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Ionic signalling in astroglia beyond calcium

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DOI: 10.1113/JP277478

Ionic signalling in astroglia beyond calcium

Alexei Verkhrastky1,2,3 , Verena Untiet2 & Christine R. Rose4

1Faculty of Biology, Medicine and Health, The University of Manchester, Manchester, M13 9PT, UK; 2Center for Basic and Translational Neuroscience, Faculty of Health and Medical Sciences, University

of Copenhagen, 2200 Copenhagen, Denmark; 3Achucarro Centre for Neuroscience, IKERBASQUE,

Basque Foundation for Science, 48011, Bilbao, Spain;

4Institute of Neurobiology, Faculty of Mathematics and Natural Sciences, Heinrich Heine University

Düsseldorf, Universitätsstrasse 1, D-40225 Düsseldorf, Germany

Running Title: Ionic Signalling in Astroglia

*Corresponding author:

Professor Alexei Verkhratsky, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK,

Telephone +44 (0)161-2757324, e-mail: [email protected]

Abstract

Astrocytes are homeostatic and protective cells of the central nervous system. Astroglial

homeostatic responses are tightly coordinated with neuronal activity. Astrocytes maintain neuronal

excitability through regulation of extracellular ion concentrations as well as assisting and modulating

synaptic transmission by uptake and catabolism of major neurotransmitters. Moreover, they support

neuronal metabolism and detoxify ammonium and reactive oxygen species. Astroglial homeostatic

actions are initiated and controlled by intercellular signalling of ions, including Ca2+, Na+, Cl-, H+ and

possibly K+. This treatise summarises current knowledge on ionic signals mediated by the major

monovalent ions, which occur in microdomains, as global events or as propagating intercellular

waves and thereby represent the substrate for astroglial excitability.



2

Key words: Astrocytes, Na+ signalling, Cl- signalling, K+ homoeostasis, H+ homeostasis

Ionic signalling

Ion gradients and the maintenance of ion homeostasis within the cytoplasm is a ubiquitous property

of all life forms on planet Earth. What was the ionic composition of the cytosol of the first cellular

ancestor, to what extent it reflected the ion concentrations of the primeval ocean (Kazmierczak et

al., 2013), what was the role of Donnan forces (Donnan, 1911) and which mechanisms (if any) this

primary cell employed to maintain cytosolic ion concentrations are all questions which remain

unanswered (Plattner & Verkhratsky, 2015, 2016, 2018). Environmental stresses (for example an

increase in osmotic pressure due to evaporation or its decrease due to rain) alter the concentrations

of extracellular ions and therefore trigger changes in intracellular ions. In this way, ions became

messengers coupling environmental challenges with cellular responses required for survival. Thus,

ionic signalling was born and it remains omnipresent in all cellular systems to this day. Cellular

physiology has always balanced the need for cytosolic ionic homeostasis (which, when

compromised, leads to rapid death) and the need to produce relevant intracellular ion changes that

trigger signalling cascades responsible for specific responses. Evolution has therefore selected both

for systems maintaining transmembrane ionic gradients and mechanisms for fast transmembrane

ion transport (which shape ionic signals), with the former enabling and regulating the latter.

Conceptually, changes in the cytosolic concentration of any ion, di- or monovalent, can contribute to

the regulation of cellular events, and hence any ion can act as a second messenger (Orlov & Hamet,

2006). Astrocytes, the electrically non-excitable homeostatic cells of the central nervous system

(CNS), utilise ionic signals as a substrate for their excitability (Verkhratsky et al., 2015; Verkhratsky &

Nedergaard, 2018). While using the same substrate (movement of ions across the plasma

membrane), glial excitability is distinct from the electrical excitability typical to neurones. Neurones

have been traditionally regarded as the only excitable cells of the nervous system because they

generate action potentials, which are conducted over large distances and can be transmitted to

other neurones through synaptic mechanisms. Research performed in the last decades, however,

has clearly established that the generation of specific, fast signals that are transmitted to other cells

is by no means an exclusive property of neurones. While not generating action potentials, astrocytes

do exhibit fast, transient fluctuations in their ionic content; moreover these ionic signals may

propagate passively or actively to neighbouring cells (Verkhratsky & Nedergaard, 2018).

Many of the acknowledged roles of astrocytes directly relate to transient changes in intracellular ion

concentrations, induced by neuronal activity. Most research has focused on Ca2+, and the signalling

role for Ca2+ in astroglia is widely accepted (Verkhratsky et al., 1998; Verkhratsky et al., 2012;

Volterra et al., 2014; Rusakov, 2015; Shigetomi et al., 2016). Astroglial Ca2+ signals result from

release of Ca2+ from intracellular stores, as well as from plasmalemmal Ca2+ influx (Shigetomi et al.,

2013; Bazargani & Attwell, 2016; Bindocci et al., 2017). In addition to Ca2+ signalling, astrocytes

experience transient changes in Na+, Cl-, K+ and H+ in response to physiological stimulation. These ion


3

transients reflect major functions that astrocytes fulfil in the nervous system. Among those is the

uptake of K+ from the extracellular space, which results in a rise in intraglial K+ concentrations ([K+]i).

