Intracellular characterization of the K⁺ indicator APG-1.

<p>(<b>A</b>) Fluorescence image of primary astrocytes loaded using APG-1 AM. Scale bar 50 µm. (<b>B</b>) <i>In situ</i> excitation and emission spectra measured by fluorescence microscopy. Intracellular spectra were ∼10 nm red-shifted compared with measurement in cuvettes. (<b>C</b>) Representative experimental trace depicting the <i>in situ</i> calibration procedure. At the time indicated by the arrow, the cell membrane was permeabilized for K⁺ using valinomycin and nigericin while the Na⁺/K⁺ ATPase was inhibited by ouabain. Solutions of different [K⁺] were then sequentially applied until stable fluorescence plateaus were obtained. (<b>D</b>) Calibration curve obtained by plotting the fluorescence plateau values measured for each known [K⁺].</p

Spectrofluorimetric characterization of the K⁺ indicator APG-1.

<p>(<b>A</b>) Emission spectra recorded in the presence of different [K⁺] in intracellular-like solutions following excitation at 515 nm. Emission maximum was ∼540 nm. (<b>B</b>) Fluorescence emission plotted as a function of [K⁺] showing a monotonic relationship of APG-1 fluorescence with increasing [K⁺] (circles). The same analysis was performed on APG-2, a related indicator with identical spectral properties (diamonds) but lower Kd for K⁺. The plots show that APG-2 fluorescence becomes saturated at [K⁺]>80 mM, which is not the case with APG-1. (<b>C</b>) Na⁺ dependency of APG-1 fluorescence measured in intracellular-like solution containing 135 mM K⁺ (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109243#pone.0109243.s001" target="_blank">Fig. S1</a>). (<b>D</b>) pH dependency of APG-1 fluorescence measured in intracellular-like solution containing 135 mM K⁺. The pH of each solution was adjusted using NMDG. This pH analysis was repeated three times. Data are presented as means ± SEM of triplicate measurements.</p

Intracellular K⁺ is modulated by [K⁺]_o level changes.

<p>(<b>A</b>) Representative single-cell [K⁺]_i trace during bath application of solutions with different K⁺ concentrations in the range 3 to 10 mM, as are found during physiological and pathological conditions. (<b>B</b>) Relationship between steady-state [K⁺]_i (measured on plateau levels) and externally applied [K⁺]_o (n = 120 cells from 12 exp). The graph indicates a steady increase in [K⁺]_i in the [K⁺]_o range 3–10 mM (plain circles), which yielded a slope of 1.04±0.06 (r = 0.82). A higher [K⁺]_o of 15 mM (open circle) failed to further increase [K⁺]_i. (<b>C</b>) Intracellular K⁺ is influenced by localized K⁺-gluconate puff applications. Representative [K⁺]_i traces (average values of 7 cells each) during puff application (black arrows) of K⁺ gluconate in close proximity to the pipette (upper trace) and at>90 µm distance (lower trace). Insets: magnification of the trace after single extracellular applications of K⁺. Average amplitude (<b>D</b>) and duration of [K⁺]_i rise (<b>E</b>) induced by K⁺ puffs (black bar) compared with responses observed in the presence of 200 µM Ba²⁺ (white bar) or 20 µM carbenoxolone (CBX, grey bar) (n = 62 cells, 5 exp). No significant changes in amplitudes were found, whereas the response duration was significantly prolonged by CBX and reduced by Ba²⁺.</p