1997 na+—ca2+ exchange currents in cortical neurons concomitant forward and reverse operation and...

8/12/2019 1997 Na+Ca2+ Exchange Currents in Cortical Neurons Concomitant Forward and Reverse Operation and Effect

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European Journal of Neuroscience, Vol. 9, pp . 1273-1281, 1997 European Neuroscience Association

Na+-Ca2+ Exchange Currents in Cortical Neurons:Concomitant Forward and Reverse Operation and Effectof GlutamateShan Ping Yu and Dennis W. ChoiCenter for the Study of Nervous System Injury and Department of Neurology, Washington University School of Medicine,St Louis, MO 63110, USAKeywords: mouse, patch clamp, intracellular calcium, glutamate toxicity

AbstractNa+-Ca2+ exchanger-associatedmembrane currents were studied in cultured murine neocortical neurons, usingwhole-c ell recording combined with in tracellular perfusion. A ne t inward current specifically associated withforward (Na+ o-Ca2+i) exchange was ev oked at -40 mV by switching external 140 mM Li+ to 140 mM N a+. Thevoltage depende nce of this current was consistent with that predicted for 3 Na+ :I Ca2+ exchange. As expected,the curre nt depended on intern al Ca2+, and co uld be bloc ked by intracellular application of the exchangerinhibitory peptide, XIP. Raising internal Na+ from 3 to 20 mM o r switching the external solution from 140 mM Li+to 30 mM Na+ activated outward currents, consistent with reverse (Na+,-Ca2+o)exchange. An external Ca2+-sensitive current was also identified as associated with reverse Na+-Ca2 + exc hange based on its internal Na+dependence and sensitivity to XIP. Com bined application of external N a+ and C a2+ in the absence of intern alNa+ triggered a 3.3-fold larger inward current than the current activated in the presen ce of 3 mM internal Na +,raising the intrigu ing possibility that Naf-Ca 2+ exchangers might concurren tly operate in both the forward andthe reverse direction, perhaps in different subcellular locations. With this id ea in m ind, we e xam ined the effect ofexcitotoxic glutamate receptor activation on exchanger operation. After 3-5 min of exposure to 100-200 pMglutamate, the forward exchanger curre nt was significantly increased even when e xternal Na+ was reduced to100 mM, and the external Ca*+-activated reverse exchanger current was eliminated.IntroductionNa+-Ca2+ exchangers are a specialized class of ion transporters with11 transmembrane segments, distributed widely in heart, kidney, brainand other tissues (Rah amimoff, 1990; Philipson and Nicoll, 19 92).In the brain, Na+-Ca2+ exchanger mUNA is abundant in the cortex,hippocampus, dentate gyrus, thalamus and cerebellum (Wroblewski,1992). The brain exchanger is similar to the heart Na+-Ca2+exchanger, and can be labelled with antibodies raised against thelatter (Furman et al., 1993; Marlier et al., 1993). Under normalconditions, Na+-Ca2' exchangers are thought to transport Na+ inand Ca 2+ out (forward, or Na+o-Ca2+i exchange), a function vitalto the maintenance of intracellular free Ca2+ ( Ca2+ Ii) homeostasis.Under some conditions, however, such as when intracellular Na+([N afIi) is increased or the m embrane is depolarized, energetics canfavour reverse operation, moving Ca2+ in and Na+ out (reverse orNa+i-Ca2+o exchange) (Mullins, 1977; DiPolo and Beau@, 1986).The physiology of Na+-Ca2+ exchangers has been extensivelystudied in cardiovascular preparations (Reeves and Sutko, 1980;Kaczorowski, 1992; Hilgemann et al., 1991; Niggli and Lederer,1993), rod outer segments (Schnetkamp et al., 1989) and squid giantaxons (Allen, 1991). The majority of Na+-Ca2+ exchangers have a

