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Neuroscience
J Physiol 594.22 (2016) pp 6627–6641
Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity Brian Roland Larsen1 , Rikke Holm2 , Bente Vilsen2 and Nanna MacAulay1 1 2
Department Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark
Key points
r Management of glutamate and K+ in brain extracellular space is of critical importance to
The Journal of Physiology
neuronal function.
r The astrocytic α2β2 Na+ /K+ -ATPase isoform combination is activated by the K+ transients occurring during neuronal activity.
r In the present study, we report that glutamate transporter-mediated astrocytic Na+ transients r r
stimulate the Na+ /K+ -ATPase and thus the clearance of extracellular K+ . Specifically, the astrocytic α2β1 Na+ /K+ -ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+ . Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+ /K+ -ATPase β-subunits.
Abstract Neuronal activity is associated with transient [K+ ]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+ /K+ -ATPase of which several subunit isoform combinations exist. The astrocytic Na+ /K+ -ATPase α2β2 isoform constellation responds directly to increased [K+ ]o but, in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+ /K+ -ATPase was not fully saturated at basal astrocytic [Na+ ]i , irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i transients associated with activity-induced glutamate transporter activity. (Received 31 March 2016; accepted after revision 23 May 2016; first published online 27 May 2016) Corresponding author N. MacAulay: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Email: [email protected] Abbreviations EnaC, epithelial Na+ channel; GLT-1, glutamate transporter 1; I/V, current/voltage; TBOA, DL-threoβ-benzyloxyasparticacid. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
DOI: 10.1113/JP272531
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Introduction Transient increases in extracellular K+ occur in the mammalian brain during neuronal network activity. The removal of the excess K+ is of critical importance with respect to preventing widespread depolarization and associated compromised neuronal signalling. The surrounding astrocytes initially take up the excess K+ and thus represent K+ sinks during the neuronal activity after which K+ returns to the neuronal structures (Ballanyi et al. 1987; Grafe & Ballanyi, 1987). Although Kir4.1-mediated spatial buffering contributes to K+ management during the peak of the activity-induced K+ transient (possibly aided by other astrocytic K+ channels (P¨asler et al. 2007; Zhou et al. 2009; Mi Hwang et al. 2014)), the Na+ /K+ -ATPase acts as a key molecular mechanism responsible for the clearance of activity-induced K+ from the extracellular space (Karwoski et al. 1989; Ransom et al. 2000; D’Ambrosio et al. 2002; Kofuji & Newman, 2004; MacAulay & Zeuthen, 2012; Larsen et al. 2014; Larsen & MacAulay, 2014; Hertz et al. 2015). The Na+ /K+ -ATPase consists of an α- and β-subunit with multiple isoforms of each subunit (α1–3 and β1–3) present in the brain (McGrail et al. 1991; Cameron et al. 1994; Peng et al. 1997; Mart´ın-Vasallo et al. 2000; Richards et al. 2007; Bøttger et al. 2011). The isoforms display distinct cellular expression patterns, with most adult neurons expressing α1, α3 and β1 and astrocytes expressing α1, α2, β1 and β2 (McGrail et al. 1991; Cameron et al. 1994; Peng et al. 1997; Mart´ın-Vasallo et al. 2000; Li et al. 2013), resulting in different temporal and spatial roles for the individual subunit isoform combinations in the management of extracellular K+ and intracellular Na+ (Ransom et al. 2000; Larsen et al. 2014; Larsen et al. 2016). Astrocytes express the α1–2 and β1–2 isoforms in unknown combinations, although the α2 form has been reported to favour interaction with β2 in mouse brain extracts (Tokhtaeva et al. 2012). At the mRNA level, α2 and β2 are the quantitatively predominant Na+ /K+ -ATPase isoforms in this cell type along with β1 (Li et al. 2013; Zhang et al. 2014; Zhang et al. 2016), although it remains unresolved whether this pattern is reproduced at the protein level. We recently identified the α2β2 combination of the Na+ /K+ -ATPase as an important regulator of extracellular K+ as a result of its low K+ affinity and thereby its ability to respond directly to elevated [K+ ] in the extracellular space (Larsen et al. 2014). Partly based on co-localization of the Na+ /K+ -ATPase with glutamate transporters (Cholet et al. 2002; Rose et al. 2009; Rose & Karus, 2013), it has in addition, been shown that Na+ /K+ -ATPase activity in primary cultures of astrocytes could be influenced by glutamate, and probably by the Na+ accumulated via the glutamate transporters (Pellerin & Magistretti, 1997). Studies observing such
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[Na+ ]i -dependent activation of the Na+ /K+ -ATPase have exposed astrocyte culture or brain slices to glutamate transporter activation or the Na+ ionophore monensin for prolonged periods of 15–20 min (Pellerin & Magistretti, 1997; Munhoz et al. 2005; Sheean et al. 2013), leaving it unresolved whether the Na+ /K+ -ATPase is acutely activated by the brief [Na+ ]i transients observed in association with neuronal activity (Langer & Rose, 2009). A Na+ -dependent increase in Na+ /K+ -ATPase activity could occur given an apparent intracellular Na+ affinity sufficiently low to prevent saturation of the Na+ /K+ -ATPase at basal intracellular Na+ concentrations. However, the influence of the β isoform on the Na+ affinity of the Na+ /K+ -ATPase has remained uncharacterized. With the majority of Na+ /K+ -ATPase Na+ affinities being determined in open membranes and with β1 as the accessory subunit (because of its ubiquitous presence in most cellular systems) (Therien et al. 1996; Holm et al. 2015), the apparent intracellular Na+ affinity of the rodent astrocytic Na+ /K+ -ATPase isoform combinations is unexplored in a sided system with intact ion gradients and membrane potential. It therefore remains unknown to what extent the different astrocytic Na+ /K+ -ATPase isoform combinations are kinetically geared towards responding to glutamate transporter-mediated [Na+ ]i increases. Glutamate is the major excitatory neurotransmitter in the CNS (Danbolt, 2001) and, as such, is released to the extracellular space during neuronal activity. Glutamate is swiftly bound and subsequently removed from the extracellular space by the glutamate transporters driven by the electrochemical Na+ gradient across the cell membrane, with one transport cycle resulting in an influx of 1 glutamate, 3 Na+ and 1 H+ , along with efflux of 1 K+ (Levy et al. 1998; Danbolt, 2001; Vandenberg & Ryan, 2013). Of the five cloned glutamate transporters, glutamate transporter 1 (GLT-1) is the dominant isoform in the rodent hippocampus (Danbolt, 2001) and has been indicated to co-localize with the α2 isoform of the Na+ /K+ -ATPase in astrocytic processes (Cholet et al. 2002). Interestingly, a knock-in familial hemiplegic migraine type 2 mouse model expressing a mutant form of Na+ /K+ -ATPase α2 (G301R) displayed reduced astrocytic glutamate uptake kinetics (Bøttger et al. 2016), emphasizing the notion of a functional interplay between these two transport mechanisms. The present study therefore aimed to determine whether Na+ /K+ -ATPase-dependent clearance of stimulus-induced K+ transients in the extracellular space of the rat hippocampus is enhanced by simultaneous activity of the astrocytic glutamate transporters, and which Na+ /K+ -ATPase isoform combinations carry the ability to respond to intracellular Na+ transients.
