Extracellular potassium and glutamate interact to modulate

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Extracellular potassium and glutamate interact to modulate mitochondria in astrocytes Theresa S Rimmele, Haissa de Castro Abrantes, Joel Wellbourne-Wood, Sylvain Lengacher, and Jean-Yves Chatton ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00124 • Publication Date (Web): 09 May 2018 Downloaded from http://pubs.acs.org on May 14, 2018

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ACS Chemical Neuroscience

Rimmele et al.

Manuscript ID: cn-2018-00124c

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Extracellular potassium and glutamate interact to modulate mitochondria in astrocytes

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Theresa S. Rimmele #,1 , Haissa de Castro Abrantes#, Joel Wellbourne-Wood#, Sylvain

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Lengacher§,2, and Jean-Yves Chatton#*

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#

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Department of Fundamental Neurosciences, University of Lausanne, 1005 Lausanne, Switzerland, §Brain and Mind Institute, EPFL, 1015 Lausanne, Switzerland

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*Corresponding author:

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Jean-Yves Chatton, PhD Dept. of Fundamental Neurosciences University of Lausanne Rue Bugnon 9 Ch-1005 Lausanne, Switzerland Tel. +41-21-692-5106 Fax: +41-21-692-5105 E-mail: [email protected]

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Medical School, Boston, MA, 02115

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Present address: F.M. Kirby Neurobiology Center, Boston Children’s Hospital, Harvard

Present address: GliaPharm, 1202 Geneva, Switzerland

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Running title: Glutamate, potassium and pH regulation of mitochondrial function

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Key words: astrocytes, glutamate transport, pH, mitochondria, extracellular potassium,

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mitochondrial potential, oxygen consumption rate, brain energy metabolism

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FOR TABLE OF CONTENTS USE ONLY

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Extracellular potassium and glutamate interact to modulate mitochondria in astrocytes

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by Theresa S. Rimmele, Haissa de Castro Abrantes, Joel Wellbourne-Wood, Sylvain

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Lengacher, and Jean-Yves Chatton

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ABSTRACT

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Astrocytes clear glutamate and potassium, both of which are released into the extracellular

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space during neuronal activity. These processes are intimately linked with energy

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metabolism. Whereas astrocyte glutamate uptake causes cytosolic and mitochondrial

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acidification, extracellular potassium induces bicarbonate-dependent cellular alkalinization.

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This study aimed at quantifying the combined impact of glutamate and extracellular

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potassium on mitochondrial parameters of primary cultured astrocytes. Glutamate in 3mM

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potassium caused a stronger acidification of mitochondria compared to cytosol. 15mM

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potassium caused alkalinization that was stronger in the cytosol than in mitochondria. While

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the combined application of 15mM potassium and glutamate led to a marked cytosolic

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alkalinization, pH only marginally increased in mitochondria. Thus, potassium and glutamate

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effects cannot be arithmetically summed, which also applies to their effects on

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mitochondrial potential and respiration. The data implies that, because of the non-linear

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interaction between the effects of potassium and glutamate, astrocytic energy metabolism

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will be differentially regulated.

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One of the principal roles played by astrocytes in the brain is to assist neurons with their

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high energy demands. A number of studies performed both in vitro1-4 and in vivo5, 6 have

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shown that during enhanced neuronal activity astrocytes engage their glycolytic metabolic

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pathway, which causes an increased glucose capture and utilization, and the subsequent

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release of lactate. This metabolic response, occurring in the presence of non-limiting oxygen

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is called aerobic glycolysis. The idea that astrocytes can favor aerobic glycolysis is puzzling,

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inasmuch as they possess a substantial density of mitochondria that enable them to

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produce ATP with a seventeen-fold higher yield. The higher glycolytic nature of astrocytes

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compared to neurons may be due to different expression levels of mitochondrial respiratory

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chain enzymes7. Mitochondria have been shown to regulate their internal ionic composition

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as the result of changes in cytosolic ion concentrations, which may adapt their metabolism

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to cellular energy demands. In previous studies our lab showed8 that rapid mitochondrial

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transients involving a localized ATP microdomain trigger a Na+-mediated mitochondrial