In this way, astrocytes buffer the rise in extracellular K+ concentration ([K+]o) and sustain neuronal

excitability (Ballanyi et al., 1987; Kofuji & Newman, 2004). Another vital function of astrocytes, the

clearance of neurotransmitters from the extracellular space, is similarly related to ion signalling. The

uptake of glutamate and GABA, for example, is mediated by Na+-dependent transporters, which also

generate transient changes in sodium concentration ([Na+]i) in astrocytes (Rose & Ransom, 1996b;

Chatton et al., 2000; Kirischuk et al., 2007). These astroglial Na+ signals have been proposed to serve

an important role in neuro-metabolic coupling (Pellerin & Magistretti, 2012). Activity-related

transient changes in the intracellular pH (pHi) of astrocytes (Chesler & Kraig, 1989) have been

implicated in the stimulation of astrocyte metabolism in response to neuronal activity (Ruminot et

al., 2011). Finally, changes in Cl- concentration ([Cl-]i), induced by activation of Cl--dependent

transporters and/or Cl- channels are linked to activity-related changes in the cellular volume of

astrocytes (Chen & Sun, 2005; Kimelberg et al., 2006; Wilson & Mongin, 2018). The properties and

functional role of Ca2+ signalling has been extensively reviewed in the past, and in this paper we shall

therefore focus on the signalling role of the other four major monovalent ions: Na+, Cl-, K+ and H+.

Astroglial Na+ signalling

Sodium ions are fundamental for electrical signalling in the nervous system and are responsible for

the regenerative depolarising phase of the action potentials: inhibition of neuronal Na+ channels

therefore effectively extinguishes nervous impulse generation and propagation (Hodgkin & Huxley,

1952; Narahashi et al., 1964). In astrocytes, the inwardly directed transmembrane Na+ gradient

provides the main electrochemical driving force for the transport of ions, neurotransmitters,

neurotransmitter precursors and many other molecules, thus supporting astroglial homeostatic

functions. Dynamic changes in astroglial Na+ in response to physiological stimulation were

discovered in late 1990s, both in vitro (Rose & Ransom, 1996a) and in situ (Kirischuk et al., 1997).

Further studies revealed their link to glutamatergic neurotransmission (Kirischuk et al., 2007; Bennay

et al., 2008; Langer & Rose, 2009), and demonstrated intra- and intercellular diffusive spread of Na+

(Bernardinelli et al., 2004; Langer et al., 2012; Langer et al., 2017). The concept of astroglial Na+

signalling, coupling neuronal activity to fast local astroglial homeostatic responses has been

subsequently formalised (Kirischuk et al., 2012; Parpura & Verkhratsky, 2012; Rose & Karus, 2013;

Verkhratsky et al., 2013; Rose & Verkhratsky, 2016).

Molecular physiology of astroglial Na+ dynamics

At rest, the cytoplasmic Na+ concentration ([Na+]i) in astrocytes is maintained at a level of ~15 - 20

mM, being thus somewhat higher than in neurones (Rose & Ransom, 1996a; Kirischuk et al., 2012;

Reyes et al., 2012, 2013; Rose & Chatton, 2016). This resting level is maintained by the balance

between Na+ extrusion, mediated almost solely by the Na+-K+-ATPase (NKA) and Na+ influx, resulting


4

from the background activity of several transporters (for example Na+-2HCO3--co-transport by the

NBCe1, or the action of the Na+-H+ exchanger) and other (yet unidentified) Na+ channels. Inhibition

of NKA with ouabain or by removal of extracellular K+ produces a fast increase in [Na+]i, revealing this

constitutive Na+ influx (Rose & Ransom, 1996a; Golovina et al., 2003a; Illarionava et al., 2014).

Astroglial NKA incorporates the 2 catalytic subunit, which bestows properties distinct from the

neuronal Na+-K+ pump (composed of 1 and 3 subunits). The affinity of the astroglial 2 subunit to

extracellular K+ is much lower as compared to neurones: the [K+]0.5 for NKA composed from 2/1

subunits is ~3.6 mM, whereas [K+]0.5 for (neuronal) NKAs assembled from 1/1, 1/2, 3/1 or

3/2 subunits lies between 0.25 and 0.65 mM (Larsen et al., 2014). As a result of this molecular

composition, the astroglial NKA activity can be increased by physiological increases in the

extracellular K+ concentration, whereas K+ binding sites of the neuronal NKA are saturated and

neuronal NKA activity can be increased solely by an elevation in [Na+]i (Hertz et al., 2015). This

functional difference defines the leading role of astroglial NKA in K+ buffering (Larsen et al., 2014;

Verkhratsky & Nedergaard, 2018). Activity of astroglial NKA is regulated by noradrenergic

innervation via -adrenoceptors (Hajek et al., 1996) and by endogenous ouabain-like molecules

(Hertz et al., 2015; Larsen et al., 2016). Loss-of-function mutations in the 2 subunit are associated

with familial hemiplegic migraine type 2; in this pathology astrocytes are deficient both in K+

buffering and glutamate clearance (Capuani et al., 2016; Stoica et al., 2017).

Sodium influx in astrocytes is mediated by plasmalemmal channels and Na+-dependent solute carrier

transporters (Fig. 1). Depending on the brain region, astrocytes may express several types of

ionotropic receptors including N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-

isoxazolepropionic acid (AMPA) type glutamate receptors, P2X purinoceptors and nicotinic

acetylcholine receptors (Verkhratsky, 2010; Verkhratsky & Nedergaard, 2018). Although some of

these receptors exhibit a Ca2+ permeability, their current is carried mainly by Na+, which dominates

the ionic composition of the extracellular milieu. The regional distribution and functional expression

of voltage-gated Na+ channels (Nav) in astrocytes in vivo remain uncertain, although astroglial

expression of Nav1.2, Nav1.3, Nav1.5, Nav1.6 channel subunits has been detected at the mRNA and

protein level, and voltage-gated Na+ currents have been characterised in a subpopulation of

astrocytes in situ in hippocampal and spinal cord slices (Pappalardo et al., 2016). Astrocytes

throughout the CNS also express transient receptor potential (TRP) channels of TRPA1, TRPC1/4/5

and TRPV1/4 varieties, with some regional differences (Doly et al., 2004; Benfenati et al., 2007a;

Malarkey et al., 2008; Butenko et al., 2012; Shigetomi et al., 2012; Verkhratsky & Parpura, 2014).

