POTASSIUM BUFFERING IN THE CENTRAL NERVOUS SYSTEM

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OTASSIUM BUFFERING IN THE CENTRAL NERVOUS SYSTEM

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. KOFUJI* AND E. A. NEWMAN

epartment of Neuroscience, University of Minnesota, 6-145 Jacksonall, 321 Church Street SE, Minneapolis, MN 55455, USA

bstract —Rapid changes in extracellular K� concentration[K�]o) in the mammalian CNS are counteracted by simpleassive diffusion as well as by cellular mechanisms of K�

learance. Buffering of [K�]o can occur via glial or neuronalptake of K� ions through transporters or K�-selective chan-els. The best studied mechanism for [K�]o buffering in therain is called K� spatial buffering, wherein the glial syncy-

ium disperses local extracellular K� increases by transfer-ing K� ions from sites of elevated [K�]o to those with lowerK�]o. In recent years, K� spatial buffering has been impli-ated or directly demonstrated by a variety of experimentalpproaches including electrophysiological and optical meth-ds. A specialized form of spatial buffering named K� si-honing takes place in the vertebrate retina, where glialüller cells express inwardly rectifying K� channels (Kirhannels) positioned in the membrane domains near to theitreous humor and blood vessels. This highly compartmen-alized distribution of Kir channels in retinal glia directs K�

ons from the synaptic layers to the vitreous humor and bloodessels. Here, we review the principal mechanisms of [K�]o

uffering in the CNS and recent molecular studies on thetructure and functions of glial Kir channels. We also discussntriguing new data that suggest a close physical and func-ional relationship between Kir and water channels in glialells. © 2004 IBRO. Published by Elsevier Ltd. All rightseserved.

ey words: potassium channel, glia, Müller cells, astrocytes,etina, aquaporin.

eurons are bathed in an extracellular fluid that is rich ina� ions and relatively poor in K� ions. The relative con-entrations of these cations are reversed inside the cells,nd the resulting chemical gradients across the cell mem-rane are crucial to many important processes, includingast electrical signaling involving Na� influx and K� efflux.ven modest neuronal K� effluxes may elicit considerablehanges in extracellular K� concentration ([K�]o), due tohe limited volume of the CNS extracellular space (Nichol-on and Sykova, 1998; Kume-Kick et al., 2002) and the lowaseline [K�]o. These [K�]o changes can impact a wideariety of neuronal processes, such as maintenance ofembrane potential, activation and inactivation of voltage-ated channels, synaptic transmission, and electrogenicransport of neurotransmitters. Thus, it is not surprising

Corresponding author. Tel: �1-612-625-6457; fax: �1-612-626-5009.-mail address: [email protected] (P. Kofuji).bbreviations: DGC, dystrophin glycoprotein complex; EK, equilibriumotential; IOS, intrinsic optic signal; [K�]o, extracellular K� concentra-

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tion; Kir, inwardly rectifying K ; Vm, glial syncytium membrane poten-ial.

306-4522/04$30.00�0.00 © 2004 IBRO. Published by Elsevier Ltd. All rights reseroi:10.1016/j.neuroscience.2004.06.008

1045

hat diverse cellular mechanisms for tight control of [K�]ore found in the CNS of vertebrates and invertebrates.hen these mechanisms are disrupted in instances such

s spreading depression, the extracellular [K�]o can reachalues as high as 60 mM (Somjen, 2001, 2002) and CNSunction is severely compromised.

In the brain, extracellular K� is kept close to 3 mMndependent of fluctuations in serum levels (Katzman,976; Somjen, 1979) although local variations in extracel-

ular K� do occur following neuronal activity changes. Forxample, light stimulation of cat retina promotes slow,ransient [K�]o increases in the primary visual cortex (Fig.A; Singer and Lux, 1975; Connors et al., 1979). Likewise,

ight stimulation of frog or cat retinas induces [K�]o in-reases in the inner and outer plexiform layers and de-reases in the outer retina and subretinal space (Fig. 1B;arwoski et al., 1985; Frishman et al., 1992). In cat spinalord, rhythmic flexion/extension of the knee joint causesK�]o to increase by as much as 1.7 mM (Heinemann et al.,990). Even higher elevations in [K�]o can be evoked byirect electrical stimulation of afferent pathways. In gen-ral, increases of neuronal activity induce local [K�]o in-reases by several millimolar until it reaches a plateau oreiling level (Heinemann and Lux, 1977; Connors et al.,982). This ceiling level is about 10–12 mM in the catomatosensory cortex (Heinemann and Lux, 1977), in theat optic nerve (Connors et al., 1982; Ransom et al., 1986),nd in the cat thalamus (Gutnick et al., 1979). The ceiling

evel is only exceeded under pathophysiological conditionsuch as in anoxia (Vyskocil et al., 1972) or in spreadingepression (Somjen, 2002).

