Inward rectifying potassium channels in human malignant glioma cells

Brain Research, 480 (1989) 249- 258 Elsevier

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Inward rectifying potassium channels in human malignant glioma cells T. Brismar and V.P. Collins Departme~.tof ClinicalNeurophysiology and the Ludwig Institutefor Cancer Research, Karolinska Hospital, Stockholm (Sweden) (Accepted 2 August 1988) Key words: Potassium channel; Inward rectification; Glial cell; Malignant glioma; Potassium homeostasis; Human

Human glioma cells obtained from established cell lines (Tp-276MG. Tp-301MG, Tp-378MG, Tp-483MG and U-2.51MG)were analyzed for the presence of ion channels with the tight-seal voltage clamp technique. The current-voltage relation revealed a marked inward rectification at hyperpolarizingvoltages, due to the presence of inward rectifyingK-channelsin cells from all studied cell lines. These channels were conducting when the membrane potential was more negative than the K-equilibriumpotential. The slope conductance for the inward K-currents (g~) was affected both by [K÷], and [K+Io.g,~ was proportional to [K~]oraised to 0.35 or 0.50, of which the larger value was measured in the presence of low [K']i (25 raM). The rectificationwas not significantlydifferent in cells perfused with Mg-free EDTA-buffered internal solution. TI+ was 3.5 times more permeant than K÷. gr, was blocked by Cs÷ (1 raM) in a voltage-dependent way (more effective in the hyperpolarized membrane), and by Na÷ (154 mM) depending on voltage and time. From measurements of unitary current events in membrane patches (outside out or cell attached) the conductance of the singleinward rectifyingchannel was estimated to be 27 _+7 pS. This type of ion channel may be important for K-uptake by glial cells and hence for the K-bomeostasisin the brain.

INTRODUCTION Several studies have indicated that glial cells are involved in the K-homeostasis in the brain. Glial cells have a membrane potential of about - 9 0 mV, which is dependent on extraceilular [K+J 27'47. Impulse activity in amphibian optic nerve fibres causes a few millivolt depolarization of the glial cells27, and similarly, in the cat visual cortex, glial cells respond with small graded depolarizations when the neighbouring neurons are excited by specific visual stimulation 24. Repolarization of neurons through an outflow of K + into the extraceUular clefts normally raises [K +] only 0.1-0.4 mM above its resting level of 3.0 mM. Much higher K-concentrations have been measured in pathological conditions such as anoxia, epileptic seizures and spreading depression 42. A pronounced and rapid uptake of 42K was found in cultured rat astrocytes exposed to elevated [K+]~. The ionic permeability of ghal cells has recentl)

been studied with the tight-seal voltage clamp technique. In retinal MOiler cells of amphibia there are different types of K-currents, one of which is 'inward rectifying' operating at normal resting potentiaP ~2. Astrocytes from newborn rats exhibit several types of ion currents, viz. small neuronal type inward Nacurrents, delayed outward K-currents, and Cl-currents 2'3'35, Ca- and Ca-activated K-currents 3°'~, all of which are turned on at larger depolarizations than encountered in glial cells. There are also transmitter (y-aminobutyric acid, glutamate and aspartate) activated channels in rat astrocytes and oligodendroc.ytes~,26. The present study was undertaken to elucidate the membrane ion channels of human glioma cells, especially with regard to the mechanism that can he responsible for K-uptake. We found that human malignant glioma cells had a high density of inward rectifying K-channels capable of large inward K-currenB at normal membrane potentials. The presence of out-

Corrmpondence: T. Brismar, Department of Oinical Neurophysiol~D,,Karoliaska Sjuklmsct, S-I040I Stockholm, Swede. 0006-8993/89/$03.50~) 1989Elsevier Science Publishers B.V. (Bion~edi¢~Division)

