Ion homeostasis in the extracellular space

Maintenance of the extracellular ion composition is of paramount importance for brain function, because every shift in ion concentrations profoundly affects the membrane properties of nerve cells and hence their excitability. Brain extracel- lular space contains high amounts of Na⁺ ([Na⁺]_o∼130 mM) and Cl⁻ ([Cl⁻]_o∼ 100 mM), whereas it is rather low in K⁺ ([K⁺]_o∼2–2.5 mM). This is reversed inside the brain cells, as the cytosol of most of neurones and glia is rich in K⁺ ([K⁺]_i∼100–140 mM) and poor in Na⁺ ([Na⁺]_i∼<10 mM). The intracellular chloride concentration is, as a rule, low in neurones ([Cl⁻]_i∼2–10 mM) and is relatively high in glial cells ([Cl⁻]_i∼30–35 mM), due to Cl⁻influx in exchange for Na⁺ and K⁺ by the Na⁺/K⁺/Cl⁻ transporters. The extracellular concentra- tion of another important cation, Ca²⁺, is relatively low in the extracellular space ([Ca²⁺]_o∼1.5–2 mM), nevertheless it is still about 20 000 times lower in the

7.7 ION HOMEOSTASIS IN THE EXTRACELLULAR SPACE 107 cytosol ([Ca²⁺]_i∼0.0001 mM). These transmembrane ion gradients together with selective plasmalemmal ion channels form the basis for generation and main- tenance of the resting membrane potential and underlie neuronal excitability.

The safeguarding of transmembrane ion gradients is the task for numerous ion- transporting systems, which allow ion movements either by diffusion (ion channels) or at the expense of energy (ion pumps and exchangers). The main ion transporters operative in astrocytes are summarized in Figure 7.7.

Apart from preserving their own transmembrane ion homeostasis, astroglial cells are heavily involved in maintenance of extracellular ion concentrations. As neuronal activity is inevitably associated with influx of Na⁺ and Ca²⁺ (depolar- ization) and efflux of K⁺(repolarization), the extracellular concentrations of these ions vary; the relative variations are especially high for K⁺ ions (due to a low [K⁺]_o and a very limited volume of the CNS extracellular space; i.e. even rela- tively modest numbers of K⁺ ions released by neurones may very substantially affect the extracellular K⁺ concentration). If the K⁺ released during action poten- tial propagation was allowed to accumulate in the extracellular space, this would cause neuronal depolarization. Small increases in [K⁺]_o would increase neuronal excitability by bringing their membrane potential closer to the action potential threshold. If K⁺ rises sufficiently to depolarize the neurones past the threshold

Figure 7.7 Ion transporting systems in the astroglial cell. The main astroglial ion trans- porters are: Na⁺/H⁺ antiporter; Na⁺/H⁺/HCO⁻₃ and Na⁺/K⁺/2Cl⁻ cotransporters; Cl⁻/HCO⁻₃ and Na⁺/Ca²⁺exchangers; Na⁺/K⁺pumps; water channels (aquaporins); K⁺and Cl⁻channels and swell-activated Cl⁻ channels. Glia also contain carbonic anhydrase, which catalyzes the conversion of H₂O + CO₂to carbonic acid (H₂CO₃, which readily dissociates to H⁺and HCO⁻₃, hence facilitating CO₂ uptake and extracellular pH regulation during neuronal activity

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for Na⁺ channel activation, neurones become inexcitable because Na⁺ channels become inactivated and there is a conduction block. Astrocytes help prevent the accumulation of extracellular K⁺, thereby stabilizing neuronal activity.

7.7.1 Astrocytes and extracellular potassium homeostasis

During intense (but still physiological) neuronal activity the extracellular potas- sium concentration may rise almost twice, from 2–2.5 mM to 4–4.2 mM; such an increase can be observed, for example, in the cat spinal cord during rhythmic and repetitive flexion/extension of the knee joint. As a rule, however, during regular physiological activity in the CNS the [K⁺]_o rarely increases by more than 0.2 to 0.4 mM. Nonetheless, locally, in tiny microdomains such as for instance occurring in narrow clefts between neuronal and astroglial membranes in perisynaptic areas, [K⁺]_omay transiently attain much higher levels. The relatively small rises in [K⁺]_o accompanying physiological neuronal activity indicate that powerful mechanisms controlling extracellular potassium are in operation. Disruption of these mecha- nisms, which do occur in pathology, results in a profound [K⁺]_o dyshomeostasis;

upon epileptic seizures, for example, [K⁺]_o may reach 10–12 mM, while during brain ischaemia and spreading depression [K⁺]_ocan transiently peak at 50–60 mM.