verview of K� regulatory mechanisms

echanisms of [K�]o buffering can be broadly categorizeds either K� uptake or K� spatial buffering (Fig. 2; New-an, 1995; Amedee et al., 1997; Somjen, 2002). In the

ase of K� uptake, excess K� ions are temporarily se-uestered into glial cells by transporters or K� channels.o preserve electroneutrality, K� influx into glial cells isccompanied by either influx of anions such as Cl� or byfflux of cations such as Na� (Fig. 2A). Eventually, K� ionsccumulated in glia are released back into the extracellularpace and the overall distribution of K� across the cellularompartments is restored. It is expected that during therocess of K� uptake, ions will accumulate within the gliand the cells will experience water influx and swelling. This

s in marked contrast to the K� spatial buffering mecha-ism (Orkand et al., 1966), in which functionally coupled,ighly K� permeable glial cells transfer K� ions from re-ions of elevated [K�] to regions of lower [K�] . The
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P. Kofuji and E. A. Newman / Neuroscience 129 (2004) 1045–10561046

lial network. The K� current is driven by the differenceetween the glial syncytium membrane potential (Vm) andhe local K� equilibrium potential (EK). In regions of [K�]oncreases, there is a net driving force causing K� to flownto the glial cells (Fig. 2B). This K� entry causes a localepolarization which propagates electrotonically throughhe glial cell network. As a result, there is a net driving forceausing K� to flow out of the glial cells. Such glial-

ig. 1. Changes in [K�]o in the cat striate cortex and frog retina with etriate cortex upon stimulation of the receptive field of hypercompleicroelectrode. Arrows represent bars of light moving down or up in a cower trace. Spike activity recorded in the reference barrel of the K�-sehanges within layers of the frog retina. A 2-s light stimulus evokes [Knd decreases of [K�]o in the subretinal space (ROS). [From Karwos

ig. 2. Diagram depicting the role of glial cells in [K�]o homeostasis.yncytium. With [K�]o equaling 3 mM, the glial syncytium has a membK�]o is increased, glial cells accumulate K� either by the activity of aechanism of [K�]o regulation, the membrane potential in the glial sync

ncreases of [K�]o produce a glial depolarization that spreads through� �
egions of elevated [K ]o and K outflow at distant regions. The intracellular curr
ons such as Na�. [From Orkand (1986), with permission.]

ediated K� movements in the K� spatial buffering mech-nism disperse local [K�]o increases with little net gain of� ions within the glial cells. The overall efficiency of thepatial buffer process will depend, in part, on the electricalpace constant of the glial cell syncytium (Newman, 1995).n certain CNS regions close to fluid reservoirs, such as theetina, K� spatial buffer currents flow within single glialells rather than through a network of cells. In these cases,

neuronal activity. (A) Upper trace. Dynamic [K�]o changes in the cat[K�]o changes were measured with a double-barreled K�-sensitiveptive field. The 1 mV scale bar corresponds to approximately 0.17 mM.icroelectrode. [From Singer and Lux (1975) with permission.] (B) [K�]oses in the inner plexiform layer (IPL) and outer plexiform layer (OPL)

1985), with permission.]

l cells are electrically coupled via gap junctions forming a functionalntial of approximately �90 mV. (A) Net K� uptake mechanism. WhenTPase or by a pathway in which K� is cotranported with Cl�. In thispatially uniform at �56 mV. (B) K� spatial buffering mechanism. Localsyncytium. The local difference in Vm and EKdrives the K� uptake in

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� influx occurs in one region of the glial cell and effluxccurs through another cell region (see “K� siphoning in

he retina,” below).Net K� uptake is mediated mainly by Na,K–ATPase

umps (sodium pumps) and Na–K–Cl cotransporters. Theodium pump is a transmembrane enzyme that acts as anlectrogenic ion transporter in the plasma membrane of allells (Kaplan, 2002; Jorgensen et al., 2003). Each cycle ofhe sodium pump activity expels three Na� ions andoves two K� ions into the cell. The primary role of the

odium pump is therefore to maintain high intracellular K�

nd low intracellular Na� (Kaplan, 2002; Jorgensen et al.,003). Upon local [K�]o increases, sodium pumps arexpected to transport K� ions into the cells in exchange fora� ions, assuming that the pump extracellular K� site isot saturated and the intracellular Na� concentration is not

imiting (Sweadner, 1995). It has been suggested that theodium pump expressed in glial cells is better suited forK�]o buffering than the neuronal isoform given observa-ions that the glial isoform has a lower affinity for extracel-ular K� (Franck et al., 1983; Reichenbach et al., 1992). Inetina, for example, glial cells (Müller cells) express aodium pump isoform with maximal activity at about 10–5 mM [K�]o while the isoform found in rod photoreceptorsaturate at [K�]o as low as 3 mM (Reichenbach et al.,992).