250 ward K-currents and small Na-currents (in one cell line) is described in the accompanying paper 6. Included is an analysis of the ion dependence of the inward K-currents, the effect of blocking agents and the single channel conductance of the inward rectifying K-channels. A brief account of some of these findings has previously been given5. MATERIALSAND METHODS Materials Established cell lines, derived from human malignant gliomas (glioblastomas). both in early (3-8) and late (>100) passages were studied. The lines Tp276MG, Tp-301MG, Tp-378MG, Tp-483MG and U251MG were examined, U-251MG only at late passages. The line U-251MG is a glial fibrillary acidic protein (GFAP)-positive, established cell line that has been widely used in glioma studies (see e.g. ref. 7). The two lines Tp-276MG an Tp-301MG are established, aneuploid, GFAP-negative glioma lines that grow as a monolayer with fascicular and fibroblastic patterns respectively7. The lines Tp-378MG and Tp-483MG have both been passaged >300 times, they grow as a monolayer culture with glial and fibroblastic growth patterns, are GFAP-positive in early (<200) passages and are aneuploid. This selection of lines may be considered representative for the many giioma lines available today although more of them show GFAP production (an astrocytic characteristic) than is usual. The cultures were routinely grown in Hams F10 medium with 10% calf serum and antibiotics7. Following detachment using trypsin (0.025%), the cells were transferred to the recording chamber. They attached and spread out during the following 16-20 h. No further enzymatic treatment was used after the cells had been transferred to the recording chamber. Current recordings Whole-cell voltage clamp and patch clamp experiments were made at room temperature (23-25 °C) with the tight-seal technique t9 using a List-Medical patch clamp EPC 7. Pipettes were pulled in two steps from soft glass (VWR micropipettes cat. no 53432921), then coated with Sylgard and fire polished. They had a resistance of about 1 MO when filled with internal solution. The series resistance compensation

was about 70%. The records of membrane current were fltered (8pole low-pass Bessel filter 2 kHz), sampled with a 12 bit, 100 kHz A/D converter (Labmaster) and stored in an IBM PC/AT for subsequent analysis. For the present analysis most recordings were made with 100 /~s sampling interval. Improved resolution was required for the detection and analysis of small Na-currents in the accompanying paper6. This was achieved by averaging many records at repeated test pulses and by the subtraction of capacitive and leak currents measured with negative or positive control pulses, like the P/4 procedure of Armstrong and Bezanilla 1. The records of data were sometimes digitally filtered by taking a weighted mean of successive current data (I,) according to the formula: ~. = (In-I + 21. + 1.+1)/4.

Cell capacitance The cell membrane capacitance was calculated from the amount of charge (= area of capacitive spike) associated with a i0 mV step in membrane potential. Very large, spread out cells and cells with long extending processes were avoided, in order to get an acceptable space clamp, which was judged from the time course of the capacitive spike. The cell diameter (or the width of the central part in spindleshaped cells) were in the range 20-30/~m and the cell capacitance was about 40 pF. In rounded cells the capacitance/calculated surface area was approximately 1/zF/cm2, which agrees with an earlier estimate on fetal human glial cells in culture45. External solutions Standard external solution (raM): NaC! 154, KC! 5.6, CaCIe 2.0, HEPES 10.0 titrated with NaOH to pH 7.4. The [K +] was made higher than in the interstitial fluid of the brain, which is 3.0 mM+2, since this facilitated the analysis of inward K-currents. 160 m M K + solution (raM): KC! 160, Ca,~ e 2.0, HEPES 10, titrated with KOH to pH 7.4. Tris solution (raM): Trizma base-HC! (pH 7.4) 154 or 160, KC! 5.6 or KCi-free as indicated ~n text, CaCi 2 2.0. Sucrose solution (raM): sucrose 310, KCI 5.6, CaCi 2 2.0, Trizma base-HCI (pH 7.4) 5.0. Cl.free solution (mM): sodium methylsulfate 154,

251 400

potassium methylsulfate 5.6 (or 5.0 when so indicated), calcium gluconate 2.0, HEPES 10, titrated with NaOH to pH 7.4. CaCI 2, CsCI or Tl-acetate was added to the abov~ solutions as indicated in text.

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lnternal solutions Standard internal solution (mM): KCI 125, KF 25.0, EGTA 10.0, CaCI 2 1.0, MgCI 2 2.0, HEPES 10.0 (titrated with KOH to pH 7.4). The whole cell recordings were generally carried out in 30 rain. No systematic studies were made on whether the presence of KF improved the survival of the cells. A solution with 125 mM Na + and 25 mM K + was made by substituting NaC! for KCI in the above composition and it was titrated with NaOH to pH 7.4. RESULTS

Membrane currents of a voltage clamped glioma cell (from the cell line U-251MG) are shown in Fig. 1.

