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

Control of Glutamate Transport by Extracellular Potassium: Basis for a Negative Feedback on Synaptic Transmission

Glutamate and K+, both released during neuronal firing, need to be tightly regulated to ensure accurate synaptic transmission. Extracellular glutamate and K+ ([K+]o) are rapidly taken up by glutamate transporters and K+-transporters or channels, respectively. Glutamate transport includes the exchange of one glutamate, 3 Na+, and one proton, in exchange for one K+. This K+ efflux allows the glutamate binding site to reorient in the outwardly facing position and start a new transport cycle. Here, we demonstrate the sensitivity of the transport process to [K+]o changes. Increasing [K+]o over the physiological range had an immediate and reversible inhibitory action on glutamate transporters. This K+-dependent transporter inhibition was revealed using microspectrofluorimetry in primary astrocytes, and whole-cell patch-clamp in acute brain slices and HEK293 cells expressing glutamate transporters. Previous studies demonstrated that pharmacological inhibition of glutamate transporters decreases neuronal transmission via extrasynaptic glutamate spillover and subsequent activation of metabotropic glutamate receptors (mGluRs). Here, we demonstrate that increasing [K+]o also causes a decrease in neuronal mEPSC frequency, which is prevented by group II mGluR inhibition. These findings highlight a novel, previously unreported physiological negative feedback mechanism in which [K+]o elevations inhibit glutamate transporters, unveiling a new mechanism for activity-dependent modulation of synaptic activity

Extracellular Potassium and Glutamate Interact To Modulate Mitochondria in Astrocytes

Astrocytes clear glutamate and potassium, both of which are released into the extracellular space during neuronal activity. These processes are intimately linked with energy metabolism. Whereas astrocyte glutamate uptake causes cytosolic and mitochondrial acidification, extracellular potassium induces bicarbonate-dependent cellular alkalinization. This study aimed at quantifying the combined impact of glutamate and extracellular potassium on mitochondrial parameters of primary cultured astrocytes. Glutamate in 3 mM potassium caused a stronger acidification of mitochondria compared to cytosol. 15 mM potassium caused alkalinization that was stronger in the cytosol than in mitochondria. While the combined application of 15 mM potassium and glutamate led to a marked cytosolic alkalinization, pH only marginally increased in mitochondria. Thus, potassium and glutamate effects cannot be arithmetically summed, which also applies to their effects on mitochondrial potential and respiration. The data implies that, because of the nonlinear interaction between the effects of potassium and glutamate, astrocytic energy metabolism will be differentially regulated

Neuronal Loss of the Glutamate Transporter GLT-1 Promotes Excitotoxic Injury in the Hippocampus

GLT-1, the major glutamate transporter in the mammalian central nervous system, is expressed in presynaptic terminals that use glutamate as a neurotransmitter, in addition to astrocytes. It is widely assumed that glutamate homeostasis is regulated primarily by glutamate transporters expressed in astrocytes, leaving the function of GLT-1 in neurons relatively unexplored. We generated conditional GLT-1 knockout (KO) mouse lines to understand the cell-specific functions of GLT-1. We found that stimulus-evoked field extracellular postsynaptic potentials (fEPSPs) recorded in the CA1 region of the hippocampus were normal in the astrocytic GLT-1 KO but were reduced and often absent in the neuronal GLT-1 KO at 40 weeks. The failure of fEPSP generation in the neuronal GLT-1 KO was also observed in slices from 20 weeks old mice but not consistently from 10 weeks old mice. Using an extracellular FRET-based glutamate sensor, we found no difference in stimulus-evoked glutamate accumulation in the neuronal GLT-1 KO, suggesting a postsynaptic cause of the transmission failure. We hypothesized that excitotoxicity underlies the failure of functional recovery of slices from the neuronal GLT-1 KO. Consistent with this hypothesis, the non-competitive NMDA receptor antagonist MK801, when present in the ACSF during the recovery period following cutting of slices, promoted full restoration of fEPSP generation. The inclusion of an enzymatic glutamate scavenging system in the ACSF conferred partial protection. Excitotoxicity might be due to excess release or accumulation of excitatory amino acids, or to metabolic perturbation resulting in increased vulnerability to NMDA receptor activation. Previous studies have demonstrated a defect in the utilization of glutamate by synaptic mitochondria and aspartate production in the synGLT-1 KO in vivo, and we found evidence for similar metabolic perturbations in the slice preparation. In addition, mitochondrial cristae density was higher in synaptic mitochondria in the CA1 region in 20–25 weeks old synGLT-1 KO mice in the CA1 region, suggesting compensation for loss of axon terminal GLT-1 by increased mitochondrial efficiency. These data suggest that GLT-1 expressed in presynaptic terminals serves an important role in the regulation of vulnerability to excitotoxicity, and this regulation may be related to the metabolic role of GLT-1 expressed in glutamatergic axon terminals