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depolarization in primary cultured mouse cortical astrocytes, which transiently enhances

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the activity of the mitochondrial respiratory chain. Glutamate transporters exhibit a

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complex stoichiometry with three Na+ and one H+ entering with glutamate in exchange for

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one K+9. Thus, glutamate uptake is coupled to the cellular entry of H+ and Na+, and is known

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to causes cytosolic acidification, observed both in cultured astrocytes10 and hippocampal

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slices11. According to the chemiosmotic coupling hypothesis, the mitochondrial matrix is

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expected to be more alkaline than the cytosol, because of the anticipated low permeability

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of the mitochondrial inner membrane and the constant extrusion of protons across

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mitochondrial inner membrane to cations by the mitochondrial respiratory chain12.

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However, the observed glutamate transport-induced cytosolic acidification was found to

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spread to mitochondria, resulting in a large pH drop in this compartment13. The acidification

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resulted in the collapse of the cytosol-to-mitochondrial matrix pH gradient.

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On the other hand, during neuronal activity, the repolarization phase of firing neurons

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leads to the release and diffusion of K+ into the extracellular space14. Astrocytes provide

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highly efficient mechanisms for rapid K+ uptake and redistribution in the brain15. They

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express both active and passive mechanisms for K+ uptake and release, such as the inwardly

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rectifying potassium channel 4.1 (Kir 4.1)16, 17, the Na+/K+/Cl- cotransporter, and the Na+/K+ 4 ACS Paragon Plus Environment

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ATPase18, 19. Astrocytes are extensively interconnected by gap junctions, forming a cellular

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syncytium, and are capable of redistributing K+ to distant sites17.

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Elevated extracellular K+ concentrations ([K+]o) mildly depolarize the surrounding

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astrocyte plasma membrane, which can cause the reversal of the Na+/bicarbonate

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exchanger (NBCe1) activity20. Thus, in bicarbonate-containing media, this effect results in an

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influx of bicarbonate into the cell leading to cytosolic alkalinization21. This mechanism was

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shown to enhance (cytosolic) glycolysis immediately and reversibly, via stimulation of the

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phosphofructokinase22.

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The aim of the present study was to investigate how glutamate and elevated [K+]o

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individually and simultaneously applied, impact metabolic responses of primary cultured

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astrocytes. We therefore studied cytosolic and mitochondrial pH, mitochondrial electrical

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potential (ΔΨmit), and cellular oxygen consumption rate (OCR) in physiological

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CO2/bicarbonate-buffered solutions, and investigated the influence of glutamate transport

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and elevated [K+]o in order to reach a better understanding of astrocyte energy metabolism

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regulation.

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RESULTS AND DISCUSSION

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We designed experiments to evaluate the respective impact of extracellular glutamate and

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K+, both known to fluctuate during network activity, on astrocyte cytosolic and

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mitochondrial pH under physiological conditions. To create conditions relevant to the in vivo

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setting, all experiments were performed in CO2/bicarbonate-buffered solutions. To

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investigate cytosolic pH changes, 2-3-week-old primary mouse cortical astrocytes were

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loaded with the pH sensitive fluorescent dye BCECF. BCECF-loaded cells (Fig. 1, a1) displayed

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intense fluorescence throughout their cytoplasm with typical bright nuclei23,

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comparison, astrocytes transfected with the MitoSypHer plasmid exhibited a fluorescent

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protein expression pattern typical of mitochondrial labeling25, with dark nuclei and punctate

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staining of rod-like structures 2-3 days after transfection (Fig. 1, a2). In both compartments

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we found a stable baseline of pH under resting conditions, which was on average 7.37±0.04

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in the cytosol and 7.25±0.07 in mitochondria.