Activation of TRP channels results in a substantial Na+ influx (Reyes et al., 2013). Additionally,

astroglial TRPC channels contribute to store-operated Ca2+ entry and thus link metabotropic

pathways and filling state of the endoplasmic reticulum with Na+ influx (Verkhratsky & Parpura,

2014). Finally, astrocytes in the subfornical organ express Nax channels activated by an increase in

extracellular Na+ concentration above 140 mM, which makes these astrocytes a part of systemic

control over body Na+ homeostasis (Noda & Hiyama, 2015).


5

Another prominent pathway for Na+ influx is associated with the activity of Na+-dependent solute

carrier (SLC) transporters (Fig. 1, 2) which are abundantly expressed in astrocytes (Verkhratsky &

Nedergaard, 2018). The Na+-dependent neurotransmitter transporters are represented by (i)

excitatory amino acid transporters types 1 and 2 (EAAT1/SLC1A3, EAAT2/SLC1A2) which are

responsible for glutamate uptake; (ii) GABA transporters type 3 (GAT-3/SLC6A12 Na+/Cl-/GABA

symporters); (iii) glycine transporters type 1 (GlyT1/SLC6A9); (iv) concentrative nucleoside

transporters CNT2/SLC28A2 and CNT3/SLC28A3 that co-transport 1 molecule of adenosine with 1

Na+ ion; (v) norepinephrine and dopamine transporters (NET/SLC6A2 and DAT/SLC6A3) and (vi) Na+-

coupled neutral amino acid transporters SNAT3/SLC38A3 and SNAT5/SLC38A5, which export

glutamine, an obligatory precursor for glutamate (Danbolt, 2001; Eulenburg et al., 2005; Eulenburg

et al., 2010; Zhou & Danbolt, 2013; Rose et al., 2018; Verkhratsky & Nedergaard, 2018). Operation of

these transporters is thermodynamically coupled to Na+ gradients and results in considerable Na+

fluxes; for example glutamate uptake through EAAT1/2 elevates [Na+]i by 10 - 20 mM (Rose &

Ransom, 1996a; Chatton et al., 2000; Kirischuk et al., 2007; Bennay et al., 2008; Langer & Rose,

2009). In addition to neurotransmitter transporters, Na+ fluxes are associated with other

homeostatic SLCs such as Na+-H+ exchanger NHE1-8/SLC9A1, Na+-bicarbonate co-transporter

NBCe1/SLC4A4, Na+-K+-Cl- co-transporter NKCC1/SLC12A2, Na+-dependent glucose transporter

SGLT1/SLC5A1 or Na+-dependent vitamin C transporter SVCT2/SLC23A2 (see (Verkhratsky &

Nedergaard, 2018) for details; Fig. 1, 2).

Astroglial Na+ and Ca2+ dynamics are coupled through the Na+-Ca2+ exchanger (NCX); of which all

three subtypes NCX1 - NCX3/SLC8A1 - A3 are operational in astrocytes. The reversal potential of the

NCX (around -80 mV) lies close to the astroglial resting membrane potential and it thus fluctuates

between forward and reverse mode. Depolarisation and increases in [Na+]i favour the reverse mode

(that is an import of Ca2+), while a rise in [Ca2+]i promotes the forward transport and Ca2+ export

(Paluzzi et al., 2007; Rojas et al., 2007; Reyes et al., 2012). The NCX thereby acts as a mechanism

coordinating Ca2+ and Na+ signalling in astroglial cells (Verkhratsky et al., 2018).

Spatial properties of astroglial Na+ signalling

Astrocyte Na+ signals can be local, that is, restricted to cellular microdomains. Such localisation was

experimentally detected in Bergmann glial cells and in hippocampal astrocytes upon weak synaptic

stimulation or with local application of glutamate to endfeet (Bennay et al., 2008; Langer & Rose,

2009; Langer et al., 2017). The exact mechanisms of the spatial localisation of Na+ signals in the

astroglial compartments remain elusive. Indeed, the cytosol does not contain known Na+-binding

proteins that may act as specific buffers limiting diffusion. Yet highly localised sub-plasmalemmal Na+

signals have also been proposed for several other cell types including cardiomyocytes (Wendt-

Gallitelli et al., 1993; Barry, 2006) and neurones (Hage & Salkoff, 2012). In astrocytes the EAATs,

NCX, NKA and Na+-permeable ionotropic receptors arguably tend to cluster in perisynaptic

processes, thus creating functional domains which may produce local [Na+]i increases (Chatton et al.,

2000; Cholet et al., 2002; Golovina et al., 2003b; Minelli et al., 2007; Rose et al., 2009). The


6

mechanism of localisation of Na+ (and any cationic) signals may be also provided by negatively

charged “traps” existing on the inner side of plasmalemma, which may promote retention of ions

close to the site of entry (Breslin et al., 2018).