C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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Na+ /K+ -ATPase and GLT-1 manage brain K+
Methods Ethical approval
The experiments were performed according to the guidelines of the Danish Veterinary and Food administration (Ministry of Environment and Food) and approved by the animal facility at the Faculty of Health and Medical Sciences, University of Copenhagen. The animal experiments conform to the principles and regulations described in (Grundy, 2015). Experiments were performed on brain tissue from male Sprague–Dawley rats (Taconic, Silkeborg, Denmark), postnatal day (P)21–P30 of age, housed in the animal facility at the Faculty of Health and Medical Sciences, University of Copenhagen. Rats were anaesthetized using gaseous 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane) (catalogue number B-4388; Sigma-Aldrich, Darmstadt, Germany) followed by decapitation. Xenopus laevis frogs were obtained from Nasco (Fort Atkinson, WI, USA) or the National Centre for Scientific Research (Paris, France) and anaesthetized with ethyl-m-aminobenzoatemethanesulfonate (tricaine) (catalogue number 103106; ICN Biomedicals, Santa Ana, CA, USA) prior to the surgical procedure. The surgical protocol, by which the oocytes were retrieved, was approved by The Danish National Committee for Animal Studies, Danish Veterinary and Food Administration (Ministry of Environment and Food). Brain slices and solutions
Following decapitation, the brain was quickly removed and placed into ice-cold cutting solution containing (in mM): 87 NaCl, 70 sucrose, 2.5 KCl, 0.5 CaCl2 , 25 NaHCO3 , 1.1 NaH2 PO4 , 7 MgCl2 and 25 D-glucose, equilibrated with gaseous 95% O2 , 5% CO2 . Oblique sagittal (transverse) hippocampal slices (400 μm) were cut with a Campden Vibrating Microtome (7000SMZ-2; Campden Instruments, Leicester, UK). Slices were transferred to the standard solution containing (in mM): 124 NaCl, 3 KCl, 2 CaCl2 , 25 NaHCO3 , 1.1 NaH2 PO4 , 2 MgCl2 and 10 D-glucose, and equilibrated with 95% O2, 5% CO2 (pH 7.4 at the experimental temperature of 33–34°C) and left to recover at 34°C for 30 min and then kept at room temperature. Ion-sensitive microelectrodes and electrophysiological recordings in slices
Electrophysiological recordings were carried out in a submerged-type recording chamber (Brain Slice Chamber 1, Scientific Systems Design; Digitimer Ltd, Welwyn Garden City, UK) with continuous superfusion at a flow rate of 2.2 ml min−1 . Recordings were performed within stratum radiatum of the CA1 region. High-frequency C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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stimulation was delivered by a concentric bipolar tungsten electrode (TM33CCNON; World Precision Instruments, Hitchin, UK) inserted into the stratum radiatum in the vicinity (500 μm) of the recording site. Stimulation trains (20–23 V at 20 Hz for 3 s) were delivered at 10 min intervals. The resulting extracellular field potentials were recorded with thin-walled filamented glass capillary microelectrodes (GC150TF-7.5; Harvard Apparatus, Cambridge, MA, USA) pulled to resistances of 15–25 M when filled with the standard solution (see above). This electrode served as reference signal for the ion-sensitive microelectrodes. Ion-sensitive microelectrodes were prepared from thin-walled non-filamented glass capillaries (GC150T-7.5; Harvard Apparatus) pulled to obtain a tip diameter in the range of 1–2 μm (Voipio et al. 1994). The capillaries were then silanized internally with gaseous N,N-dimethyltrimethylsilylamine (catalogue number 41716; Sigma-Aldrich) and baked at 180°C for 20 min prior to being backfilled with a solution containing 150 mM NaCl, 3 mM KCl followed by filling the tip of the capillary with a short column of a K+ -sensitive liquid membrane solution (IE190; World Precision Instruments). The tips of the ion-sensitive and reference electrode were placed within a few microns at the exact same depth in the core of the slice. Distance was ensured via Sensapex micromanipulators (SMX series; Sensapex, Oulu, Finland), which provide precise x, y and z co-ordinates (μm), by placing the electrode tips closely together above the slice and afterwards moving into the tissue maintaining this narrow distance. The ion-sensitive signal and the field potential signal were both recorded via an ION-01M amplifier and headstage (NPI Electronics, Tamm, Germany). All recorded signals were filtered at 0.250 kHz, sampled at 0.5 kHz and stored for offline analysis with WinEDR (courtesy of Dr John Dempster, University of Strathclyde, Glasgow, UK) and Prism, version 5.0 (GraphPad Software Inc., San Diego, CA, USA). The ion-sensitive microelectrodes were calibrated at the end of the experiments and the recorded signals were converted offline to obtain the K+ concentration (Voipio et al. 1994). For all experiments, at least three consecutive control responses were recorded at intervals of 10 min prior to bath application of drug. Time estimates of drug penetration into the recording site in the brain slice were obtained with tetramethylammonium (TMA+ ), a quartenary ion to which the K+ liquid membrane solution is highly sensitive. The exact diffusion properties within the brain slice tissue probably differ between drugs, although their transit time in the perfusion system should be identical. Using these time estimates, drugs were allowed to enter the slice and were then incubated for either 1 min (DL-threo-β-benzyloxyaspartic acid; TBOA) or 3 min (ouabain) prior to initiation of the stimulus protocol. A second response to ouabain was recorded at a 10 min interval to ascertain potential full effects.
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Heterologous expression in X. laevis oocytes
The preparation of defolliculated oocytes was carried out as described previously (Fenton et al. 2010) and the oocytes were kept in Kulori medium (in mM): 90 NaCl, 1 KCl, 1 CaCl2 , 1 MgCl2 and 5 Hepes (pH 7.4) for 24 h at 19°C prior to microinjection. Rat Na+ /K+ -ATPase α1, α2, β1 and β2 subunit isoforms (kindly provided by Gustavo Blanco, University of Kansas, Lawrence, KS, USA) and human epithelial Na+ channel (ENaC) subunits α, β and γ (kindly provided by Dan Klærke, University of Copenhagen, Copenhagen, Denmark) were subcloned into the oocyte expression vector pXOOM, linearized downstream from the poly-A segment and in vitro transcribed using T7 mMessage machine in accordance with the manufacturer’s instructions (Ambion, Austin, TX, USA). cRNA was extracted with MEGAclear (Ambion) and microinjected into defolliculated X. laevis oocytes: 10–20 ng of α1 or α2 RNA/oocyte in combination with 3–6 ng of β1 or β2 RNA/oocyte; for Na+ loading experiments, 1 ng of RNA/oocyte of each of the α, β and γ ENaC subunit was included (total ratio 10:3:1:1:1). The microinjected oocytes were kept in Kulori medium (in mM): 90 NaCl, 1 KCl, 1 CaCl2 , 1 MgCl2 and 5 Hepes (pH 7.4) for 4–5 days at 19°C prior to the experiments. Intracellular Na+ affinity measured via two-electrode voltage clamp of Xenopus oocytes
The electrophysiology recordings were carried out via conventional two-electrode voltage clamp using a CA-1B High Performance oocyte clamp (Dagan, Minneapolis, MN, USA) with a Digidata 1322A interface controlled by pCLAMP, version 9.2 (Molecular Devices, Burlingame, CA, USA). The oocyte membrane potential was voltage clamped at –50 mV. The current/voltage (I/V) curves were obtained by stepping the clamp potential from –50 mV to test potentials ranging from +40 mV to –100 mV in 20 mV increments (100 ms pulses). Recordings were low pass-filtered at 500 Hz and sampled at 2 kHz. Oocytes expressing both the Na+ /K+ -ATPase subunit isoforms of interest and the three subunits of the ENaC channel were switched to a Na+ -free medium with high K+ one day prior to electrophysiological recordings. This medium contained (in mM): 40 KCl, 62 cholineCl, 1 CaCl2 , 1 MgCl2 and 10 Hepes (pH 7.4) and was employed to deplete the oocyte of Na+ . To obtain the intracellular Na+ affinity, we employed a modified version of the methods of Crambert et al. (2000) and Horisberger and Kharoubi-Hess (2002) in which the oocytes are initially kept in a Na+ -loading medium (in mM): 10 NaCl, 92 cholineCl, 1 CaCl2 , 1 MgCl2 and 10 Hepes (pH 7.4) with 10 μM amiloride (catalogue number
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A7410; Sigma-Aldrich), a blocker of ENaC. Removal of amiloride allowed for a gradual Na+ loading via ENaC in oocytes voltage clamped to –50 mV, which terminates upon re-application of amiloride. The intracellular [Na+ ] obtained following this Na+ loading paradigm was determined by insertion of the reversal potential of the amiloride-sensitive membrane current into the Nernst equation. Immediately after re-introduction of amiloride, the oocyte was exposed to a test solution containing a saturating concentration of K+ (in mM): 10 NaCl, 10 KCl, 82 cholineCl, 1 CaCl2 , 1 MgCl2 and10 Hepes (pH 7.4). Na+ /K+ -ATPase activity is thus promoted (as a function of the increased [Na+ ]i ) and recorded as the membrane current (as described above). Further [Na+ ]i loading was obtained by repetition of the above procedure. To obtain the highest [Na+ ]i , a high-Na+ solution was employed (in mM): 100 NaCl, 2 cholineCl, 1 CaCl2 , 1 MgCl2 and 10 Hepes (pH 7.4) during the loading step and the clamp potential switched to –80 mV. The experiment was terminated by the addition of 1 mM ouabain (catalogue number O1325; Sigma-Aldrich) to the test solution followed by recording of the membrane current. This I/V curve was subtracted from the membrane currents obtained in the presence of K+ to provide the ouabain-sensitive currents attributable to the Na+ /K+ -ATPase. The obtained Na+ /K+ -ATPase activities were plotted as a function of [Na+ ]i to calculate K0.5 by means of a non-linear fit with the Hill equation (V = Vmax [Na+ ]n /(K0.5 n + [Na+ ]n ), where V is the Na+ /K+ -ATPase activity, K0.5 is the Na+ concentration giving half-maximum activation (apparent affinity), and n is the Hill coefficient) using Prism, version 5.0 (GraphPad Software Inc.). Phosphorylation assay on harvested oocyte membranes
