst software package (HiTech) to fit individual traces to single exponentials. ZnT3 null mutant mice, obtained from Dr. Richard Palmiter, University of Washington, were generated by crossing male and female heterozygotes maintained on a

C57BL/6 background ( Cole et al., 1999). The genotype of each isothipendyl animal was verified twice using PCR of genomic DNA isolated from tail before and after experiments. Mice were anaesthetized with pentobarbital and decapitated, and hippocampal slices prepared for electrophysiological study. A bipolar tungsten-stimulating electrode was placed near the junction of the granule cell layer and hilus near the midpoint of the suprapyramidal blade of the dentate. Synaptic events were evoked by a stimulus pulse; 0.2 ms monopolar square pulses were delivered at 0.033 Hz with a Digitmer constant current stimulator (DS3, Digitimer Ltd. UK). Data were collected from slices at room temperature using a Multi 700A amplifier and pClamp 9.2 software (Molecular Devices, Sunnyvale, CA). Details of field potential and whole-cell recordings for assessment of mf-LTP are provided in Supplemental Experimental Procedures.