In addition to spatially restricted Na+ transients, there is evidence that stronger synaptic stimulation

– i.e. activating more afferent fibres – results in astrocyte Na+ signals that spread within individual

cells, invading the soma as well as endfeet (Langer et al., 2017). The spread of Na+ is rapid (~100-150

µm/sec) and seems to propagate solely by passive (and un-buffered) Na+ diffusion through gap

junctions (Langer et al., 2012; Langer et al., 2017; Moshrefi-Ravasdjani et al., 2017). Propagating Na+

waves proceed not only through astrocytic networks but also enter other macroglial cells, including

oligodendrocytes and NG-2 cells. This type of co,,unication was identified in grey as well as in white

matter (Augustin et al., 2016; Moshrefi-Ravasdjani et al., 2017).

Na+ signals control astroglial physiological activity

Arguably, Na+ signals in astrocytes may occur in localised nano/microdomains associated with

perisynaptic processes that enwrap synaptic contacts (Bennay et al., 2008; Langer & Rose, 2009;

Langer et al., 2017). These perisynaptic astroglial compartments (known as synaptic cradles -

(Verkhratsky & Nedergaard, 2014)) maintain homeostasis of the synaptic cleft and support the

operation of the glutamate (GABA)-glutamine shuttle, as well as the uptake of other

neurotransmitters. To achieve this end, the membrane of astroglial perisynaptic processes (which

have an exceptionally high surface-to-volume ratio of about 25 m-1 (Grosche et al., 2002;

Reichenbach et al., 2010)) is densely packed with Na+-dependent SLC transporters that in turn are

controlled by the transmembrane Na+ gradient and hence by Na+ signals (Fig. 2).

Several examples of physiologically relevant astroglial Na+ signalling have been accumulated

recently. As described above, increases in Na+ can promote reverse activity of the NCX, resulting in

Ca2+ influx into astrocytes. There is evidence that this Na+-driven Ca2+ signalling regulates

mitochondrial mobility in astrocyte processes (Jackson & Robinson, 2018). Even relatively small

increases in [Na+]i can turn GABA or glycine transporters into the reverse mode (Eulenburg &

Gomeza, 2010; Heja et al., 2012; Unichenko et al., 2013) thus transforming astrocytes from the sink

to the source of inhibitory neurotransmitters. This physiologically relevant reversal of GABA

transporters has been demonstrated not only in vitro but also in situ in neocortical (Unichenko et al.,

2013) and hippocampal (Heja et al., 2012) slices. Of note, Na+-dependent uptake of glutamate is

safeguarded and glutamate transporters do not reverse under physiological conditions (Rose et al.,

2018; Verkhratsky & Nedergaard, 2018). Elevation of [Na+]i in astrocytes in situ directly stimulated

glutamine efflux through SNAT3/SLC38A3 Na+-dependent transporters, thus coordinating supply of

glutamine with neuronal activity (Todd et al, 2017). Influx of Na+ through Na+/HCO3- cotransporter

was recently demonstrated to be a trigger event in responses of chemosensing astrocytes of the

brain stem slices to physiological changes in PCO2/[H+] (Turovsky et al., 2016). Influx of Na+ controls


7

astroglial energy metabolism and Na+ signals trigger glucose uptake and lactate production by

astrocytes through aerobic glycolysis (Chatton et al., 2016). Moreover, the spread of Na+ between

astrocytes results in an increase in glucose uptake within the syncytium, coupling glial metabolism

with neuronal activity (Bernardinelli et al., 2014).

In addition, cytosolic Na+ may exert its action through “Na+ sensor” proteins. These may include

signalling cascades (Ono et al., 2010), enzymes (most notably glutamine synthetase (Benjamin,

1987)) or G-proteins (Rishal et al., 2003; Katritch et al., 2014). In addition, Na+ can regulate ion

channels either directly (for example Na+-dependent K+ channels (Bhattacharjee & Kaczmarek,

2005)), indirectly (spermine-induced inhibition of inward rectifier Kir 4.1 channels (Kucheryavykh et

al., 2012)), or by G-dependent inhibition of Ca2+ channels (Blumenstein et al., 2004). On an

integrative level, astroglial Na+ signals can provide long-term regulation of neurotransmission by

acting on all key elements of the glutamate (GABA)-glutamine shuttle – that is the EAATs, the

glutamine synthetase and the SNATs exporting glutamine.

Astroglial Cl- – the link to cell biology and regulator of inhibitory transmission?

Astroglial Cl- homeostasis

Besides bicarbonate (HCO3-), chloride is the main inorganic cytosolic anion, which controls numerous

cellular functions including a contribution to membrane permeability and establishment of the

membrane potential. Studies performed on astrocytes in culture indicated that the cytoplasmic Cl-

concentration in astroglia is rather high, being in the range of 20 to 50 mM, which sets the ECl at ~ -

35 mV; opening of astroglial Cl- channels hence caused Cl- efflux, inward current and depolarisation

(Kettenmann & Schachner, 1985; Bekar & Walz, 2002). In agreement with this, a radioactive Cl-

extrusion assay determined resting [Cl-]i to be 31 - 43 mM (Kimelberg, 1981), while another study

employing ion-sensitive microelectrodes indicated a resting [Cl-]i of 20 - 40 mM in astrocytes

(Kettenmann et al., 1987). Experiments in situ are few and theresults are controversial. Much lower

[Cl-]i, around 3.1-3.9 mM, was estimated in astrocytes in acute hippocampal slices based on

gramicidin patch-clamp recordings (Ma et al., 2012). In contrast, the only direct measurement of [Cl-

]i in situ performed recently performed in cerebellar Bergmann glial cells using the chloride-sensitive

dye MQAE ((6-Methoxyquinolinio)acetic acid ethyl ester bromide) and fluorescence lifetime imaging

microscopy (Untiet et al., 2017) revealed a high resting [Cl-]i of about 50 mM in 5-6 days old mice.