The X. laevis oocytes expressing various isoform constellations (α1β1, α1β2, α2β1, α2β2) were transferred to an ice-cold Na+ -free homogenization buffer consisting of (in mM): 5 MgCl2 , 1 EDTA, 80 sucrose, 20 Tris (pH 7.4) and protease inhibitors; 8 μM leupeptin and 0.4 mM pefabloc (catalogue numbers L2884 and 76307, Sigma Aldrich, Germany). Oocytes were homogenized by pipetting and the homogenate was centrifuged for 10 min at 250 g to pellet cell debris. The supernatant was transferred to a new tube and centrifuged at 16,600 g for 20 min to pellet membranes, which were subsequently re-suspended in homogenization buffer and stored at –20°C. The membrane preparation harvested from the oocytes was made leaky by the addition of 20 μl of alamethicin (7 mg ml−1 ) (catalogue number A4665; Sigma-Aldrich) to allow access of incubation media from both sides of the membrane. The phosphorylation
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Na+ /K+ -ATPase and GLT-1 manage brain K+
reaction was carried out in the presence of 20 μg ml–1 oligomycin (catalogue number O4876; Sigma-Aldrich) to prevent dephosphorylation and thus allow accumulation of phosphoenzyme. Phosphorylation of the leaky oocyte membrane suspension (10 μg total membrane protein) was carried out for 5 s at 0°C in 100 μl of standard medium containing 2 μM [γ-32 P]ATP, 20 mM Tris (pH 7.4), 3 mM MgCl2 , 1 mM EGTA and various concentrations of NaCl (as indicated) with equimolar replacement with N-methyl-D-glucamine to keep the ionic strength constant at 150 mM. The phosphorylation reaction was terminated by the addition of 1 ml (1 M) H3 PO4 /H2 PO4 − (pH 2.4) (acid quenching) and the precipitated 32 P-labelled phosphoenzyme was collected by centrifugation at 20,800 g for 25 min followed by washing with 1 ml (0.25 M) H3 PO4 /H2 PO4 − (pH 2.4) by centrifugation at 20,800 g for 15 min. The resultant phosphoenzyme was subsequently subjected to SDS-PAGE at pH 6.0. The radioactivity associated with the separated Na+ /K+ -ATPase band was quantified by phosphor imaging using a cyclone storage phosphor system (Perkin Elmer, Boston, MA, USA). The background phosphorylation was determined in the presence of 50 mM KCl without NaCl and subtracted before calculation of the phosphorylation level. The Na+ dependence of phosphorylation was fitted using SigmaPlot (SPSS, Inc., Chicago, Ltd, USA) by a single Hill function (EP = EPmax [Na+ ]n /(K0.5 n + [Na+ ]n ), where EP is the phosphorylation level, EPmax is the maximum level of phosphorylation, K0.5 is the Na+ concentration giving half-maximum activation (apparent affinity) and n is the Hill coefficient. Statistical analysis
All data are reported as the mean ± SEM. Statistical significance was tested with Student’s t test (paired or unpaired), as indicated. P < 0.05 was considered statistically significant. Statistical calculations were performed using Prism, version 5.0 (GraphPad Software Inc.). Data on brain slices and oocytes were obtained from at least three different animal preparations. Results The astrocytic Na+ /K+ -ATPase is involved in determining the peak level of extracellular K+ in rat hippocampal slices during high-frequency stimulation
To determine the regulatory role of glutamate transport on the Na+ /K+ -ATPase in the management of stimulus-induced K+ transients in the extracellular space of the mammalian brain, we approximated a native setting using acute rat hippocampal slices. [K+ ]o was recorded via ion-sensitive microelectrodes monitoring [K+ ]o . The slice was electrically stimulated in the CA1 Schaeffer C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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collaterals of hippocampus and the subsequent K+ transient monitored via the K+ -sensitive microelectrode. Prior to initiation of a pharmacological approach to resolve the importance of glutamate transporter activity on K+ clearance, we ensured that consecutive stimuli (at 10 min intervals) resulted in equal peak [K+ ]o ; a representative example is provided in Fig. 1A (n = 4). Each slice thus acted as its own control in the experiments described below. The employed electrical stimulation paradigm resulted in peak extracellular [K+ ] in the range 5.5 – 13.0 mM (n = 7) in this experimental series. To initially determine the contribution of the Na+ /K+ -ATPase isoforms with high ouabain sensitivity (α2 and α3) in a setting with functional glutamate transporters, a stimulus train was obtained (representative trace illustrated in Fig. 1B) followed by exposure to a low concentration of ouabain (5 μM). Because prolonged inhibition of the Na+ /K+ -ATPase alters ionic gradients and leads to increased extracellular [K+ ], a K+ transient was recorded after the estimated arrival of the ouabain at the recording site (3 min after introduction of the ouabain-containing test solution to the experimental chamber; see Methods) (Fig. 1B). Addition of ouabain shifted the baseline level of [K+ ]o from 3.0 to 3.17 ± 0.02 mM (at 3 min, n = 7 slices from seven rats). Because astrocytes accumulate K+ during neuronal activity (whereas neurons accumulate K+ following the end of neuronal activity; Ballanyi et al. 1987; Grafe & Ballanyi, 1987), we quantified the effect of ouabain on the K+ transients as the peak [K+ ]o amplitude (obtained from the respective baseline [K+ ]o ) and, in addition, as the area under the curve from beginning of the trace to the time point t = 8 s, after which we ascribe a fraction of the K+ clearance to the neurons, rather than the return to basal [K+ ]o (Larsen et al. 2016). After brief exposure to ouabain, stimulus-induced peak [K+ ]o amplitudes were significantly increased (to 112 ± 4% of control, n = 7 slices from seven rats, P = 0.027, normalized to their own controls) (Fig. 1C, left), as was also the case for the area under the curve (to 115 ± 6% of control, n = 7 slices from seven rats, P = 0.047, normalized to their own controls) (Fig. 1C, right). To obtain a response upon full impact of ouabain, an additional trace was obtained with an interval of 10 min, at which time the baseline [K+ ]o was increased to 3.49 ± 0.03 mM (n = 6 slices from six rats); a representative trace is provided in Fig. 1B. The peak [K+ ]o amplitude was increased (to 124 ± 6% of control, n = 6 slices from six rats, P = 0.010) after prolonged ouabain exposure. The results reveal that K+ is cleared less efficiently during neuronal activity when ouabain has affected the Na+ /K+ -ATPase isoforms α2 and α3. The slowed [K+ ]o decay phase observed post-stimulus upon prolonged (complete) ouabain exposure is in line with our previous observations (Larsen et al. 2014). In the following experimental series, we opted for the brief ouabain exposure to prevent neuronal hyperexcitability during
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the combined inhibition of Na+ /K+ -ATPase and the glutamate transporters (see below), although we may well underestimate the contribution of the Na+ /K+ -ATPase by this approach. A
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Figure 1. Inhibition of the Na+ /K+ -ATPase results in less efficient clearance of K+ during electrical stimulation Ion-sensitive microelectrodes were employed to measure extracellular [K+ ]o in rat hippocampal slices. A, electrical stimulation (3 s at 20 Hz) of stratum radiatum in CA1 of hippocampus generated field potentials resulting in transient rises in [K+ ]o . Representative traces of stimulus-evoked changes in [K+ ]o at 10 min intervals are shown to illustrate [K+ ]o transients of equal amplitudes over time (n = 4). B, representative traces of stimulus-evoked changes in [K+ ]o in control solution, after exposure to 5 μM ouabain for 3 min and 13 min. The dashed line marks the initial 8 s segment used for the area under the curve quantification summarized in (C, right panel). C, Left: peak [K+ ]o amplitude (peak [K+ ]o minus [K+ ]o baseline prior to stimulation) was normalized to its own control and summarized for the brief (3 min) ouabain exposure. Right: area under the curve for the initial 8 s (marked by the dashed line in B) was normalized to its own control and summarized for the brief (3 min) ouabain exposure (n = 7 slices from seven rats). Data are presented as the mean ± SEM and statistical significance was determined with Student’s paired t test. ∗ P < 0.05. [Colour figure can be viewed at wileyonlinelibrary.com]
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Astrocytic glutamate transporters and the Na+ /K+ -ATPase has a coupled function to control extracellular levels of K+ in rat hippocampal slices
The experimental approach of ion-sensitive microelectrodes was next employed to define the impact of glutamate transport activity on K+ management in the extracellular space. In this experimental series, electrical stimulation of the Schaeffer collaterals promoted [K+ ]o transients ranging between 5.0 and 13.5 mM (n = 8 slices from seven rats); a representative trace is provided in Fig. 2A. The non-competitive glutamate transporter inhibitor TBOA was subsequently included in the test solution to block glutamate uptake in the brain slice (200 μM, which saturates all glutamate transporter isoforms with the predominant glial glutamate transporter, GLT-1, displaying an IC50 for TBOA of 6 μM and the other glial isoform, GLAST, an IC50 of 70 μM; Shimamoto et al. 1998; Shigeri et al. 2001; Waagepetersen et al. 2001). It is critical to realize that ineffective removal of glutamate will provide an extended time frame in which glutamate acts on its respective receptors in the post-synaptic terminal. In agreement with previous studies (Campbell & Hablitz, 2004; Tsukada et al. 2005; Campbell & Hablitz, 2008; Karus et al. 2015), prolonged exposure to TBOA thus resulted in spontaneous neuronal activity associated with extracellular K+ fluctuations (data not shown). We therefore recorded the stimulus-induced K+ transient 1 min after TBOA was introduced to the experimental chamber, at which time point no spontaneous activity was detected in any of the tested slices. Although we consistently observed an effect of this brief TBOA exposure, the full concentration of 200 μM was probably not reached in the core of the slice at the time of recording. However, with the low IC50 of the predominant hippocampal glutamate transporter isoform, GLT-1, the majority of the astrocytic glutamate uptake is predicted to be prevented under the selected experimental paradigm. As expected, exposure to TBOA and the associated prolonged presence of glutamate in the extracellular space increased the duration of the field potentials (defined as beginning of electrical stimulation until return to baseline) significantly (P = 0.019) from 10.8 ± 1.2 s to 27.7 ± 6.4 s, whereas the peak amplitude of the field potential in the presence of TBOA decreased to 70.9 ± 10.9% of control (P = 0.037) (n = 7). Still, quantification of the [K+ ]o obtained during stimulation revealed a significant increase in the K+ amplitude in the presence of TBOA (representative trace provided in Fig. 2A), resulting in a peak [K+ ]o amplitude of 138 ± 15% of control (n = 8 slices from seven rats, P = 0.004) (Fig. 2B, left) and an area under the curve (0–8 s) of 140 ± 9.6% of control (n = 8 slices from seven rats, P = 0.0043) (Fig. 2B, right panel). With the TBOA-induced reduction in field potential amplitude, the elevated [K+ ]o C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