transport stoichiometry of 3Na': 1C a2+ , nd therefore are electrogenic,moving one net positive charge inward with each forward cycle. Themembrane current specifically associated with exchanger activity hasbeen demonstrated electrophysiologically in mammalian heart cells(Barcenas-Ruiz et al., 1987; Kimura et al., 1987; Hume and Uehara,1988; Mechmann and Pott, 1988; Ehara et al., 1989; Niggli andLederer, 1993), in retinal cells (Schnetkamp et al., 1989; G leasonet al., 1995) and in the squid giant axon (Caputo et al., 1989).Neuronal Na+-Ca2+ exchanger may also play important rolesin maintaining [Ca2+ Ii in normal an d pathological conditions inmammalian central neurons (Fontana et al., 1995; Reuter and Porzig,1995). The aim of the present study was to extend the electrophysiolo-gical characterization of Na+-Ca2+ exchanger current to centralneurons. A specific question was how the exchanger would respondto excitotoxic overactivation of glutamate receptors, something diffi-cult to predict a priori. On the one hand, glutamate-induced increasein [Na+]i might stimulate reverse operation of the exchanger, actingto augment intracellular Ca2+ overload and potentiate injury (Choi,1988; K iedrowski et al., 1994). On the other hand, the concurrentglutamate-induced increase in [Ca2+ ]i might stimulate the forward

Correspondence to: Dennis Choi, Department of Neurology, Box 8111, Washington University School of Medicine, 660 South Euclid Avenue St Louis,M O 63110, USAReceived 3 September 1996, revised 30 December 1996, accepted 31 January 1997


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1274 Nai-Ca2+ exchanger currents in cortical neuronsNa +-C a2+ exchange, which might serve as an important protectivemechanism, acting to restore Ca 2+ homeostasis and limit resultantcell injury (Choi, 1992). By measuring the membrane currentsassociated with forward and reverse Na+-Ca2+ exchanges respect-ively, the present experiments revealed that both exchange modesmay concurrently occur in the sam e cells, and that glutamate receptorstimulation particularly enhances the forward Na+-Ca2+ exchangeactivity even at reduced external Na'.

Materials and methodsNeurocotfical primary cell culturesMixed cortical cultures, containing neurons and confluent glial bed,were prepared as described previously (Rose et al., 1993). Dissociatedneocortices obtained from fetal mice a t 15-17 days of gestation wereplated onto a previously established glial monolayer (see below) ata density of 0.27 hemispheres per 35 mm culture dish (Falcon,Primaria), in Eagle's M inimal Essential Medium (MEM, Earle's salts)supplemented with 20 mh4 glucose (final concentration, 25 mM ), 5%fetal bovine serum and 5% horse serum. Medium was changed after1 week to MEM containing 20 mM glucose and 10% horse serum,as well as cytosine arabinoside (final concentration 10 pM) to inhibitcell division. Subsequently, cultures were fed once w eekly with ME Mcontaining 25 mM glucose plus 2 mM glutamine. Cultures were keptin a 37 C, humidified incubator in a 5 COz atmosphere. Allexperiments were performed on cultures between days 12 and20 in vitro.

Glial cultures were prepared from dissociated neocortices ofpostnatal day 1-3 m ice. Cells were plated at a density of 0.4hemispheres per 35 mm dish, in Eagle's MEM containing 25 mMglucose, 10% fetal bovine serum, 10 horse serum and 10 ng/mlepidermal growth factor; a confluent glial bed was formed in1-2 weeks.Whole-cell recording of Na+-C + exchanger currentsThe 35 mm culture dish was used as the recording chamber on thestage of an inverted microscope (Nikon). Neurons of 15-20 pmdiameter, with phase-bright cell bodies on top of the glial bed,were chosen for recordings. Neuronal identity has been previouslyconfirmed by Nissl staining and electrophysiological characterization,whereas the glial bed is immunoreactive for glial fibrillary acidicprotein (Choi et aL 1987; Rose et al. 1993).The patch electrodes had tip resistance of 5-1 0 Mi2 (fire-polished).Voltage clamp was achieved by using an EPC-7 amplifier (ListElectronic, Darmstadt, Germ any). Series resistance compensation a ndcapacitance compensation were routinely applied. Whole-cell currentwas digitally sampled at 100 or 300 ps (10 or 3.3 kHz). The currentsignals were filtered by a 3 kHz, hree-pole Bessel filter. Currenttraces were displayed and stored on a Macintosh computer (Quatra950, Apple Computer) using a data acquisitiodanalysis programPULSE (HEKA Electronik, L ambre chtPfalz, Germany). Some datawere simultaneously stored on video tape via a digital data recorder(WR-lOB, Instrutech, Great Neck, USA).lntracellular perfusionManipulation of intracellular ion concentrations was achieved byperfusion of the patch pipette and cellular dialysis as described before(Lopez 1992; Yu et al., 1994), using a modified recording electrodeholder (A058-C; E. W. Wright, Guildford, CT). The holder had twooutlets; one of them was connected with fine internal tubing of200 pm external diameter inserted close to the tip of the electrode

and the other outlet was connected to solution reservoirs. Perfusionof the internal capillary, generated by negative pressure of 4-5 p.s.i.applied to the inside of the electrode holder, replaced internal solutionin the recording electrode. Judged by coloured solution replacements,the solution in the electrode tip could be changed in