The [Cl-]i was reduced to ~35 mM in older (13 - 100 days old) animals. Such a decrease in glial [Cl-]i

parallels the neuronal Cl- switch seen during the first two postnatal weeks of development, which

enables GABA and glycine to act as inhibitory transmitters in mature brain. The glial Cl- switch is also

correlated with an up-regulation of expression of Cl- channels (Untiet et al., 2017), but the

transporters responsible for accumulation of Cl- in Bergmann glia in situ have not been identified yet.

Whether high or low levels of resting [Cl-]i are common to all astrocytes in the brain or whether

there are differences between astroglial subtypes remains to be elucidated. The only known Cl-


8

accumulating transporter, NKCC1, is highly expressed in cultured astrocytes. Its expression in situ is,

however, controversial, with data both pro (Kelly & Rose, 2010) and contra (Larsen et al., 2014); with

in vivo functional expression of NKCC1 not yet confirmed (Plotkin et al., 1997; Clayton et al., 1998).

The functional and signalling role of Cl-

Plasmalemmal Cl- permeability of astrocytes (Fig. 3) is mediated by (i) chloride channels, including

GABAA and glycine receptors; (ii) inwardly rectifying chloride channels ClC-1, -2 and -3; (iii) Ca2+-

dependent Cl- channels; (iv) anion channels of the Bestrophin (Best) family and by (v) volume-

regulated anion channels VRAC or SWELL1 (Parkerson & Sontheimer, 2004; Benfenati et al., 2007b;

Blanz et al., 2007; Park et al., 2009; Park et al., 2013; Verkhratsky & Nedergaard, 2018). In addition,

Cl- is transported across astroglial plasma membrane by SLC transporters including (i) GABA

transporters (GAT1/SLC6A1 and GAT3/SLC6A11) or glycine transporter (GlyT1/SLC6A9) with

stoichiometry of 1GABA(Gly):2Na+:1Cl− (Kavanaugh et al., 1992), (ii) chloride channels associated

with EAAT1/2 transporters (Vandenberg et al., 2008); (iii) Na+-K+-Cl- co-transporter 1

(NKCC1/SLC12A2), which moves Na+, K+ and Cl- into the cell with electroneutral stoichiometry of

1Na+ :1K+ :2Cl- (Macaulay & Zeuthen, 2012) and (iv) possibly members of K+-Cl co-transporter

(KCC/SLC12) family (Ochoa-de la Paz et al., 2005). Of note, the majority of these molecular pathways

mediate diffusional Cl- efflux, with only NKCC1 and GABA transporters capable of a (Na+-dependent)

accumulation of Cl- into the cell. The anion conductance of EAATs is not thermodynamically coupled

to the transport of glutamate but the formation of the anion pore occurs during the glutamate

transport cycle (Machtens et al., 2015).

The signalling role of Cl- is defined by multiple intracellular mechanisms and targets (Fig. 4) which

include (i) regulation of cell proliferation, cell differentiation and cell death mediated by the

apoptotic pathway; (ii) regulation of cell volume and hence morphological plasticity. How tightly

volume-regulation and [Cl-]i are coupled has been demonstrated in glioma cells. These highly

malignant tumour cells derive from astrocytes and have very high [Cl-]i (around 66 – 137 mM (Habela

et al., 2009)). It has been shown that this directly facilitates their migration and infiltration in dense

tissue (Ransom et al., 2001). (iii) modulation of several types of ion channels such as K+ channels and

TRPM7 channels through direct binding to yet uncharacterised channel protein domains and (iv)

regulation of intracellular signalling cascades such as WNK (With No lysine [K+]) and serine/threonine

protein kinases (see (Wilson & Mongin, 2018) for details and relevant literature). At the same time

astroglial [Cl-]i may play an important role in maintenance and regulation of inhibitory transmission

in the CNS. Upon strong activity of GABAergic synapses, Cl- may be depleted from the synaptic cleft.

Astrocytes, however, respond to the same GABA by Cl- efflux, and hence astroglial Cl- secretion can

supplement [Cl-]o thereby preserving the driving force for Cl- to sustain inhibitory neurotransmission

(Kettenmann et al., 1987). The role of Cl- efflux form astrocytes in maintaining GABA-ergic inhibitory

transmission was directly demonstrated in the experiments in CA1 hippocampal slices (Egawa et al.,

2013).


9

Pathophysiological consequences of disturbed Cl- homeostasis

The importance of astroglial Cl- homeostasis for inhibitory synaptic transmission is also suggested

from the analysis of a gain-of-function mutation of EAAT1 anion channels that is associated with

episodic ataxia 6 (EA6), and characterised by ataxia and epileptic seizures (Jen et al., 2005).

Operation of EAATs directly affects Bergmann glial [Cl-]i as has been shown in acute cerebellar slices

(Untiet et al., 2017). The mutated protein has been characterised in a heterologous expression

system. Cells within this system demonstrate a significant increase in anion conductance and a

decrease in glutamate transport rates (Winter et al., 2012). However, true understanding of the

pathological mechanism of this type of episodic ataxia requires further studies.

Investigation of ionic brain oedema in vitro and in situ show activation of NKCC1 mediated Cl-

accumulation followed by water influx and astrocyte swelling. The involvement of NKCC1 in cases of

brain oedema is indicated by several studies that show increased NKCC1 expression, activation and

beneficial effects of blocking NKCC1 using bumetanide. Due to the controversial data about NKCC1

expression in astrocytes and limitation of in vitro and in situ studies investigating fluid redistribution,

the role of astroglial Cl- fluxes during brain oedema need to be verified in vivo (Simard et al., 2007;

Jayakumar et al., 2011; Thrane et al., 2014).