There have been several reports in support to theotion that sodium pumps help to buffer [K�]o in the CNS.or instance, a transient accumulation of K� ions andimultaneous depletion of Na� ions in glial cells is detectedn guinea-pig cortices following electrical stimulation (Bal-anyi et al., 1987). A substantial fraction of this K� accu-

ig. 3. Differential roles of Na,K–ATPase pumps and Kir channels fontidromic stimulation induces a rise of [K�]o in area CA3 that peaksddition of the sodium pump inhibitor di-hydro-ouabain (DHO), the ba

nduces a rise of [K�]o to approximately 5.9 mM but there is no [K�]o reeriod. In the inset the two traces are shown superimposed, and the brise of [K�]o that peaked at approximately 5.2 mM followed by an un

elation to control levels and the undershoot in [K�]o following antidruperimposed, and the baseline is zeroed. [From D’Ambrosio (2002),

ulation is prevented by pharmacological blockade of so- a

ium pumps (Ballanyi et al., 1987). Likewise, in hippocam-al slices, blockade of sodium pumps increases baselineK�]o and prevents the rapid clearance of extracellular K�

ons following neuronal stimulation (Fig. 3A) (D’Ambrosiot al., 2002). In the rat optic nerve, clearance of K� accu-ulation following axonal stimulation is highly temperatureependent (Q10�2.6) as expected for a carrier-mediatedrocess, and is largely blocked by Na,K–ATPase pump

nhibitors (Ransom et al., 2000).Transient accumulation of K� ions in cultured astro-

ytes has also been linked to the activity of Na–K–Clo-transporters, which are integral membrane proteins thatransport Na, K, and Cl ions into and out of cells in anlectrically neutral manner, often with a stoichiometry ofNa:1K:2Cl (Haas and Forbush, 1998). To date, two Na––Cl cotransporter isoforms have been identified: NKCC1,hich is present in a wide variety of secretory epithelia andon-epithelial cells; and NKCC2, which is present exclu-ively in the kidney (Haas and Forbush, 1998). Both NKCC

soforms are members of a diverse family of cation-chlo-ide cotransport proteins that share a common predictedembrane topology and are sensitive to loop diuretics

uch as bumetanide and furosemide (Haas and Forbush,998). In cultured astrocytes, intracellular K� accumula-

ion following increased [K�]o can be partially prevented byurosemide or bumetanide or by removal of external Na�

nd Cl� (Kimelberg and Frangakis, 1985; Walz, 1992;ose and Ransom, 1996). More recently, the role of Na––Cl co-transporters in [K�]o homeostasis has also beenemonstrated by optical methods (MacVicar et al., 2002).

n the rat optic nerve, intrinsic optical signals (see below)evealed that increased [K�] induces astrocyte swelling,

]o regulation in rat hippocampus slice. (A) In control condition, 3-Hzimately 5.5 mM followed by a decline to approximately 4.7 mM. With

�]o increases to approximately 5.1 mM. Antidromic stimulation (3-Hz)hase. Also absent is the undershoot of [K�]o following the stimulationzeroed. (B) In control condition, 3-Hz antidromic stimulation inducedin [K�]o. With Ba2� (200 �M) in the bath, baseline [K�]o increased inulation is more pronounced. In the inset the two traces are shown

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nd bumetanide (MacVicar et al., 2002). A monoclonalntibody to the NKCC1 form of the Na–K–2Cl cotrans-orter showed that NKCC1 is expressed in astrocytes fromptic nerves (MacVicar et al., 2002), suggesting the in-olvement of this particular Na–K–Cl cotransporter isoformn K� buffering.

However, the exact role of glial K� uptake or spatialuffering in the various parts of the CNS has remainedncertain. This question is under considerable debate, andesearch has shown that the relative contributions mayary across species and between CNS regions (Newman,995). In the rat optic nerve, for example, [K�]o bufferingeems more dependent on the active uptake mechanismhan on K� spatial buffering as post-stimulus recovery ofK�]o is highly sensitive to sodium pump inhibition but noto glial K� channel blockers (Ransom et al., 2000). Byontrast, in the CA3 region of rat hippocampus, both theodium pumps and glial K� channels seem critical foretting the baseline [K�]o and the post-stimulus recoveryf [K�]o (D’Ambrosio et al., 2002; Fig. 3). In this prepara-ion, sodium pumps are necessary for the clearance ofxcess K� during afferent stimulation, whereas glial K�

hannels are necessary to prevent large [K�]o under-hoots following post-tetanus (D’Ambrosio et al., 2002). Inetina, K� spatial buffering has a major role in regulatinghe [K�]o (Newman, 1995; see below).

� spatial buffering

wo conditions are necessary for efficient K� spatial buff-ring as originally proposed by Orkand et al. (1966): 1) thelial cells should form a syncytium in which K� currentsan traverse relatively long distances; and 2) these cellshould be highly and selectively permeable to K�, whichoth enters and exits through the glial cell membranes. Asescribed in the section on “K� siphoning in the retina,”elow, spatial buffer currents can efficiently dissipate [K�]o

ncreases by flowing through single cells as well as throughsyncytium of coupled glial cells. Several lines of evidenceemonstrate that astrocytes do indeed form a functionalyncytium that allows intercellular diffusion of ions andther signaling molecules (Nagy and Rash, 2000; Rouacht al., 2002). Such extensive cellular coupling is due to theigh density of gap junctional proteins (connexins) in glialells (Dermietzel, 1998; Rouach et al., 2002). Immunocy-ochemical and in situ hybridization methods reveal thatstrocytes express multiple connexins, including Cx30,x40, Cx43 and Cx45 (Dermietzel, 1998; Dermietzel et al.,000; Zahs et al., 2003). Among these, Cx43 seems to behe most important for coupling, which is significantly re-uced in cultured astrocytes derived from Cx43 knockoutice (Dermietzel et al., 2000).