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Na+-Ca2+ exchanger currents in cortical neurons 1275TABLE . Experimental solutionsExternal solution (mM) NaCl LiC l CaC12 HEPE S BAPTA Gluc oseNaf/Ca2+ 140Li+/Ca*+Na+/O Ca2+ 140Li+/O Ca2+Internal solution (mM)3 mM Na+ 310 mM Na+20 mM Na' 200 Na+o Ca2+ 30 BAF'TA

140140NaCl13010110133130

3

22

Cs-acetate1204.2

4.24.2

130

10101010CaC12104.2101010

0.50.5HEPES102.02.02.02.0

10

BAPTAg-ATP10101010

2.0 10

2.0Voltage-activated channel blockers TEA ( 5 mM), nifedipine (10 pM), tetrodotoxin (0.5 pM) and Na+-K+ -ATPase inhibitor ouabain (20 pM) were included inall external solutions. Different Na+ concentrations were attained by substituting equimolar TEA or sucrose for N aCl. Free C a2+ in the internal 3 mM or 20mM Na+ solution was estimated as 120 nM (Yu et a l . , 1994).pH = 7.3 for all solutions.

(kindly provided by Dr R. A Colvin, Ohio State University) isa selective inhibitor of the Na+-C a2+ exchan ger, acting on anautoinhibitory site of a calmodulin binding region of the exchanger(Li e t al., 1991). It inhibits both Na+i-depend ent Ca2 + uptake (Ki -1.5 FM ) and Na+,-dependent Ca2 + efflux in sarcolemmal vesicles,as well as the Na+-Ca2+ exchange current recorded from excisedsarcolemmal patches 4 0.1 pM). It does not affect sarcolemmalCa2+ binding, Ca2+ permeability, Na+-K+ ATPase function orsarcoplasmic reticulum Ca2+-ATPase function (Li et aZ., 1991).

ResultsMembrane current linked to forward Na+-Ca2+ exchangeTo block forward N a+ -C a2 + exchange, neurons were immersed inLi+ /Ca2 + external solution (140 mM Li+, 2 mM Ca2+ , and no Naf ;Table l), with 3 mM Na+ internal solution ( 3 mM Na', 130 mMCs; Table 1). Cell capacity was 3 8.6 0.5 pF n = 12). At a holdingpotential of -40 mV, and in the presence of 0.5 pM tetrodotoxinand 10 FM nifedipine, no spontaneous currents were observed.Replacement of the Li+/Ca2+ solution with Na+/Ca2+ solution (140mM Na+, 2 mM Ca 2+; Table 1) via local application around the cellbody activated an inward current of -75.8 ? 14.9 pA n = 24) (Table2 ) ; he current density was 1.9 ? 0 .3 pNp F n = 12), similar to thecurrent density in heart cells (Kimura et al., 1987; Miura and Kimura,1989). This external Na+-activated inward current showed no time-dependent inactivation during the 0.6 s of Na+ application, andceased when Na+ application stopped (Fig. 1A). There was no gross'run-down' of this current during 10-15 min recordings (Fig. lA ,inset). The current exhibited the expected voltage-dependence: largercurrents were activated at hyperpolarized potentials (Fig. lC ), consist-ent with the known voltage-sensitivity of forward Na+-Ca2+exchange. Based on the exchange model of 3 Na+ :1Ca 2+ (Mullins,1977) the equilibrium potential of the Na+-Ca2+ exchanger, E N ~ , c ~ ,was ca lculated according to the equation:

where N^ and Eca respectively are the equilibrium potentials of Nafand Ca2+ across the membrane given by the Nernst equation. Thecalculated E N ~ , C ~n our experimental condition (140 mM externalNa+ and 120 nM [Ca2+ Ii) was +47 mV, which coincided with theexperimentally determined reversal potential of +47 2.3 mV( n = 4).