Depolarization of the membrane (pulse amplitudes 20, 40, 60 and 80 mV) from holding potential (-80 mV) caused only capacitive currents, whereas negative pulses of the same size were associated with large inward currents. These inward currents had a rapid onset and showed a slow decay at the most negative potentials. At less negative steps the inward current reached its maximum after a delay of a few milliseconds. Current-voltage (I-V) relations were plotted from the maximum amplitude of inward and outward currents. Replacement of Na + in the external solution with K + increased the inward currents considerably as illustrated (Fig. 2) by the ! - V curv¢~ obtained from another glioma cell (Tp-276MG, passage 3). Very small inward currents remained in 2C-free solutions (containing either 154 mM Na + or 154 mM Tris+); that is, the membrane currents were almost exclusively carried by K ÷ at these voltages. Not only were the inward currents larger in high [K+]o (the subscript refers to outside or/nside ion concentration), but they also appeared at more positive potenrials. In 160 mM K + the l-Vcurve bent at 0 mV, in 5.6 mM K* at -80 mV. Cells from all studied lines and passages of malignant gliomas had this type of inward K-current with activation depending on voltage and [K+]o. The sub-

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g~s Fig. 1. Membrane currents in human glioma cell (cell fire U251MG), Negative pulses ~rom holding potential (-80 mV) were associated with large inward (negative) currents; positive pulses of the same amplitudes did not eiicit the corresponding outward currents. Only at the largest positive pulses were there outward (positive) currents. Standard solutions on cell outside (154 mM Na + + 5.6 mM K ÷) and inside (150 mM K÷). Cell capacitance calculated from the size of the capacitive currents was 21 pF. Peak of capacitive transients (duration lOOps) left out in the figure. A

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external solution. Holding potential -80 mV between test pulses. Values indicate ma,~mum inward or outward currents during pulses after subtraction of capacitive cmTcnt. The measurements were made at the peak of the inward cunem, which

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extraceuuhr ion ~ (d. V'qg.5). Ceu m , m t m z e7 pF. ~ ceUfine Tp-276M~, pa=age 3.

252 sequent analysis was carried out on the cell line U251MG.

40 30

Ionic dependence of inward K-currents The inward K-current had a nearly linear voltage dependence, and it remained linear in different [K+]o . From a line drawn along the linear part of the I - V curve the slope conductance for K + (gzi) was calculated, and it was measured where this line intercepted the voltage axis. Measurements at 3 different [K+]o in several cells from the line U-251MG showed that the intercept potential was proportional to the logarithm of [K+]o (Fig. 3). Similar experiments in other cells perfused with only 25 mM K + (instead of the standard 150 mM K + in the internal solution) gave the same result, i.e. the intercept potential was related to the logarithm of [K+]o . However, the reduction of [K+]i changed the intercept voltage only by 8 mV, although the calculated effect on the K + equilibrium potential (Eg) is 39 inV. It was concluded that the critical voltage below which inward K-currents appeared corresponded to Eg only under the condition of normal (high) [g+]i . Fig. 4 shows the effect of [K*]o and [K+]i on ggi plotted on log-log scales. There was a large increase in the slope conductance (grJ when [K+]o was elevated, which can be expected from the increased concentration of current carrying ions (K +) in the conducting channels, ggi increased more than 5-fold in 160 mM K + compared to 5.6 mM K +. With 25 mM K + + 125 mM Na + internally the line fitted to the data had a slope of 0.50 which means that the gK, was

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.K-currents. [K+]o on logarithmicscale. Intercept is the potential where a line extrapolated from the steep negative part of the I-V curve crosses the voltage axis. Mean + S.E.M. is indi. cated, symbolswithout bracket had S.E.M. eg + 1.2 inV. Line fitted by eye. Measurementsin 5 cells with 125 mM Na* + 25 mM K+ and 8 cells with 150 mM K÷. Glioma cell line U251MG.

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:1: S.E.M. Values of grq from each cell at 80 mM K+ and 160 mM K+ were normalizedto the measared value of the same cell at 5.6 mM K+, then multipliedwith the mean value of$1q from all cellsat 5.6 mM K+. Lines fitted by eye.

proportional to the square-root of [K+]o. The slope was less (0.35) with 150 mM K + internal solution, ggi was also dependent on [K+]i, being larger at 150 mM K + compared to 25 mM K + + 125 mM Na +, but this difference was only present in low [K+]o. The main effect of high [K+]o on ggi came immediately after the solution change, hut there was in addition a slow increase in ggi, and the complete reversal was slow (minutes). Fig. 4 shows measurements within one minute in high [K+]o. It was noted above (Fig. 1) that the inward current had a time-dependent decline at the most negative membrane potentials. This was quite pronounced when pulses of longer duration were applied, but only in the standard external solution. There was no such decline of the current in 160 mM K + (Fig. 5). Experiments with solutions of low [K*]o, where TrisCi or sucrose substituted NaCI, revealed that the decline of the inward K-current depended on the presence of Na +, which presumably blocked the open channels.