Additionally, numerous studies have also shown thatlial cell membranes are highly and almost exclusivelyermeable to K� ions (Sontheimer, 1994). The principal� channels found in glial cells are the inwardly rectifying� (Kir) channels, which allow K� ions to flow much more

eadily in the inward than outward direction (Doupnik et al.,995; Stanfield et al., 2002). These channels have high
pen probability at the normal resting membrane potential B
nd thus allow both glial K� influx and efflux. Kir channelsn glia have been described in many CNS regions, includ-ng mammalian astrocytes from the optic nerve (Barres etl., 1990), spinal cord (Ransom and Sontheimer, 1995)nd other brain regions (Sontheimer, 1994). Although gliaay express additional types of K� channels, such as

alcium and voltage-dependent K� channels (Sontheimer,994), these other channel types are mostly inactive at theyperpolarized glial resting membrane potential (�60 to90 mV; Kuffler et al., 1966; Dennis and Gerschenfeld,969). Another important biophysical property of Kir chan-els is that their slope conductance increases with eleva-ions in [K�]o by a square root relation (Stanfield et al.,002). This unique property of Kir channels allows K�

onductance increases in glial cells, and therefore en-anced K� clearance rates, when [K�]o is raised (New-an, 1993).

Finally, an important implicit assumption in the K�

patial buffering mechanism is the low permeability of glialells to anions such as Cl� as the low anion conductancensures that net uptake of KCl will not occur upon rises ofK�]o. Unfortunately, there is no consensus concerningalues for Cl� permeability of glial cells in the native tissueWalz, 2002). While a relatively high basal Cl� conduc-ance has been reported in glial cells of guinea-pig olfac-ory cortex (Ballanyi et al., 1987) other studies have failedo demonstrate a significant Cl� conductance for glial cellsn situ (Walz, 2002).

vidence for K� spatial buffering

he first support for K� spatial buffering was provided inhe classic work by Orkand et al. (1966), who reported thatn amphibians, stimulation of the optic nerve leads to slowepolarization and repolarization of the glial cells sur-ounding the nonmyelinated axons. The slow depolariza-ion and repolarization of these glial cells was thought toeflect K� transfer by glial cells via the K� spatial bufferingechanism. Subsequently, extensive support for the K�

patial buffering hypothesis came from measurements ofxtracellular field potentials. In the case of K� spatialuffering, the transcellular transfer of K� ions from areas oflevated to lower [K�]o generates return current loops inhe extracellular space giving rise to extracellular fieldotentials. These activity-induced slow extracellular fieldotentials are generated in various CNS regions includinghe cortex and retina (Gardner-Medwin et al., 1981; Dietzelt al., 1989). In the retina, slow extracellular field potentialsslow PIII and M waves) are generated upon light stimula-ion of the retinas (Xu and Karwoski, 1997; Karwoski andu, 1999). Current source density analysis indicates that

hese waves originate from K� spatial buffering by retinallial cells (Xu and Karwoski, 1997; Karwoski and Xu,999). The transfer of K� ions by retinal glial cells, whichenerates the slow PIII wave, buffers the photoreceptor-ased light-evoked decrease in [K�]o in the outer retina.s expected, these K� spatial buffering fluxes are abol-

shed by blocking retinal glial cell K� conductance with
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Measurements of activity-dependent [K�]o changes inhe cortex and cerebellum show that [K�]o varies withepth and time in a manner consistent with transcellularransfer of K� ions (Gardner-Medwin and Nicholson,983). These [K�]o changes were not abolished by sodiumump inhibitors, indicating that they are likely due to aassive K� transport mechanism such as K� spatial buff-ring (Gardner-Medwin and Nicholson, 1983). Similar re-ults were obtained in the drone retina, where it was esti-ated that about 10 times more K� ions move as a resultf spatial buffering than by simple diffusion through thextracellular space (Coles et al., 1986).

More direct evidence for K� spatial buffering has beenrovided by optical imaging of brain slices (Holthoff anditte, 2000). When K� ions are transferred via glial cells,

here should be shrinkage of the extracellular space inreas of K� influx and swelling in areas of K� effluxDietzel et al., 1980). Changes in extracellular volumeollowing neuronal stimulation could be demonstrated byonitoring intrinsic optic signals (IOS) in brain slices. Asredicted for K� spatial buffering, stimulation of corticalreas promoted shrinkage of the extracellular space fol-

owed by swelling in the layers above and below the stim-

ig. 4. Evidences for K� spatial buffering in the rat cortex. (A) Imagef the brain slice viewed with darkfield optics. (B1-4) Time course ofOS changes upon neuronal stimulation in middle cortical layers. Redolors represent IOS increases while blue colors represent IOS de-reases. These correspond, respectively, to shrinkage and widening ofhe extracellular space. Notice the extracellular space shrinkage in theiddle cortical layers and widening in the most superficial and deep

ortical layers as predicted for a K� spatial buffering mechanism. (C)ime course of extracellular space widening in the cortical layer Ieasured independently validates the interpretation of IOS. (D) Time

ourse of [K�]o increase in layer I. (E) Time course of [K�]o increase inayer I (blue) and in layer IV (red). [From Holthoff and Witte (2000), withermission.]