TABLE. Concurrent forward and revers e Naf-C a2+ exchange~~

External solution chan ge Interna l solution A ctivated curren t n

Fomard operation of theNa+-Ca2+ exchangerLi+/Ca2+ o Na+/Ca2'Li'/Caz+ to Na+ /Ca2 + 0 Ca2+Lif/Ca 2+ to Na+/Ca*+(Na' reduce d to 100 mM)

3 mM Na'

3 mM Na+Reverse operation of theNu -Ca2+ exchangerLi/O Ca2+ to Li+/ Ca2 +Li+/O Ca2 + o Li'/Ca2+Li+/O Ca2+ o Li+/Ca2+Concurrent o peration of theNa -Cd+ exchangerLi+/O Ca2+ o N af/Ca2 +Li+/O Ca2+ to Na+/Ca 2+

3 mM Na+0 Na'o Ca*+

3 mM Na+0 Na+

(PA)

-75.8 14.9-28.0 ? 8.0*-34.6 ? 8.6*

+78.9 Z 10.2+12.0 ? 5.5*+22.7 ? 7.0*

-26.0 ? 7.9-84.7 ? 10.8*

2437

20104

53

~ ~ ___ __ ~ ~*Significant difference ( P < 0.05 by Student's t-test) from respective control(the first row in each category).

When [Na+Ii was raised from 3 mM towards 10 mM (Table 1 byintracellular perfusion, the exchanger reversal potential shifted towardsnegative potentials, as predicted by the calculated equilibrium potentialof 4 . 5 mV (Fig. 1C). The current was also expectedly sensitive toexternal Na+ and internal Ca2+. Reducing external Na' to 100 mMdecreased the size of the inward current to -34.6 8.6 pA (Table 2).When 10 mM BAPTA was included in the recording pipette, thecurrent evoked by external Na+ application was also reduced (Table 2).Further supporting the identification of this external Na+-activatedinward current as the current associated with the Na +-C a2+ exchanger,the current was largely blocked by intracellular application of theselective Na+-Ca2+ exchanger blocker, XIP (5 pM; -24 15 pAwith XIP, n = 5 ; Li e t d., 991; Wu and Colvin, 1994) (Fig. IB) .Mem brane current linked to reverse Na+-Ca2+ exchangeTo examine the reverse Na+-Ca2+ exchanger operation, we usedintracellular perfusion to manipulate [Na+Ii while blocking forwardexchan ge by substituting external Na' with Li + as above. Raising[Na+Ii from 3 to 20 mM by intracellular perfusion reversibly evoked


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1276 Nat-Ca2t exchanger currents in cortical neurons

AExternal Li+/Ca2+

180 PA0.2 sec

... ... .. . . .. . ......... ....... ... . ... ................. W Au u u L V ' II 1 min 3m in 5min 8min 10m in 15minLi+/Ca2+

+Ja+/Ca2+ Na+/Ca2+5 0 1

Control5-200-250-300

0.0 0.2 0.4 0.6 0 8 1.0 1.2 1.4 1.6Second

C

Current PA)200100

-400 1 1 1 1 1 1 1 1 \-30 -10 I 0 30 50Voltage mV)

Li+/Ca2+ a+/Ca2+

prvl XIP0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6Second

Current PA)

-40050 -30 -10 10 30Voltage mV)

3 mM [NaIi 10 mM [NaIiFIG. . Forward exchanger operation. (A) In a bath medium containing 0 Na', 140 mM LiCl and 2 mh4 CaCI2 (Li+/Ca2+ solution), local application to thecell body of a solution containing 140 mM NaCl and 2 mM CaC12 but no Li+ (Na +/C a2+ solution) reversibly activated an inward current response. Holdingpotenbal here and subsequently was 4 0 mV unless stated otherwise. (Inset) The Na+-activated inward current w as fairly stable during a 15 min controlrecorchng, a dotted line shows the baseline level. (B) Switching from Na +/C a2+ solution to a Li+/C az+ solution reversibly evoked an outward current shift thatcould be blocked by intracellular perfusion of 5 pM XIP. Notice that in the presence of XIP the baseline current moved from --125 to --55 PA, presumablydue to block of a tonic inward exchanger activity. (C) The current-voltage relationship for the Na+-a ctivated inward current shift. The current shift was triggeredby ramp voltage pulses in Na+ -free solution (Li+/Ca2+ solution) and in Na'-containing solution (Na+/C a2+ solution); internal Na + was 3 mM. The Na+-activated current disappeared at -40 mV (shown by intersection of the Na' relationship with the Li+ relationship, marked by an arrow). If intracellular Na'was increased by perfusion to 10 mM, this Na+-activated current shift was attenuated and the intersection point with the Li+ relationship shifted to morenegative potentials. Determination of the exact intersection point was hampered by an increase in the noise level, due to the higher capacitance of the specialelectrode holder needed for intracellular perfusion.

an outward membrane current (Fig. 2A). As this current was oppositein direction to that expected from an alteration of membrane surfacecharge, it probably reflected reverse Na+-Ca2+ exchange.