Effect of holding potential, Ca2+, Mg2+and pH Changes in holding potential between -100 and -20 mV lasting 1-2 rain had no significant effect on the ! - V curve (not illustrated). There was thus no evidence of conditioning or inactivation of ggi in the potential region -100 to -20 inV. Changes in [Ca2+] and pH are known to cause a shift in the ionic conductance vs potential relations in excitable tissues9. However, in malignant glioma cells (U-251MG) high

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[Ca2+]o (10 raM) or low pH (6.8) in the external solution resulted in minor changes of the slope of the 1- V curve compared to the curve in normal solution (2.0 mM Ca 2+ and pH 7.4) (Fig. 6). It has recently been described 31.~ that the presence of intracellular Mg 2+ contributes to the inward rectification in heart muscle, preventing the outward flux of K + through this channel. The effect of [Mg2+]i was tested in experiments with: (1) Mg-free internal solution buffered with 10 mM EDTA; and (2) Mgand Ca-free internal solution buffered with 10 mM EGTA. None of these solutions had any significant effe~ on the I - V curve; the rectification was not affected by these changes in [Mg2+]i or [Ca2+]i. The effect of intracellular pH changes were studied by comparison of i - V curves from experiments with different internal solutions. Changes in pH from 7.4 to 6.8 made no significant difference in the inward rectificalion

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(1.0 mM) blocked the inward K-current. The effect was complcte in the presence o~5.6 mM K + in the external solution, but not in solutl?ns ~ of 160 mM K +. In high [K*]o and 1.0 mM Cs + sonde inward current remained at -80 mV (holding potential) and -40 mV, whereas the block was stronger at -120 mV. This voltage dependence of the Cs-block is illustrated by t h e / - V c u r v e (Fig. 7). The channel for inward K-current was more permeable to T! + than K +. Comparison of inward currents (Fig. 8) in solutions of 5.0 mM Tl-acetate and

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Fig. 7. Vollage-dependcnt C~+-Idock of reward current in [K+L. Glioma cell fine U-251MG. Cell capacitance 49 pF.

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5TI Fig. 8. Inward current of Ti+ and the cffect of Cs+. The cutrents were 5 times larger in 5.0 mM Tl-acetate than in 5.0 mM potassium methylsuifate (both were O--free solutions). Addition of 1.0 mM CsCI blocked the inward Tl-currents in a voltage-dependent manner. The solutions were infused in the order: 0 K+ + 0 TI+ ([-1),5.0 mM TI" (~7), 5.0 mM T!+ + 1.0 mM Cs+ (~), 5.0 mM TI ~ (C)), and 5.0 mM K+ (A). Cell capacitance 35 pF.

5.0 mM K potassium methylsulfate in Cl-free solutions (since TICi has low solubility) showed 2 - 5 times larger currents in the presence of TI + than in K + in 4 experiments (mean 3.5 times). Only small inward currents remained in a similar solution without K + and I"1+. Cs + (1.0 mM) blocked the inward Tl-currents in a voltage-dependent fashion similar to the Cs-block of K-currents. Tetraethylammonium chloride (TEA, 10 raM) or 4-aminopyridine (4-AP, 10 mM) in the bath had no significant effect on the inward K-current (not illustrated).

Fig. 9. Inward current m outside-out patch of glioma cell membrane (cell line U-251MG). Each record shows the current associated with a step from holding potential (-80 mV) to indicared voltage. Internal solution contained 150 mM K+, external solution 160 mM K÷. One record shows the effect of 1.0 mM Cs+ added to the external solution.

disappeared. These findings agreed with the expected appearance of single channels for the ir-~'ard K-current. Since the inward K-current of the patch was 40 pA as judged from the decrease of current in presence of Cs + (and this was about 8 times the step size), there should be at least as many single channels in the patch. Visual identification of current steps, and measurements of unit size in records at repeated pulses to different potentials like those in rig. 9 provided the data used for the relation displayed in Fig. 10. A linear correlation (r = 0.89) was calculated between unit size and potential, which had a slope corresponding to a single channel conductance of 29 pS. Estimates of the single channel conductance were also obtained from recordings of membrane current in cell attached patches ~9.

Single channels for in ward K-currents After successful whole-cell recordings withdrawal of the pipette flora the cell sometimes resulted in the formation of an outside-out patch ~9. The current of the membrane patch was studied at different voltages like those used for the whole-cell recordings. When the patch was in 160 mM K + and negative voltage pulses were applied, it was possible to discern small unitary steps in the current records (Fig. 9). The steps were ca. 5.0 pA at -160 mV, larger at -200 mV and smaller at -100 mV. Cs+(l O mM) decreased the inward current, made the record noisy and the steps

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brane potential. In 5 other cells the calculated single channel conductances were 25 and 28 pS (outside-out patches with 160 mM K + inside), 20 and 29 pS (cellattached patches) and 21 pS (inside-out patch with inside exposed to 154 mM Na +, 5.6 mM K + and 2.0 mM CaZ+). These measurements of single channel conductance were all made with 160 mM K + on the outside of the membrane, and the mean value was 27 + 7pS.