lated area (Fig. 4; Holthoff and Witte, 2000). This swelling n

n the extracellular space was associated with local in-reases in [K�]o and was dependent on gap junctionaloupling (Holthoff and Witte, 2000). Such a pattern ofhanges in extracellular space volume and cellular cou-ling dependency is consistent with K� spatial buffering inhe mammalian cortex.

� siphoning in the retina

n addition to the brain, K� spatial buffering has beennvestigated in the retina, where the Müller cell is therincipal glial cell type (Newman and Reichenbach, 1996).üller cells display morphological polarization with an end-

oot process in close apposition to the vitreous and apicalicrovilli projecting into the subretinal space (Newman andeichenbach, 1996). Like many other glial cells, Müllerells can be distinguished from their neuronal counterpartsy a resting membrane potential that closely follows the EK

ollowing changes in [K�]o (Newman, 1985). This highelectivity of Müller cell membranes to K� ions is due tohe high density and localized expression of Kir channelsNewman, 1993; Kofuji et al., 2000).

Physiological studies in Müller cells provided the firstndication that K� conductances are unevenly distributedlong their plasma membrane. By focally increasing theK�]o along amphibian Müller cells and monitoring theesulting depolarizations, Newman (1984) was able to maphe subcellular distribution of K� conductance in this glialell type. He found that K� conductance was highly con-entrated in the endfoot process, with 94% of the total K�

onductance localized to this relatively small subcellularomain (Newman, 1984). Single channel patch clamp re-ordings later confirmed these findings (Brew et al., 1986).

The observation of a highly non-uniform distribution ofir channels in Müller cells led to the hypothesis thatxcess K� from the inner plexiform layer of retina, whereost retinal synapses are located, are “siphoned” byüller cells to the vitreous humor (Newman et al., 1984;ewman, 1987a). This hypothesis, called K� siphoning, isspecialized form of the spatial buffering mechanism inhich the subcellular distribution of K� channels in glialells directs clearance of K� into large reservoirs such ashe vitreous humor (Fig. 5). In mammalian retinas, theattern of Kir channel distribution is similar to that found inmphibians (Newman, 1987b). The major difference is thatdditional regions of high K� conductance are found inore distal processes in cells of species with vascularized

etina such as mouse, cat and monkey. This suggests thatüller cell’s perivascular processes may also possessigh K� conductance (Newman, 1993) and direct K� topaces near blood vessels (Newman, 1987b).

The K� siphoning hypothesis was supported by thebservation that in the amphibian retina, light flashes pro-ote increased [K�]o in both the inner plexiform layer and

he vitreous humor (Karwoski et al., 1989). These light-voked increases of [K�]o in the vitreous humor wereediated by Kir channels expressed in Müller cells, as

hown by the observation that vitreal increases werelocked by superfusion of retinas with barium, a Kir chan-
el blocker. Similarly, in cat retinas, light-evoked [K�]o

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ncreases in the inner plexiform layer are three-fold largerollowing blockade of Kir channels (Frishman et al., 1992),onfirming that glial-mediated K� spatial currents play anmportant role in curtailing large changes of [K�]o.

lial cells and K� channels

ecause Kir channels most likely underlie K� spatial buff-ring in the CNS, there is considerable interest in deter-ining their macromolecular structure, mechanisms of tar-eting and modulation by intracellular and extracellularactors. The Kir channels have been recently cloned, andver 20 genes are currently known to encode various Kirhannel subunits (Nichols and Lopatin, 1997; Stanfield etl., 2002). Site-directed mutagenesis and heterologoushannel expression have been used to identify structurallements involved in specific Kir channel functions. Thesetudies have revealed the basic Kir channel design of tworansmembrane domains and a re-entry loop (P-loop), withntracellular amino and carboxyl termini (Nichols and Lopa-in, 1997; Stanfield et al., 2002). The Kir channel subunits

ig. 5. K� siphoning in the retina. K� released in the inner plexiform layell). K� leaves the glial cell preferentially from the endfoot processes en these membrane regions. Light-evoked [K�]o decrease in the sub1996b), with permission.]

re usually categorized into seven major subfamilies (Kir1 n

o Kir7) that are diversely regulated by intracellular andxtracellular factors (Stanfield et al., 2002).