Astroglial potassium microdomains: can they bear the signalling role?

Homeostatic regulation of the interstitial concentration of potassium ions ([K+]o) is the archetypal

function of astrocytes (Larsen et al., 2016). This astroglial function (also known as K+ buffering) was

discovered in 1960, when both diffusional (Orkand et al., 1966) and energy/NKA-dependent

mechanisms were proposed (Hertz, 1965). Potassium membrane permeability of astrocytes (Fig. 5) is

determined by NKA, the main importer of K+ ions, by NKCC1, which seemingly works only in

conditions of K+ overload and may not be even functionally expressed in astrocytes in the in vivo

brain ((Larsen et al., 2014), but see (Kelly & Rose, 2010)) and by diffusion (mostly efflux) through K+

channels of which the inward rectifying Kir4.1 channel is predominant. In the physiological context,

astrocytes operate like a “K+ shuttle” accumulating K+ via NKA at the peak of neuronal activity and

releasing K+ back to neurones through Kir4.1 in the aftermath (Chever et al., 2010; Rimmele &

Chatton, 2014) and thus supporting restoration of neuronal ion gradients (Verkhratsky &

Nedergaard, 2018).

While this is a widely accepted scenario, direct experimental data on K+ signalling in astrocytes are

extremely sparse. This is largely because the available chemical indicator dyes have poor signal-to-

noise-ratio and are difficult to use. A recent study, employing the novel indicator Asante Potassium-

Green-1, reported a decrease in astrocyte [K+] by several mM in response to bath application of


10

glutamate; increasing the [K+]o on the other hand, caused an increase in astrocyte [K+]i (Rimmele &

Chatton, 2014). The proposed K+ shuttle ultimately requires retention of K+ in the perisynaptic

compartments with emergence of K+ microdomains, which may be supported by intramembrane

negative charges preventing K+ diffusion (Breslin et al., 2018). Whether these microdomains may

have any further signalling role remains an open question.

Astroglial protons – what can they do?

Regulation of astroglial pH

Astroglial pH (pHi) is usually within a range of 7.0-7.3, which is slightly more acidic than pH values

commonly found in the extracellular spaces of the brain (pHe) (Deitmer & Rose, 1996; Chesler, 2003;

Obara et al., 2008). Notably, at a pHi between 7.0 and 7.3, the free proton (H+) concentration is only

between 40 and 80 nM, and thus much lower than the free concentrations found for other main

monovalent ions, including Na+, K+ and Cl-. Astrocytes have an exceptionally high intracellular H+

buffering power of about 200 mM, provided by the enzyme carbonic anhydrase (CA), which converts

CO2 to HCO3- and H+, as well as by efficient plasma membrane transport of acid-base equivalents

(HCO3-) through the Na+-HCO3

- co-transporter NBCe1/SLC4A4 (Deitmer & Rose, 1996; Chesler, 2003;

Theparambil & Deitmer, 2015).

In the absence of a strong chemical gradient, EH+ is around -10 mV, resulting in an inwardly directed

driving force for the movement of H+ at the typical high negative membrane potential of astrocytes.

At the same time, protons may not only passively enter astrocytes, but they are also generated as a

product of cellular metabolism and ATP production, for example as a consequence of mitochondrial

respiration and generation of CO2. The latter is converted to HCO3- and H+ by CA. Astrocytes have

particularly high CA activity and it was suggested that CO2 which diffuses out of neurones is

preferentially converted into HCO3- and H+ by glial cells (Deitmer, 2002). Cellular acidification is also

promoted by ion channels, directly permeable to H+, as well as by membrane transporters that

mediate the influx of H+ such as the PMCA or Na+-dependent glutamate transporters (EAATs) (Fig. 6).

The latter couple the uptake of 1 molecule of glutamate to the influx of 3 Na+ and 1 H+ as well as to

the export of 1 K+, thereby acidifying astrocytes (Levy et al., 1998).

Astrocytes must, therefore, actively extrude H+ to counteract acidification of the cytosol during

neuronal activity (Fig. 6). The main glial acid extruder, which also plays a prominent role in the brain

pathology, is the Na+-H+ exchanger (NHE1/SLC9A1) (Deitmer & Rose, 1996; Chesler, 2003; Obara et

al., 2008; Zhao et al., 2016; Begum et al., 2018). NHE1 couples the inwardly directed Na+ gradient to

the export of H+ and its activity is thus decreased following increases in the intracellular Na+,

resulting in cellular acidification (Bondarenko et al., 2005). In addition, several transporters for HCO3-


11

are expressed by astrocytes. These include the above-mentioned NBCe1 (Brune et al., 1994;

Bevensee et al., 1997; Schrodl-Haussel et al., 2015), which is usually inwardly directed, but may

readily reverse in response to an intracellular alkalosis and thereby serve as acid loader

(Theparambil & Deitmer, 2015). Moreover, astrocytes have been reported to express Na+-dependent

and Na+-independent Cl-/HCO3--exchangers, the functional parameters of which are yet to be

characterised, as well as a vacuolar-type H+-ATPase (Deitmer & Rose, 1996; Chesler, 2003; Obara et

al., 2008). Protons extrusion is also mediated by export of lactate through monocarboxylate

transporters, MCTs (Pierre & Pellerin, 2005).

pH changes in astroglia and their signalling functions

Neuronal activity in the vertebrate brain is accompanied by substantial and complex shifts in pH in

all involved compartments: while neurones acidify and astrocytes alkalinise, the extracellular space

undergoes a biphasic alkaline-acid shift (Chesler & Kraig, 1989; Chesler, 2003; Deitmer & Rose, 2010;

Sinning & Hubner, 2013). The alkalinisation of astrocytes in response to excitatory neuronal activity

has been attributed to the accompanying rise in extracellular K+ resulting in a depolarisation of

astrocytes and a concomitant activation of inward NBCe1 activity (Brune et al., 1994; Pappas &

Ransom, 1994; Bevensee et al., 1997).