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Patch recordings can provide information about the distribution of specific ion channels on the cell surface. The pipette was always positioned in the thick central part of the cell and, therefore, the detection of inward rectifying K-channels in them patch clamp experiments imply that the central regiov, of the cell has such channels. With a whole-cell grq of about 60 nS, the channel density is ca. 2000 channels/ cell, which results in a mean density of 1 channel/2 ~m 2 (mean cell capacitance 40 pF; the relation between surface area and capacitance was described in Materials and Methods).

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time scalesand samplingintervals(100and 1000#s, respectively) in A and B. Pipette solutionwas 160 mM K+ externalsolution.

Fig. 11 shows the current records in a cell-attached patch obtained with 160 mM K + external solution in the pipette. The potential gradient over the patch membrane was changed as indicated in Fig. 11. Distinct steps of inward current measuring about 6 pA appeared when the patch was hyperpolarized by -80 mV, and smaller steps when the hyperpolarizing pulses were less. The single channel slope conductance was ca. 40 pS, calculated from the relation between change in current step size and chan~,e in mere-

Glial cells are believed to play an important role in ion homeostasis in the brain, and any such function must depend on a capacity for ion transport in the glial cell membrane. The membrane permeability was investigated in isolated human glioma cells, after cultivation for various periods in vitro. There were pronounced inward K-currents in ce!ls from all the 5 cell lines studied (both in early and ir late passages), which increased in high [K+]o or when the cell was negatively polarized. The membrane behaved like an electric rectifier since there was almost no outward current at the corresponding positive pulses. The inward K-currents depended on voltage and [K +] in a manner t.hat is typical for an inward rectifying Kchannel. The present study was restricted to cells from established lines of human gliomas. These are useful for a basic exploration of membrane ion channels in human glia since, firstly, there is no doubt that these cells are of gfial orion (3 of the 5 lines express glial fibrillary acid protein in culture), and secondly, cclk of

256 identical genetic origin can be investigated repeatedly under controlled conditions. A similar type of inward current has already been identified in preliminary studies of normal glial cells in primary culture (Brismar and Collins, unpublished observations). Are the findings in glioma cell lines relevant for the understanding of glial cells in situ? It is well-known that glial or glioma cells may lose their phenotypical characteristics in culture, and it is quite possible that there are quantitative differences in ion channel density of cultured cells and astrocytes in situ. It is, however, unlikely that channels for inward rectifying Kcurrents appear in the cytolemma of each of a series of different glioma cell lines, that would not be expressed under normal conditions. To our knowledge there are no similar studies on human glial or glioma cells. In astrocytes from newborn rat cerebral hemispheres inward K-rectification has not been described, but there are other types of voltage- or transmitter-activated ion channels 2,3,25' 26,35.39. However, inward rectification was described in retinal glial cells (Miiller cells) of the turtle s and the Miiller cells of amphibia exhibit several types of K-currents, one of which was inward rectifying 4'32. Inward, or anomalous, rectification has been found in several different preparations, viz. skeletal muscle 23, cardiac muscle cells TM, central neurons of Aplysia22, and in starfish eggs 16. The characteristic feature, found here and in earlier studies, is the dependence on both voltage and [K+]o for activation. Experiments with high [K+]i and various [K+]o showed that the channel opens below a critical voltage, which is close to the equilibrium potential for K. Changes in [K+]i had, however, only minor effects on the potential dependence of inward rectification, which agrees with the results of the perfusion experiments on starfish eggs 17. The inward rectification has an intricate dependence on the cationic composition of the external medium. (1) The inward K-current ~iepe,lded mainly on [K+]o . The slope conductance (gKi) increased with [K+]o raised to 0.50 or less, when [K+]i was high. A square-root dependence on [K+]o was found in starfish eggs t6 and in measurements of siqgle channel conductances in tunicate eggs 36 and guinea pig heart muscle cells4°. (2) In high [Na+]o the inward K-current showed a voltage-dependent decay, which was more pronounced and rapid at very negative poten-