Of these family members, immunocytochemical and initu hybridization studies demonstrate that the Kir4.1 chan-el is broadly expressed in brain (Poopalasundaram et al.,000; Higashi et al., 2001), and different reports haveuggested that it is expressed only in glial cells (Higashi etl., 2001) or in both neurons and glia (Li et al., 2001).ir4.1 immunoreactivity can be demonstrated in cultured

Li et al., 2001) and in situ astrocytes (Poopalasundaram etl., 2000; Higashi et al., 2001). In the olfactory bulb, Kir4.1

mmunoreactivity is detected in about half of the glial fibril-ary acidic protein-positive astrocytes, but not in neuronsHigashi et al., 2001). Immunogold microscopic examina-ion reveals that Kir4.1 channels are enriched in the pro-esses of astrocytes enveloping synapses and blood ves-els (Higashi et al., 2001). In addition to Kir4.1 channels,ther Kir channel subunits may also be expressed in var-

ous glial cell types. Kir2.2 channels are expressed inergman glial cells and astrocytes in the cerebellum (Leo-

pon neuronal stimulation enters the principal glial cell of retina (Müllerg blood vessels and the vitreal endfoot, as K� conductance is maximalace (SRS) lead to K� efflux from apical processes. [From Newman

er (IPL) unvelopin

oudakis et al., 2001), and Kir2.1 channels are found in

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strocytes and oligodendrocytes in the forebrain (Stone-ouse et al., 1999). Finally, single cell in situ PCR exper-

ments in astrocytes from mouse hippocampal slices iden-ified transcripts for Kir2.1, Kir2.2, Kir2.3 and Kir4.1 chan-els (Schroder et al., 2002). Thus, a large variety of Kirhannel subtypes may be expressed in glial cells and thisay explain the wide range of single channel Kir conduc-

ances reported in glial cells (Sontheimer, 1994).The properties of specific Kir channel subunits have

een assessed in heterologous expression systems suchs Xenopus oocytes or transfected cells. Kir2.1 currentshow steep inwardly rectifying current-voltage relation-hips with minimal outward currents at membrane poten-ials positive to EK (Kubo et al., 1993). In contrast, Kir4.1hannels are weakly rectifying, allowing substantial out-ard currents (Takumi et al., 1995). A further complexity isrovided by the fact that Kir channels are tetrameric pro-eins, and in heterologous expression systems Kir subunitsan form either homomeric or heteromeric channels (Stan-eld et al., 2002). The expression of Kir5.1 subunits inenopus oocytes or mammalian cell lines does not result

n functional channels, but co-expression with Kir4.1 chan-els leads to formation of heteromeric channels that areighly sensitive to intracellular changes in pH (Tucker etl., 2000).

As in retinal Müller cells (see below), the distribution of� conductance in astrocytes seems to be non-omogenous. Freshly dissociated salamander astrocytesave an approximately 10-fold higher conductance in theirndfeet than other cell regions (Newman, 1986). So far,nly Kir4.1 channels have been mapped to astrocytic end-eet (Higashi et al., 2001). In conclusion, although it is clearhat K� conductance in astrocytes is likely mediated by Kirhannels, further investigations are required to determinehe precise molecular composition of such channels.

ir channel subtypes expressed in Müller cells

n contrast to astrocytes in the brain, retinal Müller cells areelatively homogenous and are thus an attractive model fortudying Kir channel composition. Recently, severalroups have examined the distribution of Kir channels inüller cells using immunocytochemical and molecular bi-logical techniques (Ishii et al., 1997; Kofuji et al., 2000).hese studies demonstrate highly concentrated expres-ion of the weakly rectifying Kir4.1 channels in Müller cellndfeet and on the processes enveloping blood vesselsFig. 6A, C, E), a distribution that correlates well with thereviously mentioned electrophysiological studies (New-an, 1987b, 1993). Several additional lines of evidencergue that Kir4.1 channels are the principal Kir channelubtype in Müller cells: 1) patch clamp recordings in rabbitüller cells and in transfected 293 cells expressing Kir4.1

hannels show similar single channel conductances andpen probabilities (Tada et al., 1998); 2) genetic ablation ofir4.1 channels in mice decreases the membrane conduc-

ance of Müller cells by 10-fold (Kofuji et al., 2000); and 3)he slow PIII wave of the electroretinogram, which is gen-rated by K� fluxes through Müller cells, is absent in Kir4.1
nockout animals. This effect was not caused by overall s
mpairment of neuronal function as indicated by the facthat the a- and b-waves, associated with neuronal activity,ere not decreased in the knockout animals (Kofuji et al.,000).

Other Kir channels, such as the strongly rectifyingir2.1 channels, may also be expressed in Müller cells. Inouse Müller cells, Kir2.1 channels are expressed along

he plasma membrane in a uniform manner that does notesemble the clustered distribution seen for Kir4.1 chan-els (Kofuji et al., 2002). Such differential expression ofir2.1 and Kir4.1 channels may enhance the efficiency of� siphoning in the retina (Kofuji et al., 2002). Expressionf weakly rectifying Kir4.1 channels in selective membraneomains would allow K� ions to leave Müller cells and betored in extracellular sinks such as the vitreous humorhereas expression of strongly rectifying Kir2.1 channelsould allow greater influx of K� in the synaptic layers