Na+ /K+ -ATPase and GLT-1 manage brain K+
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connection with glutamate transport, we carried out a parallel series of experiments with combined blockage of the Na+ /K+ -ATPase and the glutamate transporters. A control trace was initially obtained upon electrical stimulation (peak [K+ ]o in the range 5.0–10.0 mM, n = 6 slices in six rats; representative trace provided in Fig. 2C). The pharmacological approaches above (Fig. 1B and 2A) were subsequently combined in a fashion mimicking the individual experiments (i.e. ouabain was
O A
should not result from increased stimulus-induced neuronal activity because of inefficient glutamate removal from the synaptic areas but could, in part, be assigned to reduced K+ clearance from the extracellular space. The K+ released into the extracellular space via the Na+ -K+ -coupled glutamate transporter is, in the presence of TBOA, absent and, if anything, should reduce [K+ ]o . To obtain the fraction of the K+ transient managed by the ouabain-sensitive isoforms of the Na+ /K+ -ATPase in
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Figure 2. The glutamate transporters and the Na+ /K+ -ATPase work in concert to manage extracellular K+ during electrical stimulation Ion-sensitive microelectrodes were employed to measure extracellular [K+ ]o in rat hippocampal slices, essentially as shown in Fig. 3. A, representative traces of stimulus-evoked changes in [K+ ]o prior to and after exposure to 200 μM TBOA for 1 min. The dashed line marks the initial 8 s segment used for the area under the curve quantification. B, peak [K+ ]o amplitude (left) or the area under the curve of the initial 8 s (right) in the presence of TBOA was normalized to the control and summarized (n = 8 slices from seven rats). C, representative traces of stimulus-evoked changes in [K+ ]o during control conditions and after exposure to a combination of 200 μM TBOA (for 1 min) and 5 μM ouabain (for 3 min). The dashed line marks the initial 8 s segment used for the area under the curve quantification. D, peak [K+ ]o amplitude (left) or the area under the curve of the initial 8 s (right) obtained in the presence of TBOA and ouabain was normalized to the control and summarized (n = 6 slices from six rats). Data are presented as the mean ± SEM and statistical significance determined with Student’s paired t test. ∗∗ P < 0.01; ∗∗∗ P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com] C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
B. R. Larsen and others
Intracellular Na+ affinity in an intact cell system
Glutamate transport-induced Na+ /K+ -ATPase activation, as observed above, requires that the Na+ binding sites on the intracellular face of the Na+ /K+ -ATPase are not saturated at the basal intracellular Na+ concentration. Astrocytes express the catalytic isoforms α1 and α2 and the associated isoforms β1 and β2 of the Na+ /K+ -ATPase
(McGrail et al. 1991; Cameron et al. 1994; Li et al. 2013), for which the apparent Na+ affinities are not fully characterized. To determine the apparent Na+ affinity of the rat astrocytic Na+ /K+ -ATPase isoform combinations (α1β1, α1β2, α2β1, α2β2) in an intact cell +
K+K+
Glutamate
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in ba
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introduced 3 min prior to the recording and TBOA 1 min before). Electrical stimulation at this exact time point provided robust increases in peak [K+ ]o amplitude (249 ± 40% of control, n = 6 slices from six rats, P < 0.001; representative trace provided in Fig. 2C; normalized values summarized in Fig. 2D, left) and area under the curve (0–8 s: 276 ± 50% of control, n = 6 slices from six rats, P < 0.001; normalized values summarized in Fig. 2D, right). Any indirect effects on extracellular K+ conferred by TBOA are predicted to be similar to those of the experiment described above, irrespective of whether ouabain is present or not. Thereby these effects, possibly including reduced astrocytic K+ clearance, contribute to the 40% increase in stimulus-induced [K+ ]o observed in Fig. 2B. To obtain the part of the K+ management assigned to the (predominantly glial) Na+ /K+ -ATPase activity, the ouabain-sensitive alteration in peak [K+ ]o was determined by deducting the response obtained in the presence of ouabain and TBOA (Fig. 2D, left) from that obtained with TBOA alone (Fig. 2B, left). To visualize the impact of glutamate transport on Na+ /K+ -ATPase activity, data from Fig. 1 and 2 are combined in Fig. 3 to obtain the ouabain-sensitive fraction of the K+ transients in the absence and presence of the glutamate transport inhibitor. The hatched part of the bars represents the excess peak [K+ ]o amplitude observed upon inhibition of the α2/α3 Na+ /K+ -ATPase. This fraction increases by 9-fold under conditions of abolished glutamate transport (n = 6–8) (and by 10-fold when calculated from the area under the curve, 0–8 s), indicating a synergistic effect of inhibitor combination vs. addition of the effects of the single exposures (P = 0.03, Student’s t test) and infers reduced Na+ /K+ -ATPase-mediated K+ clearance in the absence of parallel astrocytic glutamate transport. This fraction may be an underestimate because part of the increase in stimulus-induced [K+ ]o in the presence of TBOA alone can probably be assigned to slowed Na+ /K+ -ATPase-mediated K+ clearance in the absence of parallel glutamate transport-mediated [Na+ ]i transients. This synergistic effect of the transport inhibitors on K+ management suggests that the two transport mechanisms work in concert: the glutamate transporter-dependent increase in [Na+ ]i further activates the glial α2 isoform of the Na+ /K+ -ATPase and thereby augments its ability to remove K+ from the extracellular space during neuronal activity.
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peak [K+]o amplitude (% of control)
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Figure 3. Glutamate-induced intracellular Na+ elevation increases Na+ /K+ -ATPase activity During neuronal activity, K+ and glutamate are released into the synaptic space and are initially removed by the glial Na+ /K+ -ATPase and glutamate transporters. Inhibition of the ouabain-sensitive isoforms of the Na+ /K+ -ATPase increases the stimulus-evoked peak [K+ ]o , indicated as the hatched part of the column marked ‘ouabain’ in the histogram (signifies compromised K+ clearance by Na+ /K+ -ATPase). Inhibition of the glutamate transporters affects the stimulus-evoked peak [K+ ]o as a result of prolonged activation of the post-synaptic glutamate receptors and the blockade of the glutamate transporter-mediated release of K+ during its transport cycle. Inhibition of both the ouabain-sensitive isoforms of the Na+ /K+ -ATPase and the glutamate transporters yielded a larger stimulus-evoked peak [K+ ]o . The ouabain-sensitive K+ increase is indicated as the hatched part of the bar marked ‘TBOA + ouabain’. Comparison of these hatched areas (the ouabain-sensitive compromised K+ clearance without or with TBOA) illustrates the 9-fold larger Na+ /K+ -ATPase activity (presumably α2 as a result of it residing in the same compartment as the glutamate transporters) that is glutamate transporter-dependent. [Colour figure can be viewed at wileyonlinelibrary.com] C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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Na+ /K+ -ATPase and GLT-1 manage brain K+
system, these were heterologously expressed in Xenopus oocytes. This expression system displays negligible endogenous Na+ /K+ -ATPase activity (
Neuroscience
J Physiol 594.22 (2016) pp 6627–6641
Glutamate transporter activity promotes enhanced Na+/K+-ATPase-mediated extracellular K+ management during neuronal activity Brian Roland Larsen1 , Rikke Holm2 , Bente Vilsen2 and Nanna MacAulay1 1 2
Department Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark Department of Biomedicine, Aarhus University, Aarhus, Denmark
Key points
r Management of glutamate and K+ in brain extracellular space is of critical importance to
The Journal of Physiology
neuronal function.
r The astrocytic α2β2 Na+ /K+ -ATPase isoform combination is activated by the K+ transients occurring during neuronal activity.
r In the present study, we report that glutamate transporter-mediated astrocytic Na+ transients r r
stimulate the Na+ /K+ -ATPase and thus the clearance of extracellular K+ . Specifically, the astrocytic α2β1 Na+ /K+ -ATPase subunit combination displays an apparent Na+ affinity primed to react to physiological changes in intracellular Na+ . Accordingly, we demonstrate a distinct physiological role in K+ management for each of the two astrocytic Na+ /K+ -ATPase β-subunits.