Alternatively, reverse Na+-Ca2+ exchange current could be trig-gered by switching the external solution from Lif/Ca2+ to a lowlevel of Na+/Ca2+ (30 mM) (Table 1). Instead of the inward current


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Na+-Ca2+ exchanger currents in cortical neurons 1277was due to block of tonically active reverse Na f- Ca 2+ exchange, itwas blocked by prior intracellular dialysis with Na+-free solution(Fig. 2B). Such internal Na'-dependence argues against the alternativepossibility that the current shift reflects activation of a Ca2+-sensitiveCl- channel by Ca2+ removal (Taleb et al. 1992). In addition, thecurrent could be blocked by intracellularly applied 10 pM XIP (-2.9

4.7 pA with XIP, n = 5).Yet another explanation for the current shift produced by applicationof external Naf/O Ca2+ solution might be an increase in forwardexchange. External Ca2+ might compete with Na+ for the externalbinding site of the Na+-Ca2+ exchanger (Miura and Kimura, 1989),so removing external Ca2 + might enhance forward Na+-Ca2+exchange due to increased binding of external Na+ to the exchanger.To eliminate this possibility, experiments were performed in externalNa' -free solutions which block forward exchan ge. Withdrawal ofexternal Ca2 + (from Li+/Ca2' to Li+/ 0 Ca2+ solution) induced asimilar inward current shift, and application of Ca2 + (from Li+ /0Ca2+ to Li+/Ca2+ solution) triggered an outward current shift(Table 2). Again, prior intracellular dialysis with Na' -free solutionsuppressed these current shifts (Table 2), consistent with the idea thatthese currents reflected reverse Na+-Ca2+ exchange. Lastly, sinceNa+-Ca2 + exchanger activation depends on the availability of [Ca2+],for an internal regulatory site (DiPolo and BeaugC, 1986; Kimuraet al., 1986), we verified that reduction of [Ca2+], (with 10 mMBAPT A in Ca2+-free pipette solution) reduced the outw ard currentshift evoked by the external solution switch fro m Li +/0 Ca 2+ to Li'/Ca2+ (Table 2).

A20 mM Na' 20 mM Na'3mM Na'Solution --

I PA40 sec

BNaO Caz+ Na+/O C&Na*/Ca2+ N a 7 E Na?Ca2'-...............................................

3 mM Internal Na' No Internal NatM P A.3 C

FIG.2. Reverse exchanger operation. (A) An outward current was generatedby raising intracellular Na'. The ex tracellular solution contained 0 Na', 140mMLi+ and 2 mM Ca 2+ , onditions disabling forward operation of the Na+-Ca exchanger. Raising internal Na' to 20 mM by intracellular perfusionreversibly activated an outward current shift. Similar results were obtained inthree ex eriments. (B) In the presence of 3 mM internal Na', switching fromcurrent shift ( lef t), that was dependent upon the presence of in tracellu lar Na'(righ t). The same results were obtained in three experiments. A dotted lineshows the zero current baseline.

Na+/Ca3+ solution to Na+/O Ca2 + solution reversibly induced an inward

TABLE . Effects of glutamate on Nat-Ca2+ exchanger currentsExternal solution change Control Glutamate n

(PA) (PA)Forward operationLi+/Ca2+ o Na f/C a2+ -83.8 19.2 -196.2 60.8* 5Li+/Ca2+ o Na+/Ca2+(Na' reduced to 100 mM) -37.0 7.2 -105.8 ? 24.9* 12Reverse operationLi'/Ca*+ to Na +/C a2+(Na' reduced to 30 mM) +63.8 13.7 +94.0 22.4 4LiIO Ca2' to Li'/Ca2+ +76 .4 16.5 -18.9 6.2* 9Forward and reverse exchange currents were activated by the specifiedextracellular solutions before (control) and 1-3 min after glutamate treatment(100-200 pM in Na+/Ca2+ olution for 3-5 min). BAPTA was omitted fromthe pipette solution to permit [Ca2+Ii to increase normally. Negative currentreflects inward current (forward exchange) and positive current reflects outwardcurrent (reverse exchange).*Significant difference from control (P < 0.01).

triggered by shifting from Lif/Ca2+ to 140 mM Na+/Ca2+ (seeabove), an outward current appeared (Table 3). This apparent reversalof exchange current is consistent with negative shift in exchangereversal potential predicted by the 3Na': 1Ca2 + exchan ge model(Mullins, 1977; Ehara et al. , 1989).