Kofuji et al., 2002).A recent additional study suggests the expression of

ir5.1 channels in the soma and stalks of Müller cells;mmunoprecipitation assays show that a fraction of their4.1 subunits in retina are coassembled with Kir5.1 sub-nits (Ishii et al., 2003). Because heterologously ex-ressed Kir4.1 and Kir5.1 form heteromeric Kir channelshat are highly sensitive to physiological changes in intra-ellular pH, it has been suggested that expression of Kir5.1ubunits in Müller cells promotes the coordinated couplingetween acid–base regulation and K� buffering in theetina (Ishii et al., 2003). In this hypothesis, increases inK�]o and the resulting glial depolarization would increasehe activity of the electrogenic Na�-HCO3� co-transporterNewman, 1996a). The increased influx of HCO3� wouldhen cause intracellular alkalinization and subsequent in-reases in the activity of heteromeric Kir4.1/Kir5.1 chan-els, ultimately enhancing K� uptake into Müller cells (Ishiit al., 2003).

In summary, investigations in Müller cells have pro-ided compelling evidence demonstrating a major role forir4.1 channels in retinal K� buffering. Kir4.1 channelsppear to have the functional and anatomical distributionshat best match the physiological studies performed inüller cells over the past decade. Biochemical and immu-ocytochemical work also suggests that other Kir chan-els, including Kir2.1 and Kir5.1, may be involved in differ-ntial modification of K� entry pathways into these cells.

ir channel accessory proteins in Müller cells:ocalization and function

he focal aggregation of Kir4.1 channels in Müller cellsaises the intriguing question of how these channels areargeted to such precise glial subcellular domains. This isn important question, as the efficiency of the retinal K�

iphoning process is highly dependent on the clusterednd non-homogenous distribution of Kir channels in Müllerells (Newman, 1995). It has recently been shown that theater channel aquaporin 4 (AQP4) is also highly enriched

n the endfeet and perivascular processes of Müller cellsNagelhus et al., 1999). At this time, the physiological
ignificance of such channel co-localization is not precisely

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nown. Transfer of K� ions by K� siphoning in the retina isxpected to generate osmotic imbalances and parallel wa-er fluxes may be needed to dissipate such imbalances. Its possible that the highly asymmetric distribution of AQP4hannels allows funneling of water to the appropriate com-artments in the retina, from Müller cells into the vitreousumor and regions near blood vessels (Nagelhus et al.,999).

This spatial overlap of Kir and aquaporins, two highlyon-homologous channel types, suggests that there maye a common molecular mechanism for their subcellularistribution and targeting. Although the Kir4.1 and AQP4hannels are highly divergent in their primary sequences,hey share a key -S-X-V-COOH motif in their C-termini.his sequence is able to bind to PDZ domains, which areodular amino acid motifs implicated in many protein-

ig. 6. Kir4.1 channel localization in wild type and mdx3Cv (dystrophinembrane (arrow) and to processes around blood vessels (arrowheads

hroughout the retina with a reduction in staining at the inner limiting marrowheads). The Müller-specific marker glutamine synthetase (C, Dcale bar�25 �m (A). [From Connors and Kofuji (2002), with permiss

rotein interactions (Hung and Sheng, 2002). Proteins s

ossessing these domains are abundantly expressed inhe nervous system and include post-synaptic densityrotein-95, Chapsyn-110/PSD-93, SAP-102 and hDlg/AP97 (Hung and Sheng, 2002). Positioning of severalroteins in the postsynaptic density in excitatory synapses

s critically dependent on their interactions with PDZ-do-ain containing proteins (Sheng and Sala, 2001).

Although specific PDZ domain-containing protein(s)ave yet to be unequivocally identified in Müller cells, theAP97 protein, which is present in Müller cells, has beenhown to increase Kir4.1 currents in heterologous systemsHorio et al., 1997). Another candidate is the PDZ domain-ontaining adapter protein, �-syntrophin. In tissues where-syntrophin is present, it is localized to the cell membraney its association with the multi-protein dystrophin glycop-otein complex (DGC; Ahn and Kunkel, 1995). The DGC

t) mouse retinal sections. (A) Kir4.1 is concentrated at the inner limitingtype retina. (B) In the mdx3Cv mouse, Kir4.1 is more evenly distributed

(arrow) and no apparent enrichment of Kir4.1 around blood vesselsrged images (E, F) suggest the localization of Kir4.1 to Müller cells.

knockou) in wild-embrane

pans the cell membrane, forming a molecular bridge be-

taIcftTbmtdmpceinrD

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ween basal lamina proteins in the extracellular space andn array of signaling molecules in the intracellular domain.mmunolocalization studies in retina revealed that the DGComponents, �-dystroglycan and the short dystrophin iso-orm, Dp71, appear to be localized in a fashion very similaro that of Kir4.1 in Müller cells (Claudepierre et al., 2000b).he role of Dp71 in the localization of Kir4.1 has recentlyeen investigated in the dystrophin null mutant mouse,dx3Cv (Connors and Kofuji, 2002). Immunohistochemis-

ry experiments revealed that the polarized subcellularistribution of Kir4.1 is altered in Müller glial cells fromdx3Cv mice, displaying a more homogeneous distributionattern (Fig. 6B, D, F), though immunoblotting and wholeell patch clamp experiments revealed that the channel isxpressed at normal levels at the plasma membrane and

ts electrophysiological properties are unchanged (Con-ors and Kofuji, 2002). Similar findings have also beeneported for a null mouse line for the dystrophin isoformp71 (Dalloz et al., 2003).