The so-called “depolarisation-induced alkalinisation” of astrocytes effectively results in an

extracellular acidification (Chesler & Kraig, 1989). Because a decrease in extracellular pH dampens

neuronal activity (Sinning & Hubner, 2013), this was long viewed as a mechanism by which

astrocytes might modulate the excitability of neurones (Ransom, 1992, 2000). An exception is found

in areas of the brainstem that are responsible for sensing CO2 and pH and control breathing patterns

(Marina et al., 2018). Here, a CO2-induced acidification was accompanied by activation of astrocyte

NBCe1, resulting in secondary activation of NCX, an increase in Ca2+ and release of ATP from

astrocytes, exciting respiratory circuits (Gourine et al., 2010; Kasymov et al., 2013; Turovsky et al.,

2016).

In forebrain astrocytes, several laboratories have now convincingly established that NBCe1 plays a

key role in neuro-metabolic coupling (Chatton et al., 2016). Activation of NBCe1 results in

stimulation of a soluble adenylyl cyclase and production of cAMP in astrocytes, which enhances

glycolysis (Choi et al., 2012). Another major player, coupling astrocytic depolarisation-induced

alkalinisation (and NBCe1) to metabolism, is the phosphofructokinase, a key enzyme of glycolysis. It

is strongly pH-dependent and is efficiently stimulated by alkalinisation, resulting in an activation of

astrocyte glycolysis in response to increases in extracellular K+ (Bittner et al., 2011; Ruminot et al.,

2011). The pH-related increase in glycolysis was suggested to play a main role in the increased

production of lactate by astrocytes, driving the so-called “astrocyte-neurone lactate shuttle”

(Pellerin & Magistretti, 2012; Chatton et al., 2016). While this concept is still under debate (Dienel,

2014; Bak & Walls, 2018; Barros & Weber, 2018), a recent study found that NBCe1 also regulates the


12

NADH/NAD+ redox state of astrocytes, again confirming its central role in adapting astrocytic

metabolism to the energy requirements of active neurones (Kohler et al., 2018).

Recapitulation and Conclusion

Astroglial excitability and astroglial physiological responses are mediated and regulated by highly

coordinated changes in intracellular ion concentrations, which affect astroglial biochemistry (mostly

through Ca2+ and Cl-) and astroglial membrane transporters (mostly Na+ and K+). Ionic fluxes, which

form the backbone for this type of “cytosolic ionic excitability”, are mediated through membrane

channels and transporters which couple translocation of these ions. Notably, the NKA is central to

the transport of all major ions: it directly controls [Na2+]i and [K+]i and indirectly affects fluxes of Ca2+

(through NCX), of Cl- (through K+/Cl- and K+/Na+/Cl- co-transporters) and of H+ (through NHE and

NBC). The molecules responsible for ionic signalling are concentrated in astroglial perisynaptic

processes where they provide for coordination of the astrocytic homeostatic response with neuronal

activity.

The concept of astroglial Ca2+ excitability is well defined and intimate mechanisms have been

revealed. Molecular cascades responsible for signalling mediated by other ions are, in contrast, in

statu nascendi, and numerous fundamental properties of ionic intracellular dynamics are yet to be

described. In particular, the organisation of micro- and nano-domains for different ions has to be

characterised, and relevant molecular mechanisms revealed. Similarly, molecular targets and

physiological outcomes of signals mediated by different ions have to be elucidated and placed in

context of nerve tissue operation in physiological and pathological conditions.

Competing interests

The authors disclose any conflict of interest.

Author contributions

All authors have contributed to the conception and drafting of the text and figures. Moreover, all

authors contributed its revision and approved the final version of the manuscript. All authors qualify

for authorship, and all those who qualify for authorship are listed.

Funding

Work in the laboratory of CRR is supported by the Deutsche Forschungsgemeinschaft (DFG, Ro

2327/8-2 and 13-1).

Acknowledgements

We are grateful to Lisa Felix, Institute of Neurobiology, Heinrich Heine University Düsseldorf, for

proofreading the manuscript.


13

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Figure legends

Figure 1. Molecules mediating Na+ fluxes in the plasmalemma of astrocyte.

Influx of Na+ occurs though (i) Na+-permeable channels which include ionotropic receptors (AMPAR,

MNDAR, P2XR - AMPA, NMDA glutamate receptors and ionotropic purinoceptors); channels of the

transient receptor potential (TRP) family (TRPC1/4/5 channels that operate as a part of store-

operated Ca2+ entry and hence generate Na+ influx in response to the depletion of endoplasmic

reticulum Ca2+ stores; as well as TRPA and TRPV channels); voltage-dependent Nav channels and

[Na+]o-activated Nax channels; (ii) through Na+-dependent SLC transporters that include excitatory

amino acid transporters EAAT1,2, GABA transporters GAT 1,3, glycine transporters GlyT,

noradrenaline transporters NET or concentrative adenosine transporters CNT2/3. The main pathway

for Na+ exit is provided by Na+-K+ pump, NKA. The Na+-Ca2+ exchanger NCX fluctuates between

forward and reverse mode and couples Na+ and Ca2+ signalling.