We also identified a membrane current sensitive to external Ca2+as the reverse Na+-Ca2+ exchange current. When Na+ /Ca2 + externalsolution was switched to a Ca2+-free solution (Na+/O Ca2+ solution;Table l , an inward current shift was reversibly evoked (-84.0 ?20.7 PA, n = 4; Fig. 2B). Supporting the idea that this current shift

Concomitant forward and reverse Na+-C + exchanges in thesame cellsBoth external N a+- and external Ca2+-activated membrane currentswere ob served in the same cells. In each of six cells tested, we observedboth a baseline reverse exchange current (outward current blocked byremoval of external solution switch from Li+/C a2+ o Li+ /0 Ca2+) anda subsequent forward exchange current (inward current immediatelyactivated by a second switch from Li+/O Ca2+ to Nai/O Ca2 +) Fig. 3).These data led us to the hypothesis that both forward and reverseNaf-C a2+ exchan ge activities might exist simultaneously, perhaps indifferent parts of same cells. To test this hypothesis, we examinedwhether forward exch ange activation combined with reverse exchangeblockage wo uld evoke a larger inward current than the activation with-out blocking reverse exchange. Indeed, activation of forward exchan geby external solution switch from Li/0 Ca2+ to Na+ /Ca2 + n the absenceof Na + in the recording pipette evoke d a 3.3-fold larger inward currentthan that activated by the sam e solution switch in cells where the pipettesolution contained 3 mM Na' (Table 2 ) .Effect of glutamateon forward and reverse Na+-Ca2+exchange currentsWith baseline characterization accomplished, we examined whethertoxic exposure to glutamate would reduce, or even reverse, normalforward Na+-Ca2 + exchange. Cells were initially immersed in Li/Ca2+external solution and the inward current associated with forward Na +-Ca2+ exchange was activated by local application of Na+/C a2+ olutionas described above. Glutamate (100-200 pM fo r 3-5 min) exposurewas carried out in N a+/C a2+ solution, zero internal BAFTA, and novoltage clamp, to approxim ate physiological conditions (Tab le 1). Themembran e potential depolarized from a baseline value of -5 1 ? 2 mV( n = 45) to -14 ? 2 mV ( n = 16), measured during a 5 min applicationof 200 p M glutamate; it returned to -35 % 2 mV measured 1-10 minafter washing out glutamate n = 16). Brief checking under voltage


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1278 Nat-Ca2' exchanger currents in cortical neuronsLi+/OCa2+Li+/Ca2+ Na'lO Ca2+

100-1:::i0

0 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4Time second)

FIG.3. Both forward and reverse operation of the exchanger can be measuredin the same cell. This experiment was started with an external solutioncontaining Li' (140 mM) and Ca2+ (2 mM), and an internal solutioncontaining 3 mM Na' and 120 nM Ca2 +. Withdrawal of ex ternal Ca2+produced an inward current sh ift, probably representing blockade of ongoingreverse exchanger operation. When this Ca2+-fre e external solution wasswitched from Li+- to Na+-containing solution, an additional inward currentshift appeared, due to activation of forward exchanger current. Similar resultswere observed in an additional five experiments.

clamp (-70 mV) during glutamate exposure revealed that glutamateinduced an inward current of -806 108 pA ( n = 13).When the Naf -activated (140 mM) forward Na+-Ca2 + exchangecurrent was reassessed 1-3 min after glutamate exposure (at 110 mVholding potential), it was observed to be more than doubled (2.4-foldincrease; Fig. 4A, Table 3), despite a negative shift in exchanger reversalpotential to +20 ? 2.9 mV (n = 3; Fig. 4C). We considered thepossibility that failure to detect reductionheversa1 of forward Na+-Ca2+ exchange was due to artificially high levels of external Na'.After intense glutamate receptor activation in vivo, one might expect areduction in external Na + concentration d ue to massive influx of N a+(Arens e t al. 1992). However, even when the above experiment wasrepeated with external Na+ reduced t o 100 mM (external Na + duringglutamate exposure was maintained at 140mM), the same basic resultwas obtained (2.8-fold increase; Table 3). Reduction of external Na'to 30 mM, a condition which produced reversal of forward exchangecurrent under normal conditions (see above) still triggered reverseexchange cu rrent shifts (Table 3).Accompanying the increased forward exchange current, glutamateexposure eliminated the external Ca2+-sen sitivecurrent shift associatedwith reverse exchange (Fig. 4B), suggesting that the normal baselinereverse Na+-Ca2+ exchanger activity had been eliminated. Indeed,after glutamate exposure a small opposite membrane current w as notedin several experiments upon removal of external Ca2 + Fig. 4B). Similarresults were obtained when the reverse Na'-Ca2+ exchanger wasprobed with the external solution switch from Li+/O Ca2+ to Lif/C a2+(Table 3). Even w hen tested at 0mV holding potential, further favouringreverse Na f-C a2+ exchange, no Ca2+-sensitive everse exchanger cur-rent was observed after glutamate exposure (-3.6 ? 2.2 PA; n = 5).