These results strongly suggest that the DGC is impor-ant to the localization of Kir4.1 in Müller cells. It is possiblehat the DGC targets the Kir channels to the membraneomains facing the vitreous or blood vessels by binding ofxtracellular portions of the DGC to the basal lamina of

hese regions (Fig. 7). This assumes the existence of anntermediate protein containing a PDZ domain. As men-ioned previously, the best candidate for such an adaptorrotein is �-syntrophin. �-Syntrophin is expressed inüller cells and is putatively part of the Müller cell-specificGC (Claudepierre et al., 2000a). In addition, �-syntrophinas been shown to interact with AQP4 in astrocytes in aDZ-dependent manner, and is required for the membrane

ig. 7. Schematic representation of the glial cell DGC and associatedetween a syntrophin isoform, Kir4.1 and AQP4. [From Kofuji and Co

xpression and localization of AQP4 (Neely et al., 2001). e

herefore, �-syntrophin could underlie the co-localizationf Kir4.1 and AQP4 seen in Müller cells.

In astrocytes, the direct participation of DGC proteinsn the targeting of Kir4.1 and AQP4 channels has beenemonstrated in an �-syntrophin knockout mouse line. Instrocytes, as in Müller cells, AQP4 and Kir4.1 are stronglyxpressed in glial membranes (endfeet) that are in directontact with capillaries and the pia (Nielsen et al., 1997;igashi et al., 2001). Quantitative immunoelectromicros-opy has shown that in the hippocampus of �-syntrophinnockout mice, the expression of AQP4 in astrocyte end-eet is greatly diminished, while expression of Kir4.1 chan-els is less affected (Amiry-Moghaddam et al., 2003).hese results indicate that �-syntrophin is critical for the

argeting and clustering of AQP4 channels to astrocyticndfeet, but is perhaps not as vital for targeting of Kir4.1hannels. Further work will be needed to establish whetherir4.1 channels are indeed linked to the DGC in astrocytesia syntrophin isoforms.

Despite the apparent lack of major rearrangements inir4.1 channel localization and expression in hippocampusstrocytes, the clearance of extracellular K� following neu-onal stimulation was significantly impaired in hippocam-us slices from �-syntrophin knockout mice (Amiry-oghaddam et al., 2003). Such studies provide the firstvidence for a direct functional coupling between AQP4nd Kir4.1 channels. Perhaps, as suggested by Amiry-oghaddam et al. (2003), the efficiency of K� spatialuffering is decreased by impairment of the accompanyingater flux. This tantalizing observation suggests that im-aired targeting or function of AQP4 or/and Kir4.1 chan-els may have clinical relevance in conditions such as

water channels. The complex is shown with the putative interactions03), with permission.]

pilepsy or brain edema. It remains to be seen, however,

wt[Kq

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prbet

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hether Kir4.1 channels play an important role in K� spa-ial buffering in the brain and in the overall buffering ofK�]o. Future studies in mice with genetic inactivation ofir4.1 channels should provide an answer to this importantuestion.

CONCLUSIONS

t has been almost 40 years since the initial proposal of K�

patial buffering as a mechanism for [K�]o buffering in theNS (Orkand et al., 1966). Since then, it has become clear

hat [K�]o regulation involves both net uptake of K� ionsnd K� spatial buffering. The relative importance of these� homeostatic mechanisms may vary from site to site in

he CNS. K� spatial buffering has been best investigatedn the retina, where it is called K� siphoning to reflect theirected K� transport from the plexiform layers to theitreous and to blood vessels (Newman et al., 1984). Mo-ecular studies indicate that Kir4.1 channel localization isritical to the highly asymmetric K� conductance found inüller cells (Kofuji et al., 2000). Furthermore, Kir2.1 andossibly Kir5.1 are also expressed in Müller cells (Kofuji etl., 2002). K� buffering in the retina may be facilitated byoncerted action of the strongly rectifying Kir2.1 channels,hich allow K� entry into Müller cells, and the weakly

ectifying Kir4.1 channels, which allow K� exit to largeinks such as the vitreous humor (Kofuji et al., 2002).oreover, recent reports indicate that dystrophin andystrophin-associated proteins may promote the clusteringnd subcellular distribution of Kir4.1 channels and theater channel AQP4 in astrocytes (Amiry-Moghaddam etl., 2003). The co-localization of water and K� channels inlial cell membranes suggests that K� buffering and waterux are tightly coupled in the brain.

Although significant progress has been made in theast decade, we still do not have a coherent picture of theelative importance of the various mechanisms for [K�]ouffering in the CNS. Given recent advances in optical,lectrophysiological and genetic methods, it is plausiblehat this picture will gain greater clarity in the near future.

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(Accepted 4 June 2004)(Available online 28 October 2004)

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