Abstract Neuronal activity is associated with transient [K+ ]o increases. The excess K+ is cleared by surrounding astrocytes, partly by the Na+ /K+ -ATPase of which several subunit isoform combinations exist. The astrocytic Na+ /K+ -ATPase α2β2 isoform constellation responds directly to increased [K+ ]o but, in addition, Na+ /K+ -ATPase-mediated K+ clearance could be governed by astrocytic [Na+ ]i . During most neuronal activity, glutamate is released in the synaptic cleft and is re-absorbed by astrocytic Na+ -coupled glutamate transporters, thereby elevating [Na+ ]i . It thus remains unresolved whether the different Na+ /K+ -ATPase isoforms are controlled by [K+ ]o or [Na+ ]i during neuronal activity. Hippocampal slice recordings of stimulus-induced [K+ ]o transients with ion-sensitive microelectrodes revealed reduced Na+ /K+ -ATPase-mediated K+ management upon parallel inhibition of the glutamate transporter. The apparent intracellular Na+ affinity of isoform constellations involving the astrocytic β2 has remained elusive as a result of inherent expression of β1 in most cell systems, as well as technical challenges involved in measuring intracellular affinity in intact cells. We therefore expressed the different astrocytic isoform constellations in Xenopus oocytes and determined their apparent Na+ affinity in intact oocytes and isolated membranes. The Na+ /K+ -ATPase was not fully saturated at basal astrocytic [Na+ ]i , irrespective of isoform constellation, although the β1 subunit conferred lower apparent Na+ affinity to the α1 and α2 isoforms than the β2 isoform. In summary, enhanced astrocytic Na+ /K+ -ATPase-dependent K+ clearance was obtained with parallel glutamate transport activity. The astrocytic Na+ /K+ -ATPase isoform constellation α2β1 appeared to be specifically geared to respond to the [Na+ ]i transients associated with activity-induced glutamate transporter activity. (Received 31 March 2016; accepted after revision 23 May 2016; first published online 27 May 2016) Corresponding author N. MacAulay: Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen N, Denmark. Email: [email protected] Abbreviations EnaC, epithelial Na+ channel; GLT-1, glutamate transporter 1; I/V, current/voltage; TBOA, DL-threoβ-benzyloxyasparticacid. C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
DOI: 10.1113/JP272531
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Introduction Transient increases in extracellular K+ occur in the mammalian brain during neuronal network activity. The removal of the excess K+ is of critical importance with respect to preventing widespread depolarization and associated compromised neuronal signalling. The surrounding astrocytes initially take up the excess K+ and thus represent K+ sinks during the neuronal activity after which K+ returns to the neuronal structures (Ballanyi et al. 1987; Grafe & Ballanyi, 1987). Although Kir4.1-mediated spatial buffering contributes to K+ management during the peak of the activity-induced K+ transient (possibly aided by other astrocytic K+ channels (P¨asler et al. 2007; Zhou et al. 2009; Mi Hwang et al. 2014)), the Na+ /K+ -ATPase acts as a key molecular mechanism responsible for the clearance of activity-induced K+ from the extracellular space (Karwoski et al. 1989; Ransom et al. 2000; D’Ambrosio et al. 2002; Kofuji & Newman, 2004; MacAulay & Zeuthen, 2012; Larsen et al. 2014; Larsen & MacAulay, 2014; Hertz et al. 2015). The Na+ /K+ -ATPase consists of an α- and β-subunit with multiple isoforms of each subunit (α1–3 and β1–3) present in the brain (McGrail et al. 1991; Cameron et al. 1994; Peng et al. 1997; Mart´ın-Vasallo et al. 2000; Richards et al. 2007; Bøttger et al. 2011). The isoforms display distinct cellular expression patterns, with most adult neurons expressing α1, α3 and β1 and astrocytes expressing α1, α2, β1 and β2 (McGrail et al. 1991; Cameron et al. 1994; Peng et al. 1997; Mart´ın-Vasallo et al. 2000; Li et al. 2013), resulting in different temporal and spatial roles for the individual subunit isoform combinations in the management of extracellular K+ and intracellular Na+ (Ransom et al. 2000; Larsen et al. 2014; Larsen et al. 2016). Astrocytes express the α1–2 and β1–2 isoforms in unknown combinations, although the α2 form has been reported to favour interaction with β2 in mouse brain extracts (Tokhtaeva et al. 2012). At the mRNA level, α2 and β2 are the quantitatively predominant Na+ /K+ -ATPase isoforms in this cell type along with β1 (Li et al. 2013; Zhang et al. 2014; Zhang et al. 2016), although it remains unresolved whether this pattern is reproduced at the protein level. We recently identified the α2β2 combination of the Na+ /K+ -ATPase as an important regulator of extracellular K+ as a result of its low K+ affinity and thereby its ability to respond directly to elevated [K+ ] in the extracellular space (Larsen et al. 2014). Partly based on co-localization of the Na+ /K+ -ATPase with glutamate transporters (Cholet et al. 2002; Rose et al. 2009; Rose & Karus, 2013), it has in addition, been shown that Na+ /K+ -ATPase activity in primary cultures of astrocytes could be influenced by glutamate, and probably by the Na+ accumulated via the glutamate transporters (Pellerin & Magistretti, 1997). Studies observing such
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[Na+ ]i -dependent activation of the Na+ /K+ -ATPase have exposed astrocyte culture or brain slices to glutamate transporter activation or the Na+ ionophore monensin for prolonged periods of 15–20 min (Pellerin & Magistretti, 1997; Munhoz et al. 2005; Sheean et al. 2013), leaving it unresolved whether the Na+ /K+ -ATPase is acutely activated by the brief [Na+ ]i transients observed in association with neuronal activity (Langer & Rose, 2009). A Na+ -dependent increase in Na+ /K+ -ATPase activity could occur given an apparent intracellular Na+ affinity sufficiently low to prevent saturation of the Na+ /K+ -ATPase at basal intracellular Na+ concentrations. However, the influence of the β isoform on the Na+ affinity of the Na+ /K+ -ATPase has remained uncharacterized. With the majority of Na+ /K+ -ATPase Na+ affinities being determined in open membranes and with β1 as the accessory subunit (because of its ubiquitous presence in most cellular systems) (Therien et al. 1996; Holm et al. 2015), the apparent intracellular Na+ affinity of the rodent astrocytic Na+ /K+ -ATPase isoform combinations is unexplored in a sided system with intact ion gradients and membrane potential. It therefore remains unknown to what extent the different astrocytic Na+ /K+ -ATPase isoform combinations are kinetically geared towards responding to glutamate transporter-mediated [Na+ ]i increases. Glutamate is the major excitatory neurotransmitter in the CNS (Danbolt, 2001) and, as such, is released to the extracellular space during neuronal activity. Glutamate is swiftly bound and subsequently removed from the extracellular space by the glutamate transporters driven by the electrochemical Na+ gradient across the cell membrane, with one transport cycle resulting in an influx of 1 glutamate, 3 Na+ and 1 H+ , along with efflux of 1 K+ (Levy et al. 1998; Danbolt, 2001; Vandenberg & Ryan, 2013). Of the five cloned glutamate transporters, glutamate transporter 1 (GLT-1) is the dominant isoform in the rodent hippocampus (Danbolt, 2001) and has been indicated to co-localize with the α2 isoform of the Na+ /K+ -ATPase in astrocytic processes (Cholet et al. 2002). Interestingly, a knock-in familial hemiplegic migraine type 2 mouse model expressing a mutant form of Na+ /K+ -ATPase α2 (G301R) displayed reduced astrocytic glutamate uptake kinetics (Bøttger et al. 2016), emphasizing the notion of a functional interplay between these two transport mechanisms. The present study therefore aimed to determine whether Na+ /K+ -ATPase-dependent clearance of stimulus-induced K+ transients in the extracellular space of the rat hippocampus is enhanced by simultaneous activity of the astrocytic glutamate transporters, and which Na+ /K+ -ATPase isoform combinations carry the ability to respond to intracellular Na+ transients.