DiscussionIdentification of neuronal Na+-Ca2+ exchanger currentsTo our knowledge, the present study is the first to describe the memb ranecurrent associated with Na+ -Ca2 + exchange activity in brain neurons.

As reviewed above, previous reports have described this current insquid axons, retinal cells and cardiac muscles.Our identification of the external Na+-activated inward current asgenerated by forward Na+,,-Ca2+i exchan ge is supported by its depend-ence on Na+ gradient and internal Ca2 +,characteristic voltage depend-ence, and its sensitivity to the specific Na t-C a2+ exchanger inhibitorXIP. As expected, the current persists in the presence of Ca 2+ , Na+ andK + channel blockers, plus a Na+-K+-ATPase inhibitor. In addition,the measured reversal potential of this inward curren t matches the valuecalculated from the 3Na': 1Ca2 +exchanger model. The external Ca2 +-activated membrane current is internal Na+-d epend ent, consistent withreverse Na+-Ca2+ exchange; its XIP-sensitivity and intracellular Ca 2+dependence further support its association with the Naf-Ca2+exchanger.Concomitant forward and reverse Na+-C + exchangeactivitiesThe present study also provides preliminary evidence suggesting thatboth forward and reverse exchanger operations can take place concur-rently in a single neuron, most likely in different parts of the cell. Na+-Ca2+ exchangers have been detected on neuronal cell bodies as well ason dendrites (Jung et al. 1994; Reuter and Porzig, 1995). Whole-cellcurrent can be expected to reflect a summation of currents from thesedifferent cellular locations. Concurrent forward and backward Na+-Ca2 + exchange m ight be caused by differences in the distribution ofintracellular Ca2+ in different submembrane areas (Mu ller and Connor,1992; Niggli and Lipp, 1993). Forward exchange m ight occur in loca-tions where local [Ca2+Ii s high, such as in dendrites, while reverseexchange might occur w here local [C a2+]i is low, such as in the cellbody. We acknowledge that our holding potential of -40 mV mayhave accentuated reverse exchange over what would occur at morehyperpolarized resting conditions, but in any case our data providesupport for the idea that the exchanger can achieve a high degree ofdynamic flexibility, responding differently to different conditionswithin the sam e cell. Further investigation into the po ssibility of concur-rent bidirectional Na+-Ca2 + exchanger operation in both neurons andnon-neuronal c ell types is warranted.Effect of toxic glutamate exposure on Na+-C + exchangecurrentsAfter exposure to toxic concentrations of glutamate (Choi, 1988),the inward current associated with forward Na+-Ca2+ exchangeincreased, and the outward current associated with reverse Na+-Ca2+exchan ge was eliminated or even shifted to a small net inward currentof uncertain aetiology. This was true even when the membraneholding potential was shifted positively to 0 mV (above the levelattained during the glutamate exposure itself), suggesting that theNa f-C a2 + exchanger favours restoration of C a2+ homeostasis follow-ing glutamate-induced toxic C a2+ overload.Favouring Ca 2+ over Na' homeostasis may in part reflect theknown asymmetry of Na+-Ca2+ exchanger activation, as Ca2+ canactivate the exchanger with higher affinity >10-fold) from insidethe membrane than from outside (DiPolo and BeaugB, 1990, 1991).Further increasing the impact of glutam ate-induced [Ca2+ ], elevationupon the exchanger, [Ca 2+]i may rise transiently higher in theimmediate submembrane region than in the cytoplasm as a whole,due to hindered diffusion (Zucker and Stockbridge, 19 83; Nowyckyand Pinter, 1993). In contrast, entering Na' probably diffuses awayfrom the submembrane region more rapidly than C a2+, and thus mayhave a smaller potentiating effect on reverse exchange than enteringCa2 + does on forward exchange.