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. In

Glutamate transport by astrocytes was shown to cause a cytosolic pH decrease as a result

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of the proton co-transported with glutamate10,

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glutamate transport on cytosolic and mitochondrial pH, respectively, in physiological

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CO2/bicarbonate-buffered solutions by applying glutamate (200µM) in low (3mM) K+

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containing solutions. In these and following experiments, the application of glutamate (or

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K+, see below) was maintained until a stable response (plateau) was achieved before

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returning to the control solution. The time of application therefore somewhat varied

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according the cells' responses in individual experiments. The application of glutamate

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caused cytosolic acidification (-0.07±0.003 pH units) as depicted in Fig. 1, b1. Glutamate-

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induced mitochondrial acidification was found to be stronger (Fig. 1, b2) (-0.18±0.03 pH

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units). A possible explanation for this effect could be a lower H+ buffering capacity of

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mitochondria in comparison to the cytosol, as was proposed before25. Another factor could

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be facilitated uptake of H+ into mitochondria. H+ can potentially enter this subcellular

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compartment via several mechanisms, including mitochondrial uncoupling proteins26 or

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cation–proton exchangers (e.g. Na+/H+ exchanger or K+/H+ exchanger)27. Overall, these

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results indicate that glutamate transporter activity causes cellular acidification in

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physiological CO2/bicarbonate buffered salines, and that this acidification is transmitted to

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mitochondria, similar to what was described before in HEPES-buffered solutions13.

. We first examined the effects of


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Figure 1. Respective effects of glutamate and [K+]o on intracellular pH. Cytosolic (left panels) and mitochondrial (right panels) pH in astrocytes were monitored using the fluorescent indicator BCECF (a1) and the genetically encoded MitoSypHer fluorescent protein targeted to the mitochondrial matrix (a2). Scale bars, 20µm. The effects of 200µM glutamate and 15mM K+ applied separately or in combination in bicarbonate-containing solutions are compared. Example traces of effects of glutamate application are shown for cytosolic pH (b1, n=32 cells, 4 exp) and mitochondrial pH (b2: n=9 cells, 8 exp), as well as the 15mM K+ effect on these parameters (c1: n=33 cells, 4 exp; c2: n=12 cells, 7 exp). Combined effects of high K+ and glutamate on cytosolic pH (d1: n=33 cells, 4 exp) and mitochondrial pH (d2: n=12 cells, 7 exp) are shown. (e) Quantitative effects of glutamate and [K+]o on cytosolic and mitochondrial pH. (i) Mean amplitudes of pH changes (± SEM) were compiled from experiments presented above. (ii) Raw projection of the expected pH changes in cytosol and mitochondria resulting from the arithmetic summation of acidification/alkalinization individually caused by 200µM glutamate and 15mM [K+]o. (iii) Projection taking into account the inhibitory action of elevated [K+]o on glutamate glial transport activity, as previously described34.

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A cellular alkalinizing effect of elevated [K+]o on astrocytes was described before28-31. We

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then investigated the effect of high [K+]o. We chose to apply a [K+]o of 15mM, close to the

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previously described [K+]o ceiling level32, as the highest concentration expected to be found

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in the brain. This concentration allows us to compare results to those of previous studies20,

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[K+]o was switched from 3 to 15mM (Fig. 1, c1), which is consistent with a response involving

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the NBCe1 activity as described other studies20,

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bicarbonate-free HEPES-buffered solutions (not shown). The alkalinization caused by the

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[K+]o switch was found to occur in mitochondria as well, with an amplitude of 0.12±0.02 pH

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units (Fig. 1, c2). Thus, the effect of [K+]o on pH was found to be stronger in the cytosol in

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comparison to mitochondria. pH in both compartments returned rapidly to their resting

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level after switching back to baseline [K+]o. Among possible mechanisms underlying the

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mitochondrial alkalinization is the K+ transport to mitochondrial matrix performed by the

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H+/K+ exchanger33.

. We observed a rapid and marked alkalinization of the cytosol (0.25±0.01 pH units) when

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. Alkalinization was not observed in

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In vivo, neuronal glutamate release and K+ extrusion into the extracellular space are two

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processes expected to overlap in time and space. We therefore combined the application of

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15mM K+ and 200µM glutamate in the next experiments. The addition of glutamate and

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high K+ resulted in a net alkalinization of cytosol (0.18±0.009 pH units) (Fig. 1, d1).


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Interestingly, we found mitochondrial pH to undergo only slight alkalinization (0.05±0.01 pH

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units) (Fig. 1, d2).