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Figure 2. Molecular targets of Na+ signalling in astroglia.

Abbreviations: ASCT2 - alanine-serine-cysteine transporter 2; ASIC - acid sensing ion channels; CNT2,

concentrative nucleoside transporters; EAAT - excitatory amino acid transporters; ENaC - epithelial

sodium channels; GAT - GABA transporters; GS - glutamine synthetase, GlyT1-glycine transporter.

iGluRs - ionotropic glutamate receptors; Nax - Na+ channels activated by extracellular Na+; NAAT -

Na+-dependent ascorbic acid transporter; NBC - Na+/HCO3- (sodium-bicarbonate) co-transporter;

NCX - Na+/Ca2+ exchanger; NCLX - mitochondrial Na+/Ca2+ exchanger; NHE - Na+/H+ exchanger; NKCC1

- Na+/K+/Cl- co-transporter, NET - norepinephrine transporter; MCT1 - monocarboxylase transporter

1; P2XRs - ionotropic purinoceptors; SN1,2 - sodium-coupled neutral amino acid transporters which

underlie exit of glutamine; TRP - transient receptor potential channels; ROS - reactive oxygen

species; VRAC - volume-regulated anion channels.

See text for further explanation.

Modified from (Verkhratsky & Nedergaard, 2018).


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Figure 3. Molecular pathways of Cl- fluxes in astroglia

Transmembrane Cl- fluxes are mediated by (i) ionotropic GABAA and glycine (Gly) receptors, (ii) by

anionic channels, including volume regulated anion channels VRAC, chloride channels ClC1,2,3 and

bestrophine1(Bes1) channels; (iii) Cl- may diffuse through plasmalemma via Cl- channels associated

with (but not thermodynamically coupled to) glutamate transporters EAAT1,2; (iv) Cl- flux is

thermodynamically coupled to the transport of GABA by GAT1,3 transporters, which may operate in

forward and reverse modes; (v) Cl- efflux is mediated by K+-Cl- co-transporter and (vi) Cl-

accumulation is associated with the activity of Na+-K+-Cl- co-transporter NKCC1.

Figure 4. Targets of Cl- intracellular signalling

The signalling role of Cl- is defined by multiple intracellular targets. Regulation of cell volume is

mediated by volume regulating anion channel (VRAC), hence morphological plasticity and cell

migration. As signalling molecule, Cl- activates K+ channels and TRPM7 channels, cSLO-2 and NBCe1.

Intracellular signalling cascades as for example WNK kinases are regulated by Cl- changes.

Programmed cell death is accompanied by apoptotic volume decrease and thus Cl- efflux.

Furthermore, regulation of cell proliferation and differentiation are targets of Cl- signalling.


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Figure 5. Molecular pathways of K+ fluxes in astroglia.

Potassium fluxes are mediated by (i) K+ channels of which the most abundant is inward rectifying

Kir4.1 channel; (ii) by K+-Cl- co-transporter (iii) Na+-K+-Cl- co-transporter NKCC1 and by (iv) Na+-K+

pump, NKA

Figure 6. Molecular pathways of H+ fluxes in astroglia.

Abbreviations: EAAT1,2 - Excitatory amino acid transporters 1, 2; NHE - Na+ H+ exchanger, PMCA -

plasmalemmal Ca2+ ATPase; VH+ pump - vacuolar H+ pump localised in plasmalemma; MCT -

monocarboxylate transporter; NBC - Na+-bicarbonate exchanger.

Modified from (Verkhratsky & Nedergaard, 2018).


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NAME: Alexei Verkhratsky

BIOGRAPHICAL NOTE:

Professor Alexei Verkhratsky, PhD, D.Sc, Member of Academia Europaea (2003), Member of the

German National Academy of Sciences Leopoldina (2013), Member of Real Academia Nacional de

Farmacia of Spain (2012), member of Polish Academy of Sciences (2017); member of The Dana

Alliance for Brain Initiatives (2012), is an internationally recognised scholar in the field of cellular

neurophysiology. His research is concentrated on the mechanisms of inter- and intracellular

signalling in the CNS, being especially focused on two main types of neural cells, on neurones and

neuroglia. He made important contributions to understanding the chemical and electrical

transmission in reciprocal neuronal-glial communications and on the role of intracellular Ca2+ signals

in the integrative processes in the nervous system. Many of his studies are dedicated to

investigations of cellular mechanisms of neurodegeneration. He was the first to perform intracellular

Ca2+ recordings in old neurones in isolation and in situ, which provided direct experimental support

for “Ca2+ hypothesis of neuronal ageing”. In recent years he studies glial ageing and gliopathology in

age-related brain diseases, including Alzheimer disease as well as in neuropsychiatric diseases. He

authored a pioneering hypothesis of astroglial atrophy as a general mechanism of cognitive brain

disorders including neurodegenerative and psychiatric diseases.

Scientometry: Prof Verkhratsky authored and edited 12 books and published ~ 400 papers and

chapters. His papers were cited >21000 times, H-index 79 (Scopus, 11/2018).

He was among 30 Most cited European neuroscientists in 2016

http://www.labtimes.org/labtimes/ranking/2016_01/index2.lasso


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Ionic signalling in astroglia beyond calcium...Ionic signalling in astroglia beyond calcium DOI: 10.1113/JP277478 Document Version Accepted author manuscript Link to publication record