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Methods Ethical approval
The experiments were performed according to the guidelines of the Danish Veterinary and Food administration (Ministry of Environment and Food) and approved by the animal facility at the Faculty of Health and Medical Sciences, University of Copenhagen. The animal experiments conform to the principles and regulations described in (Grundy, 2015). Experiments were performed on brain tissue from male Sprague–Dawley rats (Taconic, Silkeborg, Denmark), postnatal day (P)21–P30 of age, housed in the animal facility at the Faculty of Health and Medical Sciences, University of Copenhagen. Rats were anaesthetized using gaseous 2-bromo-2-chloro-1,1,1-trifluoroethane (halothane) (catalogue number B-4388; Sigma-Aldrich, Darmstadt, Germany) followed by decapitation. Xenopus laevis frogs were obtained from Nasco (Fort Atkinson, WI, USA) or the National Centre for Scientific Research (Paris, France) and anaesthetized with ethyl-m-aminobenzoatemethanesulfonate (tricaine) (catalogue number 103106; ICN Biomedicals, Santa Ana, CA, USA) prior to the surgical procedure. The surgical protocol, by which the oocytes were retrieved, was approved by The Danish National Committee for Animal Studies, Danish Veterinary and Food Administration (Ministry of Environment and Food). Brain slices and solutions
Following decapitation, the brain was quickly removed and placed into ice-cold cutting solution containing (in mM): 87 NaCl, 70 sucrose, 2.5 KCl, 0.5 CaCl2 , 25 NaHCO3 , 1.1 NaH2 PO4 , 7 MgCl2 and 25 D-glucose, equilibrated with gaseous 95% O2 , 5% CO2 . Oblique sagittal (transverse) hippocampal slices (400 μm) were cut with a Campden Vibrating Microtome (7000SMZ-2; Campden Instruments, Leicester, UK). Slices were transferred to the standard solution containing (in mM): 124 NaCl, 3 KCl, 2 CaCl2 , 25 NaHCO3 , 1.1 NaH2 PO4 , 2 MgCl2 and 10 D-glucose, and equilibrated with 95% O2, 5% CO2 (pH 7.4 at the experimental temperature of 33–34°C) and left to recover at 34°C for 30 min and then kept at room temperature. Ion-sensitive microelectrodes and electrophysiological recordings in slices
Electrophysiological recordings were carried out in a submerged-type recording chamber (Brain Slice Chamber 1, Scientific Systems Design; Digitimer Ltd, Welwyn Garden City, UK) with continuous superfusion at a flow rate of 2.2 ml min−1 . Recordings were performed within stratum radiatum of the CA1 region. High-frequency C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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stimulation was delivered by a concentric bipolar tungsten electrode (TM33CCNON; World Precision Instruments, Hitchin, UK) inserted into the stratum radiatum in the vicinity (500 μm) of the recording site. Stimulation trains (20–23 V at 20 Hz for 3 s) were delivered at 10 min intervals. The resulting extracellular field potentials were recorded with thin-walled filamented glass capillary microelectrodes (GC150TF-7.5; Harvard Apparatus, Cambridge, MA, USA) pulled to resistances of 15–25 M when filled with the standard solution (see above). This electrode served as reference signal for the ion-sensitive microelectrodes. Ion-sensitive microelectrodes were prepared from thin-walled non-filamented glass capillaries (GC150T-7.5; Harvard Apparatus) pulled to obtain a tip diameter in the range of 1–2 μm (Voipio et al. 1994). The capillaries were then silanized internally with gaseous N,N-dimethyltrimethylsilylamine (catalogue number 41716; Sigma-Aldrich) and baked at 180°C for 20 min prior to being backfilled with a solution containing 150 mM NaCl, 3 mM KCl followed by filling the tip of the capillary with a short column of a K+ -sensitive liquid membrane solution (IE190; World Precision Instruments). The tips of the ion-sensitive and reference electrode were placed within a few microns at the exact same depth in the core of the slice. Distance was ensured via Sensapex micromanipulators (SMX series; Sensapex, Oulu, Finland), which provide precise x, y and z co-ordinates (μm), by placing the electrode tips closely together above the slice and afterwards moving into the tissue maintaining this narrow distance. The ion-sensitive signal and the field potential signal were both recorded via an ION-01M amplifier and headstage (NPI Electronics, Tamm, Germany). All recorded signals were filtered at 0.250 kHz, sampled at 0.5 kHz and stored for offline analysis with WinEDR (courtesy of Dr John Dempster, University of Strathclyde, Glasgow, UK) and Prism, version 5.0 (GraphPad Software Inc., San Diego, CA, USA). The ion-sensitive microelectrodes were calibrated at the end of the experiments and the recorded signals were converted offline to obtain the K+ concentration (Voipio et al. 1994). For all experiments, at least three consecutive control responses were recorded at intervals of 10 min prior to bath application of drug. Time estimates of drug penetration into the recording site in the brain slice were obtained with tetramethylammonium (TMA+ ), a quartenary ion to which the K+ liquid membrane solution is highly sensitive. The exact diffusion properties within the brain slice tissue probably differ between drugs, although their transit time in the perfusion system should be identical. Using these time estimates, drugs were allowed to enter the slice and were then incubated for either 1 min (DL-threo-β-benzyloxyaspartic acid; TBOA) or 3 min (ouabain) prior to initiation of the stimulus protocol. A second response to ouabain was recorded at a 10 min interval to ascertain potential full effects.
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Heterologous expression in X. laevis oocytes
The preparation of defolliculated oocytes was carried out as described previously (Fenton et al. 2010) and the oocytes were kept in Kulori medium (in mM): 90 NaCl, 1 KCl, 1 CaCl2 , 1 MgCl2 and 5 Hepes (pH 7.4) for 24 h at 19°C prior to microinjection. Rat Na+ /K+ -ATPase α1, α2, β1 and β2 subunit isoforms (kindly provided by Gustavo Blanco, University of Kansas, Lawrence, KS, USA) and human epithelial Na+ channel (ENaC) subunits α, β and γ (kindly provided by Dan Klærke, University of Copenhagen, Copenhagen, Denmark) were subcloned into the oocyte expression vector pXOOM, linearized downstream from the poly-A segment and in vitro transcribed using T7 mMessage machine in accordance with the manufacturer’s instructions (Ambion, Austin, TX, USA). cRNA was extracted with MEGAclear (Ambion) and microinjected into defolliculated X. laevis oocytes: 10–20 ng of α1 or α2 RNA/oocyte in combination with 3–6 ng of β1 or β2 RNA/oocyte; for Na+ loading experiments, 1 ng of RNA/oocyte of each of the α, β and γ ENaC subunit was included (total ratio 10:3:1:1:1). The microinjected oocytes were kept in Kulori medium (in mM): 90 NaCl, 1 KCl, 1 CaCl2 , 1 MgCl2 and 5 Hepes (pH 7.4) for 4–5 days at 19°C prior to the experiments. Intracellular Na+ affinity measured via two-electrode voltage clamp of Xenopus oocytes
The electrophysiology recordings were carried out via conventional two-electrode voltage clamp using a CA-1B High Performance oocyte clamp (Dagan, Minneapolis, MN, USA) with a Digidata 1322A interface controlled by pCLAMP, version 9.2 (Molecular Devices, Burlingame, CA, USA). The oocyte membrane potential was voltage clamped at –50 mV. The current/voltage (I/V) curves were obtained by stepping the clamp potential from –50 mV to test potentials ranging from +40 mV to –100 mV in 20 mV increments (100 ms pulses). Recordings were low pass-filtered at 500 Hz and sampled at 2 kHz. Oocytes expressing both the Na+ /K+ -ATPase subunit isoforms of interest and the three subunits of the ENaC channel were switched to a Na+ -free medium with high K+ one day prior to electrophysiological recordings. This medium contained (in mM): 40 KCl, 62 cholineCl, 1 CaCl2 , 1 MgCl2 and 10 Hepes (pH 7.4) and was employed to deplete the oocyte of Na+ . To obtain the intracellular Na+ affinity, we employed a modified version of the methods of Crambert et al. (2000) and Horisberger and Kharoubi-Hess (2002) in which the oocytes are initially kept in a Na+ -loading medium (in mM): 10 NaCl, 92 cholineCl, 1 CaCl2 , 1 MgCl2 and 10 Hepes (pH 7.4) with 10 μM amiloride (catalogue number
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A7410; Sigma-Aldrich), a blocker of ENaC. Removal of amiloride allowed for a gradual Na+ loading via ENaC in oocytes voltage clamped to –50 mV, which terminates upon re-application of amiloride. The intracellular [Na+ ] obtained following this Na+ loading paradigm was determined by insertion of the reversal potential of the amiloride-sensitive membrane current into the Nernst equation. Immediately after re-introduction of amiloride, the oocyte was exposed to a test solution containing a saturating concentration of K+ (in mM): 10 NaCl, 10 KCl, 82 cholineCl, 1 CaCl2 , 1 MgCl2 and10 Hepes (pH 7.4). Na+ /K+ -ATPase activity is thus promoted (as a function of the increased [Na+ ]i ) and recorded as the membrane current (as described above). Further [Na+ ]i loading was obtained by repetition of the above procedure. To obtain the highest [Na+ ]i , a high-Na+ solution was employed (in mM): 100 NaCl, 2 cholineCl, 1 CaCl2 , 1 MgCl2 and 10 Hepes (pH 7.4) during the loading step and the clamp potential switched to –80 mV. The experiment was terminated by the addition of 1 mM ouabain (catalogue number O1325; Sigma-Aldrich) to the test solution followed by recording of the membrane current. This I/V curve was subtracted from the membrane currents obtained in the presence of K+ to provide the ouabain-sensitive currents attributable to the Na+ /K+ -ATPase. The obtained Na+ /K+ -ATPase activities were plotted as a function of [Na+ ]i to calculate K0.5 by means of a non-linear fit with the Hill equation (V = Vmax [Na+ ]n /(K0.5 n + [Na+ ]n ), where V is the Na+ /K+ -ATPase activity, K0.5 is the Na+ concentration giving half-maximum activation (apparent affinity), and n is the Hill coefficient) using Prism, version 5.0 (GraphPad Software Inc.). Phosphorylation assay on harvested oocyte membranes