tials. Solutions with different [Na+]o and [K+]o have similar effect on the inward K-currents in skeletal muscle 44. (3) Cs +, even at low concentration, blocked the inward K-currents in a voltage-dependent way, being more effective at very negative potentials, which agrees with the findings in starfish egg cells 15 and skeletal muscle It. (4) Low outside pH or high [Ca2+]o caused a minor decrease in the inward rectification, but there was no shift in the I - V curve as is typical for other voltage-dependent ion channels. Similar small effects on inward rectification of high [Ca2+]o and low pH has been reported in egg cells 14, and no blockade of inward K-currents was detected in 10 mM Ca 2+ in skeletal muscle 43. (5) TI + was 3.5 times more permeant than K +. Sta~sh egg cells are 1.5 times more permeable to TI + than K + (ref. 16). The single channel conductance of the inward rectifying channel in tunicate egg cells is about 5 pS in 50 mM K + measured from unitary steps in patch clamp recordings 1° and 10 pS in frog skeletal muscle studied with current fluctuation measurements at. The single channel conductance was higher (27 pS) in guinea pig heart cells studied with cell-attached patches in 145 mM K + outside, indicating that the mammalian heart ventricle possesses a different channel species 40. A similar value (27 + 7 pS) was calculated in records from both cell-attached and cell-free patches in external solutions containing 160 mM K +, and it is thus possible that glial cells and heart cells have the same kind of inward rectifying channel. Definite conclusions on this point await a detailed characterization of the single channel events.

Siqnificance of inward rectifying K-channels Several studies hart. indicated that glial cells have a large K-permeability that plays an important role for glial uptake of K + and the K-homeostasis in the brain. First, Orkand et al. ~ established the concept of 'spatial buffering' by glial cells based on their findings in amphibian optic nerve. Gardner-Medwin 12 demonstrated that fluxes of K + in rat brain ,,re much greater than could be attriUuted to an extracellular flow, and he concluded that K + can pass through the cells (largely glial cells). The K-permeable membrane of glial cells allows extracellular K + to distribute intracellularly, which increases the distribution volume for K + relative to non-permeable ions.

257 In addition, there may be regional differences in [K+! outside a cell which makes K + move through the cell along its electrochemical gradient. Cytoplasmic uptake, extracellular diffusion and spatial buffering all contribute to the K-buffering capacity in the brain. Their relative importance depends on the cellular geometry and the time of the [K +] perturbation t3. In retinal glial (Miiller) cells and in astrocytes from salamander optic nerve, the endfeet have especially high sensitivity ~o extracellular K + (refs. 32, 33), which has been related to a higher density of inward rectify ing K-channels in the endfeet 4. Suc;'i differences in the K-conductance will determine the pathway taken by the K-current and can distribute K* to large sinks like the vitreous fluid and the blood vessels32'33. A multitude of K-channels has been found in excitable and other cells2n, but of these only the inward rectifying channel is fully activated at resting potential levels, which makes them suitable for K-transport into glial cells. Here we have demonstrated inward K-currents in voltage clamped ceils, where the membrane was held at different voltages by the feedback circuit. Under normal conditions there can only be a passive net inward flux of K ÷ when the resting potential is more negative than the K-equilibrium potential. This requires either: (1) that there is an active electrogenic pump transporting K* out of the glial cell or some other mechanism which makes the resting potential more negative; or (2) that the extraceliular medium is inhomogenous with locally elevated [K +] (more positive K-equilibrium potential) not affecting the whole-cell resting potential very much. The latter alternative is equivalent with the spatial buffering mechanism. What could be the specific nutpose of a rectifying K-channe~ nn glial cells as compared to channel without a directional preference?

REFERENCES 1 Armstrong, C.M. and 13¢zanilla,F., Charge movement associated with the opening and dosing of the activationgates of the Na channels, J. Gen. Physiol., 63 (1974) 553-552. 2 Bevan, S. and Raft, M., Voltage-dependentpotassiumcurrents in cultured astrocytes, Nature (Lond.L 315 (1985) 229-232. 3 Bevan, S., Chiu, S.Y,, Gray, P.T.A. and Ritchie, J.M., The pgesence of voltage-gated sodium, potassium and chloride channels in rat cultured astrocytes, Proc. R. Soc. Lond. Set. B, 225 (1985) 299-313. 4 Brew, H., Gray, P.T.A., Mobbs. P.and Attwell, D., End-

Like a valve it w i l l prevent the immediate back-flow of K + into the intercellular space, and it can lock K ÷ in glial cells next to the neurons from where it originated. This may facilitate the energy-requiring process of pumping K* back into the neurons for the long-term K-homeostasis. A failing K-homeostasis has been observed in epileptic loci, where the intense neuronal activity has increased the K-load beyond the capacity of the K-buffeting mechanisms. In anoxia, inhibition of energyrequiring transport systems may depolarize both glial cells and neurons, thereby causing abno:'mally high extracellular [K+r .2 because ot !ncreased K-release as well as inhibited glial uptake of K* (since the inward K-currents require a maintained resting potential). The phenomenon of spreading depression '~ is characterized by a local accumulation of K*, driving extracellular [K +] above 10-12 mM, which may be due to a primary glial dysfunction~. Although spreading depression has only been shown experimentally in animals and not been found in the human cortex, recent cerebral blood flow studies have inferred that spreading depression might be involved in the pathophvsioiogy of migraine ~'37. it is intriguing :hat functioning or failing glial inward rectifying Kchannels may have a role in different disease mechanisms in the brain.