The major system which removes K⁺from the extracellular space is located in astrocytes and is represented bylocal K⁺ uptake andK⁺ spatial buffering. The local K⁺ uptake occurs in the individual cells and is mediated by K⁺ channels and transporters (Figure 7.8). The resting membrane potential (V_m) of astrocytes (∼ –90 mV) is determined almost exclusively by high K⁺ permeability of glial plasmalemma and therefore it is very close to the K⁺ equilibrium potential, E_K. Increases in [K⁺]_o would instantly shift the E_K towards depolarization, which would generate an inflow of K⁺ ions (as the V_m<E_K). However, this inward K⁺ current rapidly depolarizes the membrane and soon V_mbecomes equal to E_K, and K⁺ influx ceases. Therefore, K⁺ channels contribute only a little towards local K⁺uptake. Most of it is accomplished via Na/K pumps and Na/K/Cl transporters.

The Na/K pump expels Na⁺out of the cell and brings K⁺ into it. The glial Na/K pumps are specifically designed for the removal of K⁺from the extracellular space when [K⁺]_o is increased, as they saturate at around 10–15 mM [K⁺]_o; in contrast neuronal Na/K pumps are fully saturated already at 3 mM [K⁺]_o. In addition, K⁺ uptake is assisted by Na/K/Cl cotransport, in which Cl⁻ influx balances K⁺ entry. However, the capacity for local K⁺ uptake is rather limited, because it is accompanied with an overall increase in intracellular K⁺ concentration; water follows and enters the cells, resulting in their swelling.

A much more powerful and widespread mechanism for the removal of excess extracellular K⁺ is spatial buffering, a model proposed in the 1960s by Wolf- gang Walz and Richard Orkand. In this case, K⁺ ions entering a single cell are redistributed throughout the glial syncytium by intercellular K⁺ currents through gap junctions. After this spatial redistribution, K⁺ions are expelled into either the

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Figure 7.8 Astrocytes provide for local and spatial potassium buffering: Buffering of extracel- lular potassium occurs through astroglial inward rectifier potassium channels K_ir(local potassium buffering). Potassium is released into the extracellular space during neuronal activity (K⁺efflux underlies the recovery phase – repolarization – of the action potential). Astrocytes take up excess K⁺through K_ir, redistribute the K⁺through the astroglial syncytium via gap junctions (spatial potassium buffering), and release K⁺through K_ir. See the text for further details

interstitium or perivascular space, where they are removed into the blood. In spatial, K⁺buffering, the K⁺ions are transported across the membranes through K⁺chan- nels. Local K⁺entry depolarizes the cell, which creates an electrical and chemical gradient between this cell and neighbouring astrocytes connected via gap junctions.

This provides the force for K⁺ions to diffuse into the syncytium, preventing local membrane depolarization (thus maintaining K⁺ influx) and dispersing K⁺ ions through many cells, so that the actual elevation in cytoplasmic K⁺concentration is minimal (Figure 7.8). The principal K⁺channels responsible for spatial buffering are inwardly rectifying channels of K_ir4.1 type. These channels are only mildly rectifying; i.e. they allow both inward and outward K⁺ movements at the resting membrane potential levels. This is important, as K⁺ is finally expelled from the glial syncytium also through the K_ir channels. Another important feature of K_ir channels is that their conductance is directly regulated by the [K⁺]_o levels; the conductance increases as a square root of increase in [K⁺]_o. In other words, local increases in [K⁺]_o augments the rate of K⁺ accumulation of glial cells. The K_ir channels are clustered in perisynaptic processes of astroglial cells and in their endfeet (where the density of K_ir channels can be up to 10 times larger that in the rest of the cell membrane). This peculiar distribution facilitates K⁺ uptake

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around areas of neuronal activity and K⁺ extrusion directly into the vicinity of blood vessels.

Sometimes, K⁺spatial buffering may take place within the confines of an indi- vidual glial cell. A particular example of this process, known as K⁺ siphoning, was described in retinal Müller cells by Eric Newman in the 1980s (Figure 7.9).