The X. laevis oocytes expressing various isoform constellations (α1β1, α1β2, α2β1, α2β2) were transferred to an ice-cold Na+ -free homogenization buffer consisting of (in mM): 5 MgCl2 , 1 EDTA, 80 sucrose, 20 Tris (pH 7.4) and protease inhibitors; 8 μM leupeptin and 0.4 mM pefabloc (catalogue numbers L2884 and 76307, Sigma Aldrich, Germany). Oocytes were homogenized by pipetting and the homogenate was centrifuged for 10 min at 250 g to pellet cell debris. The supernatant was transferred to a new tube and centrifuged at 16,600 g for 20 min to pellet membranes, which were subsequently re-suspended in homogenization buffer and stored at –20°C. The membrane preparation harvested from the oocytes was made leaky by the addition of 20 μl of alamethicin (7 mg ml−1 ) (catalogue number A4665; Sigma-Aldrich) to allow access of incubation media from both sides of the membrane. The phosphorylation
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reaction was carried out in the presence of 20 μg ml–1 oligomycin (catalogue number O4876; Sigma-Aldrich) to prevent dephosphorylation and thus allow accumulation of phosphoenzyme. Phosphorylation of the leaky oocyte membrane suspension (10 μg total membrane protein) was carried out for 5 s at 0°C in 100 μl of standard medium containing 2 μM [γ-32 P]ATP, 20 mM Tris (pH 7.4), 3 mM MgCl2 , 1 mM EGTA and various concentrations of NaCl (as indicated) with equimolar replacement with N-methyl-D-glucamine to keep the ionic strength constant at 150 mM. The phosphorylation reaction was terminated by the addition of 1 ml (1 M) H3 PO4 /H2 PO4 − (pH 2.4) (acid quenching) and the precipitated 32 P-labelled phosphoenzyme was collected by centrifugation at 20,800 g for 25 min followed by washing with 1 ml (0.25 M) H3 PO4 /H2 PO4 − (pH 2.4) by centrifugation at 20,800 g for 15 min. The resultant phosphoenzyme was subsequently subjected to SDS-PAGE at pH 6.0. The radioactivity associated with the separated Na+ /K+ -ATPase band was quantified by phosphor imaging using a cyclone storage phosphor system (Perkin Elmer, Boston, MA, USA). The background phosphorylation was determined in the presence of 50 mM KCl without NaCl and subtracted before calculation of the phosphorylation level. The Na+ dependence of phosphorylation was fitted using SigmaPlot (SPSS, Inc., Chicago, Ltd, USA) by a single Hill function (EP = EPmax [Na+ ]n /(K0.5 n + [Na+ ]n ), where EP is the phosphorylation level, EPmax is the maximum level of phosphorylation, K0.5 is the Na+ concentration giving half-maximum activation (apparent affinity) and n is the Hill coefficient. Statistical analysis
All data are reported as the mean ± SEM. Statistical significance was tested with Student’s t test (paired or unpaired), as indicated. P < 0.05 was considered statistically significant. Statistical calculations were performed using Prism, version 5.0 (GraphPad Software Inc.). Data on brain slices and oocytes were obtained from at least three different animal preparations. Results The astrocytic Na+ /K+ -ATPase is involved in determining the peak level of extracellular K+ in rat hippocampal slices during high-frequency stimulation
To determine the regulatory role of glutamate transport on the Na+ /K+ -ATPase in the management of stimulus-induced K+ transients in the extracellular space of the mammalian brain, we approximated a native setting using acute rat hippocampal slices. [K+ ]o was recorded via ion-sensitive microelectrodes monitoring [K+ ]o . The slice was electrically stimulated in the CA1 Schaeffer C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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collaterals of hippocampus and the subsequent K+ transient monitored via the K+ -sensitive microelectrode. Prior to initiation of a pharmacological approach to resolve the importance of glutamate transporter activity on K+ clearance, we ensured that consecutive stimuli (at 10 min intervals) resulted in equal peak [K+ ]o ; a representative example is provided in Fig. 1A (n = 4). Each slice thus acted as its own control in the experiments described below. The employed electrical stimulation paradigm resulted in peak extracellular [K+ ] in the range 5.5 – 13.0 mM (n = 7) in this experimental series. To initially determine the contribution of the Na+ /K+ -ATPase isoforms with high ouabain sensitivity (α2 and α3) in a setting with functional glutamate transporters, a stimulus train was obtained (representative trace illustrated in Fig. 1B) followed by exposure to a low concentration of ouabain (5 μM). Because prolonged inhibition of the Na+ /K+ -ATPase alters ionic gradients and leads to increased extracellular [K+ ], a K+ transient was recorded after the estimated arrival of the ouabain at the recording site (3 min after introduction of the ouabain-containing test solution to the experimental chamber; see Methods) (Fig. 1B). Addition of ouabain shifted the baseline level of [K+ ]o from 3.0 to 3.17 ± 0.02 mM (at 3 min, n = 7 slices from seven rats). Because astrocytes accumulate K+ during neuronal activity (whereas neurons accumulate K+ following the end of neuronal activity; Ballanyi et al. 1987; Grafe & Ballanyi, 1987), we quantified the effect of ouabain on the K+ transients as the peak [K+ ]o amplitude (obtained from the respective baseline [K+ ]o ) and, in addition, as the area under the curve from beginning of the trace to the time point t = 8 s, after which we ascribe a fraction of the K+ clearance to the neurons, rather than the return to basal [K+ ]o (Larsen et al. 2016). After brief exposure to ouabain, stimulus-induced peak [K+ ]o amplitudes were significantly increased (to 112 ± 4% of control, n = 7 slices from seven rats, P = 0.027, normalized to their own controls) (Fig. 1C, left), as was also the case for the area under the curve (to 115 ± 6% of control, n = 7 slices from seven rats, P = 0.047, normalized to their own controls) (Fig. 1C, right). To obtain a response upon full impact of ouabain, an additional trace was obtained with an interval of 10 min, at which time the baseline [K+ ]o was increased to 3.49 ± 0.03 mM (n = 6 slices from six rats); a representative trace is provided in Fig. 1B. The peak [K+ ]o amplitude was increased (to 124 ± 6% of control, n = 6 slices from six rats, P = 0.010) after prolonged ouabain exposure. The results reveal that K+ is cleared less efficiently during neuronal activity when ouabain has affected the Na+ /K+ -ATPase isoforms α2 and α3. The slowed [K+ ]o decay phase observed post-stimulus upon prolonged (complete) ouabain exposure is in line with our previous observations (Larsen et al. 2014). In the following experimental series, we opted for the brief ouabain exposure to prevent neuronal hyperexcitability during
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Figure 1. Inhibition of the Na+ /K+ -ATPase results in less efficient clearance of K+ during electrical stimulation Ion-sensitive microelectrodes were employed to measure extracellular [K+ ]o in rat hippocampal slices. A, electrical stimulation (3 s at 20 Hz) of stratum radiatum in CA1 of hippocampus generated field potentials resulting in transient rises in [K+ ]o . Representative traces of stimulus-evoked changes in [K+ ]o at 10 min intervals are shown to illustrate [K+ ]o transients of equal amplitudes over time (n = 4). B, representative traces of stimulus-evoked changes in [K+ ]o in control solution, after exposure to 5 μM ouabain for 3 min and 13 min. The dashed line marks the initial 8 s segment used for the area under the curve quantification summarized in (C, right panel). C, Left: peak [K+ ]o amplitude (peak [K+ ]o minus [K+ ]o baseline prior to stimulation) was normalized to its own control and summarized for the brief (3 min) ouabain exposure. Right: area under the curve for the initial 8 s (marked by the dashed line in B) was normalized to its own control and summarized for the brief (3 min) ouabain exposure (n = 7 slices from seven rats). Data are presented as the mean ± SEM and statistical significance was determined with Student’s paired t test. ∗ P < 0.05. [Colour figure can be viewed at wileyonlinelibrary.com]
J Physiol 594.22
Astrocytic glutamate transporters and the Na+ /K+ -ATPase has a coupled function to control extracellular levels of K+ in rat hippocampal slices