ACKNOWLEDGEMENTS This work has been supported by the Swedish Medical Research Council (project n o 14X-4255) and the Foundations of Karolinska lnstitutet. Mrs. Liz Fjelkestam provided excellent technical assistance.

feet of retinal ghal cells have higher densities of Ion channels that mediate K" buffering,Nature (Lond.), 324 0986) 46f~-468.

5 Brismar, T. and Collins. V.P., lmvard K-currentin human malignant glioma cclh - - possible ~ for K-homeostasis in :Se brain. Acta Physiol. $cand., 132 (19~8)

259-260. 6 Brismar. T. and Collim, V.P., Polassiumand sodinmchlmnels in human i~oma cells. Brain R~seun~, 481} 0989) 259-267. 7 Collins.V.P.. Cultured human ~

and gJioma cells.In

G.W. Richter and M.A. ~ (Eds.), ~ Review o / E w e n m e n ~ PaOu~V'. tot. 24. Aca,ku~, 1983,

258 pp. 135-202. 8 Conner, J.D., Detwiler, P.B. and Sarthy, P.V., Ionic and electrophysiologicalproperties of retinal Miiller (glial) cells of the turtle, J. Physiol. l Lond. ), 362 (1985) 79-92. 9 Frankenhaeuser, B. and Hodgkin, A.L., The action of calcium on the elec0rical properties of squid axons, J. Physiol. (Lond.), 137 (1957) 218-244. 10 Fukushima, Y.. Singi~ channel potassium currents of the anomalous rectifier, Nature ILond.), 294 (198 !) 368- 371. II Gay, L.A. and Stanfield, P.R., Cs* causes a voltage-dependent block of inward K currents ir restin8 skeletal muscle fibres, Nature (Lond.), 267 0977) 169-170. 12 Gardner-Medwin, A.R., A study of the mechanism by which potassium moves through brain tissue in the rat, J. Physiol. (Lond.), 335 (1983) 353-374. 13 Gardner-Medwin, A.R., A new fran~work for assessment of potassium-buffering mechanisms, Ann. IV. Y. Acad. Sci., 481 (1986) 287-302. 14 Hagiwara, S., Miyazaki, S., Moody, W. and Patlak, J., Blocking effects of barium and hydrogen ions on the potassium current during anomalous rectification in the starfish egg , J. Physiol. (Lond. j 279 (1978) 167-185. 15 Hagiwara, S., Miyazaki, S. and Roscnthal, N.P., Potassium current and the effect of cesium on this current during anomalous rectification of the egg cell membrane of a starfish,J. Can. Physiol., 67 (1976) 621-638. 16 Hagiwara, S and Takahashi, K., The anomalous rectification and cation selectivity of the membrane of the starfish eg~ cell, J. Membr. Biol., 18 (1974) 61-80. 17 Hagiwara, S. and Yoshii, M., Effects of internal potassium alld sodium on the anomalous rectification of the starfish egg as examined by internal perfusion, J. Physiol. (Lond.), 292 (1979) 251-265. 18 Hall, A E . , Hurter, O.F. and Noble, D., Current-voltage relations of Purkinje fihre~ in ¢odium-deficient solutions, J. Physiol. (Lon.~.), 16o (1963) 225-240. 19 HamiU, O.P., Marry, A., Neher, E., Saxmann, B. and Sigworxh, F., Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane pa:ches, Pfl~gers Arch., 391 (1981)85-100. 20 Hertz, L . An intense potassium uptake into astrocytes, its further enhancement by high concentrations of potassium, and its possible involvement in potassium homeostasis at the cellular level, Brain Research, 145 (1978) 202-208. 21 [lille, B.. lol, Channels of Excitable Membranes, Sinaaer, Sunderland, MA, 1984, p. 220. 22 Kand¢l, E.R., Tauc, L., Anomalous rectification in the metacere~ral giant cells and its consequences for synaptic rransr~ission, ]. Physiol. (Lond.), lg3 (1966)28"-304. 23 Katz, B., Les constantes ilectriques de la membrane du musch', Arch. Sci. Physiol., 3 (1949) 285-300. 24 Kelly, J.P. and Vat. Essen, D.C., Cell structure and function in the visual cortex of the cat, J. Physiol. (Lond.), 238 (1974) 515-547. 25 Kcttermann, H., Backus, K.H and Schachner, M., 7Aminobutyric acid opens CI-channels in cultured astrocyt~s, Brain Resea~rh, 404 (1987) I-9. 26 Ketten~ann, H. and ~hachner, M., Pharmacological properti:s of gamma-aminobutyric acid-, glutamate-, and aspartat,:-induced depolarizations in cultured astrocytes, J. Neurosci., 5 (1985) 3295-3301. 27 Kuffler S.W., Ntcholls, J.G. and Orkand, R.K., Physiological properties of glial cells in the central nervous syst'~m of amphibia, J. Neurophvsiol., 29 (1966) 768-787.

