Figure 1.
Spectrofluorimetric characterization of the K+ indicator APG-1.
(A) Emission spectra recorded in the presence of different [K+] in intracellular-like solutions following excitation at 515 nm. Emission maximum was ∼540 nm. (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. (C) Na+ dependency of APG-1 fluorescence measured in intracellular-like solution containing 135 mM K+ (see also Fig. S1). (D) 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.
Figure 2.
Intracellular characterization of the K+ indicator APG-1.
(A) Fluorescence image of primary astrocytes loaded using APG-1 AM. Scale bar 50 µm. (B) In situ excitation and emission spectra measured by fluorescence microscopy. Intracellular spectra were ∼10 nm red-shifted compared with measurement in cuvettes. (C) Representative experimental trace depicting the in situ 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. (D) Calibration curve obtained by plotting the fluorescence plateau values measured for each known [K+].
Figure 3.
Intracellular K+ is modulated by [K+]o level changes.
(A) 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) 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. (C) 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 (D) and duration of [K+]i rise (E) 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+.
Figure 4.
Intracellular K+ is modulated by glutamate application.
(A) Application of 200 µM glutamate induced a rapid and reversible [K+]i decrease with a amplitude of 2.3±0.1 mM (B) The [K+]i response to glutamate application in the range 0.1 µM to 10 mM followed a Michaelis and Menten kinetics with an apparent EC50 of 11.1±1.4 µM. (C) Simultaneous Na+ and K+ monitoring using SBFI and APG-1, respectively. SBFI was sequentially excited at 340 and 380 nm, and its fluorescence excitation ratio computed, and APG-1 was excited at 490 nm. Glutamate application (200 µM) is indicated in the graph. Exemplar single cell trace out of three experiments. (D) The fluorescent dye Fura-2 co-loaded with APG-1 was excited at 360 nm, its Ca2+-insensitive wavelength, whereas APG-1 was excited at 490 nm. Whereas 200 µM glutamate application caused a decline of the APG-1 fluorescence response, that of Fura-2 at 360 nm remained unperturbed (Exemplar trace out of two experiments).