Müller cells have contacts with virtually all the cellular elements of the retina. The main endfoot of the Müller cell closely apposes the vitreous space, whereas the apical part projects into the subretinal space; Müller cells also send perivascular processes, which enwrap retinal capillaries. Importantly, the endfoot and perivas- cular processes contain very high densities of K_ir channels. Potassium buffering mediated by Müller cells occurs primarily in the inner plexiform layer of the retina, which contains most of the retinal synapses. The K⁺ions released during synaptic activity enter the cytosol of the glial cell, through which they are rapidly equi- librated. Subsequently, the excess of potassium is expelled through K_ir channels located in the endfoot into the vitreous humour or through K_ir channels located in perivascular processes into the perivascular space. Some of the K⁺ ions may also be released through apical processes, where light induces a decrease in [K⁺]_o in the subretinal space.

7.7.2 Astrocytes and chloride homeostasis

Astrocytes contribute to overall chloride homeostasis by activation of anion chan- nels. As the cytosolic concentration of Cl⁻ in astroglial cells is high, opening of anion channels will cause Cl⁻ efflux. This efflux is activated during hypo- osmotic stress. Alternatively astrocytes can accumulate chloride by Na⁺/ K⁺/2Cl⁻ cotransporter.

7.7.3 Astrocytes and extracellular Ca²⁺

Calcium concentration in small extracellular compartments and particularly in perisynaptic compartments may fluctuate rather substantially, as Ca²⁺is accumu- lated by neurones when the invading action potential activates Ca²⁺channels. The actual [Ca²⁺]_ocan decrease below 1 mM, which may affect generation of Ca²⁺

signals in the terminal, and hence neurotransmission. The lowering of extracel- lular Ca²⁺concentration to∼0.5 mM triggers Ca²⁺signalling in astrocytes, which originates from InsP₃-driven intracellular Ca²⁺ release from the ER stores. This may, in principle, help restore [Ca²⁺]_o, as Ca²⁺ can leave the astrocyte through either plasmalemmal Ca²⁺pump or sodium–calcium exchanger. Extracellular Ca²⁺

concentration may plunge much deeper (to 0.01–0.1 mM) under ischaemic condi- tions, which in turn can initiate seizures (see Chapter 10).

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Figure 7.9 ‘Potassium siphoning’ in retinal Müller glial cell. Potassium buffering in the retina is provided by Müller glial cells: K⁺ ions enter the cytosol of the Müller cell in the inner plexiform layer; K⁺then equilibrates within the glial cytosol and excess K⁺is expelled through K_ir channels located in the endfoot into the vitreous humour or through K_ir channels located in perivascular processes into the perivascular space. Some of the K⁺ ions may be also released through apical processes, where light induces a decrease in [K⁺]_o in the subretinal space. The same process of K⁺siphoning can occur in astrocytes, taking up K⁺via their perisynaptic or perinodal processes and releasing K⁺via their perivascular endfeet, where it could help regulate blood flow

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7.7.4 Astrocytes and regulation of pH

The pH in both the extracellular space and the cytoplasm of neural cells is the subject of tight control by numerous buffering systems. The extracellular pH (pH_o) varies between 7.1 and 7.3, whereas intracellular pH in both neurones and glia lies in a range of 6.8 to 7.5. This means that the concentration of free protons, H⁺, is quite low, being somewhere around 50 nM in the extracellular milieu and 30–160 nM in the cytosol of neural cells.

Maintenance of extracellular pH is physiologically important, as even small fluctuations of pH_o may significantly affect synaptic transmission and neuronal excitability. Lowering of pH below 7.0, for example, almost completely inhibits NMDA receptors; in addition, acidification of the extracellular space can activate proton-sensitive cationic channels (known as ASICs – Acid-Sensitive Ion Chan- nels) present in many types of neurones. Neurones and neuronal terminals are the main source of protons in the brain. Neurones, as the main consumers of energy, produce CO₂, which is an end product of oxidative metabolism. The CO₂, by reacting with water, produces protons (CO₂+H₂O↔H2CO₃↔HCO⁻₃+H⁺). Further- more, protons are released in the course of synaptic transmission, as synaptic vesicles are acidic (with pH∼5.6). These changes, at least in part, are counterbal- anced by bicarbonate and proton transporters present in astroglial cells; particularly important is the Na⁺ /HCO⁻₃ cotransporter (NBC), which can operate in both directions, either supplying or removing HCO⁻₃ from the extracellular space.