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The quantitative pH effects of glutamate and high K+ on astrocytes in bicarbonate-

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containing solutions are summarized in Fig. 1e, panel i). The figure displays the acidifying

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effects of glutamate on both cytosol (pH 7.24±0.01) and mitochondria (pH 7.02±0.05) and

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the alkalinization caused by high [K+]o in the two compartments (cytosol pH 7.68±0.01),

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mitochondria (pH 7.51± 0.06). The figure also indicates that the overall effect of combined

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application of glutamate and K+ on pH was a strong cytosolic alkalinization (pH 7.53±0.03),

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whereas mitochondria were only marginally alkalinized (pH 7.20± 0.03). In this condition,

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their combined effect on cytosolic pH was relatively similar, although lower, to the value

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estimated from the summation of the effect of glutamate and 15mM K+ individually (Fig. 1e,

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panel ii). It can be seen, however, that the pH change in mitochondria did not match the

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arithmetic projection of the effects in isolation (Fig. 1e, panel iii), meaning the acidifying and

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alkalinizing effects of glutamate and high [K+]o respectively did not add up in a linear

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manner.

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This observation indicates that glutamate and high [K+]o do not behave as independent

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variables, but somehow influence each other. Indeed, we recently demonstrated that [K+]o

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exerts tight control over the kinetics of the glutamate transporter, in which increasing [K+]o

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over the physiological range has an immediate and reversible inhibitory action on glutamate

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transporters34. To verify whether this interaction at the level of glutamate transporters

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could provide an explanation for this unanticipated results on mitochondrial pH, we

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considered the fact that the glutamate transport activity in 15mM [K+]o is actually reduced

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to 37% of its activity in 3mM [K+]o34. We postulated that the amplitude of the actual

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glutamate-mediated acidification in these high [K+]o conditions is correspondingly lower. Fig.

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1e (panel iii) shows that the calculated summation of glutamate- and [K+]o-mediated effects

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on pH becomes very close to the values observed experimentally (Fig. 1e, panel i) when the

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inhibitory effect of elevated [K+]o on glutamate transport is taken into account.

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[K+]o elevations have been shown to moderately depolarize the astrocyte plasma

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membrane, and because it is taken up by astrocytes, cytosolic K+ in turn also transiently

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increases24. On the other hand, astrocyte glutamate transporters (GLAST and GLT-1) operate

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with a complex stoichiometry of three Na+ and one H+ entering with glutamate in exchange 9 ACS Paragon Plus Environment


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for one K+9. Thus, this counter-transport of K+ was shown to cause measurable cytosolic K+

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decreases24. As cytosolic K+ fluctuations could impact ΔΨmit and their ability to produce ATP,

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we investigated their respective impact on the mitochondrial potential, which is estimated

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to be around -180mV under resting conditions. Glutamate transport was found to mildly

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hyperpolarize ΔΨmit, whereas uncoupling agents, for instance carbonyl cyanide-4-

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(trifluoromethoxy)phenylhydrazone (FCCP), rapidly depolarize mitochondria and abolish

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ΔΨmit35.

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To assess the impact of [K+]o alone and in combination with glutamate on ΔΨmit, we used

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the mitochondrial potential fluorescent indicator Rhodamine 123 (Rh123). We found that

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application of glutamate led to a slight decrease in Rh123 fluorescence (Fig. 2a,b), which

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corresponds to a hyperpolarization of ΔΨmit. Conversely, superfusion with 15mM K+ led to

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substantial increase in Rh123 fluorescence, i.e. depolarization of ΔΨmit. The figure shows

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that the combined application of glutamate and 15mM [K+]o caused a depolarization of

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ΔΨmit, however less efficiently than 15mM K+ alone. ΔΨmit responses between conditions

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overall followed the same pattern in HEPES-buffered solutions and in bicarbonate solutions

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(Fig. 3c). There was however a statistical difference between HEPES and bicarbonate

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conditions for the ΔΨmit response to 15mM K+ application (p=0.04) and the one to 15mM K+

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plus glutamate (p

Extracellular potassium and glutamate interact to modulate

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