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Na+-Ca2+ exchanger currents in cortical neurons 1279Na+/Ca2+ Na+/Ca2+

Li+/Ca2+ Li+/Ca2+Li+/Ca*+

Control

Li /O Ca2+Li+/Ca2+

.......................................

300 QA0.3 ecAfter G lutamate

Li+/O Ca2+Li+/Ca2+i+/Ca2+

. . ...................................................

ontrol After Glutamate

C

10050

a

2w -50

100-150-200

Control w

60 -40 -20 0 20 40 60Voltage (mV)FIG.4. Glutamate exposure enhanced the forward exchanger current and blocked the reverse exchanger current. (A) Changing from Li+-containing solution toNaf-containing solution activated the forward exchanger current. After 3 min of exposure to 200 FM glutamate in Na''Ca2+ external solution this forwardexchanger current was markedly increased. BAPTA was omitted from the intracellular solution to permit increases in [Ca2'Ii (as in B). A dotted line shows thezero current baseline (as in B). (B) The external Ca2+-sensitive current linked to the reverse Naf-Ca2' exchange was blocked by glutamate exposure. Unlikein Figure 2B, the current was triggered in 0 Na' extracellular solution, showing its independence of external Na'. The inward current due to block of thereverse Na'-Ca2+ exchange was totally abolished after exposure to 200 yM glutamate for 3 min. Instead, a small outward current appeared. (C) The current-voltage relationship of the Na+-activated forward exchanger current before and after glutamate exposure. Glutamate exposure increased the slope of the current-voltage relationship and shifted the reversal potential to the left.

The glutamate-induced changes in exchanger current observed inthe present experiments are unlikely to be due to direct effects ofglutamate upon the exchanger, as current measurements were per-formed after washout of applied glutamate. However, it is quitepossible that the glutamate exposure produced a lasting alteration inexchanger behaviour mediated by intracellular signalling pathways,for example an overall up-regulation of exchanger function due toprotein phosphorylation (Blaustein et al. 1996).

The idea that glutamate exposure preferentially enhances forwardexchan ge is consistent with findings that the Na'-Ca2' exchan gerin hippocampal neurons contributes to recovery of [Ca2+]i followingK+ depolarization and glutamate receptor activation (Koch andBarish, 1994), and that inhibition of Na+-Ca2' exchan ge followingtoxic glutamate exposure increases the incidence of death of cerebellargranule cells (Andreeva et al. , 1991). However, the alternativepossibility, that toxic glutamate exposure p e r se increases [Na+]i


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1280 N a f - C a 2 + e x c h a n g e r c u rr e nt s in cortical neuronssufficiently to overcome the [Ca2']i increase and shift the exchangertowards in ju ry-promoting reverse opera t ion (Choi , 1988; Kiedrowskiet al., 1994) , may occur under certain condit ions. For example, i nthe i schaem ic bra in in vivo mass ive ce l lu la r Na' in f lux may lowera], t o 38-50 mM (Hansen , 1985; Sies jo , 1992; Xie et al., 1994) ,

a l eve l near tha t which a lone increased reverse exchange in thepresen t exper iments . R eversa l of the Naf-Ca2+ exchanger has beenimplicated in Ca2+-loading myocardial cel l s durin g ischaem ia (Allenet al. 1993) and in anoxic in ju ry to op t ic nerve (S tys et al., 1 9 9 2 ) o rhepa tocy tes (Gasbanin i et al., 1993) . However , upon res to ra t ion ofb lood f low to the i schaemic bra in , ce l lu la r Na+ grad ien ts recoverqu ick ly (Hansen an d Nedergaard , 1988; E le f f et al., 1991) , pe rhapssh i ft ing the Na+-Ca2+ exchanger back to cytoprotective forwardoperat ion.

AcknowledgementsThe authors express their gratitude to Dr R. A. Colvin for kindly providingthe N a+-C a2+ exchanger inhibitory peptide XIP. The study was supported inpart by Natio nal Institutes of Health (USA ) grant 50788 (to D. W. C).

AbbreviationsBAF'TA 1,2-bis-(O-aminophenoxy)ethane-N,N,N ,N -tetraaceticcidMEM Minimal Essential MediumTEA tetraethylammonium

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