28 Lauritzcn, M., Cortical spreading depression as a putative migra;ne mechanism, Trends Neurosci., 10 (1987) 8-13. 29 L¢~o, A.A.P., Spreading depression of activity in the cerebral cortex, J. Neurophysiol., 7 (1944) 359-300. 30 MacVicar, B.A., Voltage dependent calcium channels in giial cells, Science, 226 (1984) 1345-1 t47. 31 Matsuda, H., Saigusa, A. and Irisawa, H., Ohmic conduc. lance through the inwardly rectifying K channel and blocking by internal M82.. Nature (Lond.), 325 (1987) 156-159, 32 Newman, E.A., Voltage-dependent calcium and potassium channels in retinal [,lied cells, Nature (Lond.), 317 (1985) 809-811. 33 Newman, E.A., High potassium conductance in astmcyte endfeet, Science, 233 (1986) 453-454. 34 Nicholson, C. and Kraig, R.P., The behavior of extracellular ions during spreading depression. In Zeuthen led.), The Applicatioa of Ion.Selective Microelectrodes, Elsevier 1981 pp. 217-237. 35 NJwak, L., Ascher, P and Berwald-Netter, Y., Ionic channels in mouse astrocytes in culture, J. Neurosci., 7 (1987) 101-109. 36 Ohmori, H., lnactivatio, kinetics and steady state noise in the anomalous rectifier of tunicate egg cell membranes, J. Physiol. (Lond.), 281 (1978) 77-99. 37 Olesen, J. Migraine and regional cerebral blood flow, Trends Neurosci., 8 (1985) 318-322. 38 Orkand, R.K., Nicholls, J.G. and Kuffler, S.W., Effect of nerve impulses on the membrane potential of glial cells in the ceittral nervous system of amphibia, J. Neurophysiol.. 29 (1966) 78S-806. 39 Ouandt, F.N. and MacVicar, B.A., Calcimn activated potassium channels in cultured astrocytes, Neurosclence, 19 (1986) 29-41. 40 Sakmann, B. and Trube, G., Conductance properties of single inwardly rectifying potassium channels in ventricular cells from guinea-pig heart, J. Physiol. (Lond.), 347 (1984) i-657. 41 Schwarz, W., Neumcke, B. and Palade, P.T., K.current fluctuations in inward-rectifying channels of frog skeletal muscle, J. Membr. Biol., 63 (1981) 85-92. 42 Somjen, G.C., Aitken, e.G., ";iacchino, J.L. and McNamars, J.O., Interstitial ion concentrations and pawxysmal discharges in hippocampal formation and spinal cord, Adv. Neurol., 44 (1986) 663-680. 43 Standen, N.B. and Stanfield, P.R., A potential- and timedependent blockade of inward rectification in frog skeletal muscle fibres by barium and strontium ions, J. Physiol. (Lond.), 280 (1978) 169-191. 44 Standen, N.B. and Stanfield, P.R., Potassium depletion and sodium block of potassium currents under hyperpolarization in frog sartorius muscle, J. Physiol. (Lond.), 294 (1979) 497-520. 45 Trachtenherg, M.C., K'ornblith, P.L. and Htluptli, J:, Biophysical properties of cultured human glial cells, Brain Re. search, 38 (1972) 279-298. 46 Vandenberg, C., Inward rectification of a potassium channel in cardiac ventricular cells depends on internal magnesium ions, Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 2560-2564. 47 Walz, W., Wuttke, W. and Hertz, L., Astrocytes in primary cultures: membrane potential characteristics reveal exclusive potassium conductance and potassium accumulator properties, Brain Research, 292 (1984) 367-374.