The experimental approach of ion-sensitive microelectrodes was next employed to define the impact of glutamate transport activity on K+ management in the extracellular space. In this experimental series, electrical stimulation of the Schaeffer collaterals promoted [K+ ]o transients ranging between 5.0 and 13.5 mM (n = 8 slices from seven rats); a representative trace is provided in Fig. 2A. The non-competitive glutamate transporter inhibitor TBOA was subsequently included in the test solution to block glutamate uptake in the brain slice (200 μM, which saturates all glutamate transporter isoforms with the predominant glial glutamate transporter, GLT-1, displaying an IC50 for TBOA of 6 μM and the other glial isoform, GLAST, an IC50 of 70 μM; Shimamoto et al. 1998; Shigeri et al. 2001; Waagepetersen et al. 2001). It is critical to realize that ineffective removal of glutamate will provide an extended time frame in which glutamate acts on its respective receptors in the post-synaptic terminal. In agreement with previous studies (Campbell & Hablitz, 2004; Tsukada et al. 2005; Campbell & Hablitz, 2008; Karus et al. 2015), prolonged exposure to TBOA thus resulted in spontaneous neuronal activity associated with extracellular K+ fluctuations (data not shown). We therefore recorded the stimulus-induced K+ transient 1 min after TBOA was introduced to the experimental chamber, at which time point no spontaneous activity was detected in any of the tested slices. Although we consistently observed an effect of this brief TBOA exposure, the full concentration of 200 μM was probably not reached in the core of the slice at the time of recording. However, with the low IC50 of the predominant hippocampal glutamate transporter isoform, GLT-1, the majority of the astrocytic glutamate uptake is predicted to be prevented under the selected experimental paradigm. As expected, exposure to TBOA and the associated prolonged presence of glutamate in the extracellular space increased the duration of the field potentials (defined as beginning of electrical stimulation until return to baseline) significantly (P = 0.019) from 10.8 ± 1.2 s to 27.7 ± 6.4 s, whereas the peak amplitude of the field potential in the presence of TBOA decreased to 70.9 ± 10.9% of control (P = 0.037) (n = 7). Still, quantification of the [K+ ]o obtained during stimulation revealed a significant increase in the K+ amplitude in the presence of TBOA (representative trace provided in Fig. 2A), resulting in a peak [K+ ]o amplitude of 138 ± 15% of control (n = 8 slices from seven rats, P = 0.004) (Fig. 2B, left) and an area under the curve (0–8 s) of 140 ± 9.6% of control (n = 8 slices from seven rats, P = 0.0043) (Fig. 2B, right panel). With the TBOA-induced reduction in field potential amplitude, the elevated [K+ ]o C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
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connection with glutamate transport, we carried out a parallel series of experiments with combined blockage of the Na+ /K+ -ATPase and the glutamate transporters. A control trace was initially obtained upon electrical stimulation (peak [K+ ]o in the range 5.0–10.0 mM, n = 6 slices in six rats; representative trace provided in Fig. 2C). The pharmacological approaches above (Fig. 1B and 2A) were subsequently combined in a fashion mimicking the individual experiments (i.e. ouabain was
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should not result from increased stimulus-induced neuronal activity because of inefficient glutamate removal from the synaptic areas but could, in part, be assigned to reduced K+ clearance from the extracellular space. The K+ released into the extracellular space via the Na+ -K+ -coupled glutamate transporter is, in the presence of TBOA, absent and, if anything, should reduce [K+ ]o . To obtain the fraction of the K+ transient managed by the ouabain-sensitive isoforms of the Na+ /K+ -ATPase in
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J Physiol 594.22
Figure 2. The glutamate transporters and the Na+ /K+ -ATPase work in concert to manage extracellular K+ during electrical stimulation Ion-sensitive microelectrodes were employed to measure extracellular [K+ ]o in rat hippocampal slices, essentially as shown in Fig. 3. A, representative traces of stimulus-evoked changes in [K+ ]o prior to and after exposure to 200 μM TBOA for 1 min. The dashed line marks the initial 8 s segment used for the area under the curve quantification. B, peak [K+ ]o amplitude (left) or the area under the curve of the initial 8 s (right) in the presence of TBOA was normalized to the control and summarized (n = 8 slices from seven rats). C, representative traces of stimulus-evoked changes in [K+ ]o during control conditions and after exposure to a combination of 200 μM TBOA (for 1 min) and 5 μM ouabain (for 3 min). The dashed line marks the initial 8 s segment used for the area under the curve quantification. D, peak [K+ ]o amplitude (left) or the area under the curve of the initial 8 s (right) obtained in the presence of TBOA and ouabain was normalized to the control and summarized (n = 6 slices from six rats). Data are presented as the mean ± SEM and statistical significance determined with Student’s paired t test. ∗∗ P < 0.01; ∗∗∗ P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com] C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
B. R. Larsen and others
Intracellular Na+ affinity in an intact cell system
Glutamate transport-induced Na+ /K+ -ATPase activation, as observed above, requires that the Na+ binding sites on the intracellular face of the Na+ /K+ -ATPase are not saturated at the basal intracellular Na+ concentration. Astrocytes express the catalytic isoforms α1 and α2 and the associated isoforms β1 and β2 of the Na+ /K+ -ATPase
(McGrail et al. 1991; Cameron et al. 1994; Li et al. 2013), for which the apparent Na+ affinities are not fully characterized. To determine the apparent Na+ affinity of the rat astrocytic Na+ /K+ -ATPase isoform combinations (α1β1, α1β2, α2β1, α2β2) in an intact cell +
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introduced 3 min prior to the recording and TBOA 1 min before). Electrical stimulation at this exact time point provided robust increases in peak [K+ ]o amplitude (249 ± 40% of control, n = 6 slices from six rats, P < 0.001; representative trace provided in Fig. 2C; normalized values summarized in Fig. 2D, left) and area under the curve (0–8 s: 276 ± 50% of control, n = 6 slices from six rats, P < 0.001; normalized values summarized in Fig. 2D, right). Any indirect effects on extracellular K+ conferred by TBOA are predicted to be similar to those of the experiment described above, irrespective of whether ouabain is present or not. Thereby these effects, possibly including reduced astrocytic K+ clearance, contribute to the 40% increase in stimulus-induced [K+ ]o observed in Fig. 2B. To obtain the part of the K+ management assigned to the (predominantly glial) Na+ /K+ -ATPase activity, the ouabain-sensitive alteration in peak [K+ ]o was determined by deducting the response obtained in the presence of ouabain and TBOA (Fig. 2D, left) from that obtained with TBOA alone (Fig. 2B, left). To visualize the impact of glutamate transport on Na+ /K+ -ATPase activity, data from Fig. 1 and 2 are combined in Fig. 3 to obtain the ouabain-sensitive fraction of the K+ transients in the absence and presence of the glutamate transport inhibitor. The hatched part of the bars represents the excess peak [K+ ]o amplitude observed upon inhibition of the α2/α3 Na+ /K+ -ATPase. This fraction increases by 9-fold under conditions of abolished glutamate transport (n = 6–8) (and by 10-fold when calculated from the area under the curve, 0–8 s), indicating a synergistic effect of inhibitor combination vs. addition of the effects of the single exposures (P = 0.03, Student’s t test) and infers reduced Na+ /K+ -ATPase-mediated K+ clearance in the absence of parallel astrocytic glutamate transport. This fraction may be an underestimate because part of the increase in stimulus-induced [K+ ]o in the presence of TBOA alone can probably be assigned to slowed Na+ /K+ -ATPase-mediated K+ clearance in the absence of parallel glutamate transport-mediated [Na+ ]i transients. This synergistic effect of the transport inhibitors on K+ management suggests that the two transport mechanisms work in concert: the glutamate transporter-dependent increase in [Na+ ]i further activates the glial α2 isoform of the Na+ /K+ -ATPase and thereby augments its ability to remove K+ from the extracellular space during neuronal activity.
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Figure 3. Glutamate-induced intracellular Na+ elevation increases Na+ /K+ -ATPase activity During neuronal activity, K+ and glutamate are released into the synaptic space and are initially removed by the glial Na+ /K+ -ATPase and glutamate transporters. Inhibition of the ouabain-sensitive isoforms of the Na+ /K+ -ATPase increases the stimulus-evoked peak [K+ ]o , indicated as the hatched part of the column marked ‘ouabain’ in the histogram (signifies compromised K+ clearance by Na+ /K+ -ATPase). Inhibition of the glutamate transporters affects the stimulus-evoked peak [K+ ]o as a result of prolonged activation of the post-synaptic glutamate receptors and the blockade of the glutamate transporter-mediated release of K+ during its transport cycle. Inhibition of both the ouabain-sensitive isoforms of the Na+ /K+ -ATPase and the glutamate transporters yielded a larger stimulus-evoked peak [K+ ]o . The ouabain-sensitive K+ increase is indicated as the hatched part of the bar marked ‘TBOA + ouabain’. Comparison of these hatched areas (the ouabain-sensitive compromised K+ clearance without or with TBOA) illustrates the 9-fold larger Na+ /K+ -ATPase activity (presumably α2 as a result of it residing in the same compartment as the glutamate transporters) that is glutamate transporter-dependent. [Colour figure can be viewed at wileyonlinelibrary.com] C 2016 The Authors. The Journal of Physiology C 2016 The Physiological Society
J Physiol 594.22
Na+ /K+ -ATPase and GLT-1 manage brain K+
system, these were heterologously expressed in Xenopus oocytes. This expression system displays negligible endogenous Na+ /K+ -ATPase activity (
