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J Physiol. Oct 15, 1998; 512(Pt 2): 449–457.
PMCID: PMC2231210

Sodium pump evokes high density pump currents in rat midbrain dopamine neurons

Abstract

  1. Patch pipettes contained various concentrations of Na+ ([Na+]pip) in order to record strophanthidin-sensitive currents under voltage clamp in dopamine neurons in slices of rat substantia nigra and ventral tegmental area.
  2. When [Na+]pip was 40 mm and the external K+ concentration ([K+]o) was 2.5 mm, strophanthidin (10 μm) evoked 461 ± 121 pA of inward current. This effect was concentration dependent, with an EC50 of 7.1 ± 2.6 μm. At potentials of −60 to −120 mV, strophanthidin-induced currents were not associated with significant changes in chord conductance.
  3. Strophanthidin (10 μm) evoked 234 ± 43 pA of inward current when [Na+]pip was 0.6 mm, and 513 ± 77 pA when [Na+]pip was 80 mm. Despite higher pump currents with greater [Na+]pip, the strophanthidin EC50 was not significantly different for any of six different [Na+]pip.
  4. Sodium pump currents were half-maximal when the [Na+]pip was about 1.3 mm. Maximum pump current was estimated at 830 pA (29 μA cm−2) at concentrations of intracellular Na+ that were assumed to be saturating (50–100 mm).
  5. Strophanthidin currents were smaller in a reduced [K+]o (EC50= 0.2 mm).
  6. These data show that intracellular Na+ loading evokes relatively large pump currents. Our results are consistent with the physiological role of the sodium pump in burst firing in midbrain dopamine neurons

The sodium pump, also known as Na+-K+-ATPase, is an ATP-dependent enzyme that extrudes intracellular Na+ in exchange for extracellular K+. Of the three subunits that comprise the sodium pump, it is the α-subunit that possesses ATP catalytic activity and contains the binding site for cardiac glycosides (Fambrough et al. 1994). Three isoforms of the α-subunit have been cloned thus far (Shull et al. 1986), and each differs somewhat in its affinity for Na+ and K+ (Jewell & Lingrel, 1991). The α1-subunit in rat is prominently expressed in heart (Emanuel et al. 1987) and is resistant to inhibition by cardiac glycosides (Price & Lingrel, 1988). In contrast, α2- and α3-subunits, which are expressed with α1 in brain (Urayama et al. 1989), are more sensitive to cardiac glycosides (Berrebi-Bertrand et al. 1990). Because the sodium pump extrudes three Na+ in exchange for two K+, its activity causes membrane hyperpolarization which is essential for maintaining the resting membrane potential in excitable cells. Because it also maintains the transmembrane gradient for Na+, it is also essential for many metabolic processes that are needed to sustain life (Lees, 1991).

Our previous work in midbrain dopamine neurons showed that the electrogenic sodium pump also causes the rhythmic hyperpolarizations that underlie the burst firing of action potentials in the presence of N-methyl-D-aspartate (NMDA) receptor stimulation (Johnson et al. 1992). This action of the sodium pump may be important because burst firing has been shown to release more dopamine at nerve terminals than does the same number of evenly spaced spikes (Gonon & Buda, 1985). Furthermore, the increased dopamine release that occurs during burst firing has been linked to behavioural phenomena such as motivation, attention and reward seeking (Schultz & Romo, 1990). Utilization of sodium pump currents during burst firing is relatively unusual, having been described thus far only in dopamine neurons, neonatal spinal cord motoneurons (Ballerini et al. 1997) and the L3 Aplysia neuron (Willis et al. (1974). Therefore, one may wonder if pump current in dopamine neurons is unusual in some way that enables it to participate in burst firing.

A previous attempt to quantify pump current in dopamine neurons was complicated by the fact that Na+ loading evokes a large sulphonylurea-sensitive K+ conductance (Seutin et al. 1996). In the present study, we added K+ channel blockers to patch pipette solutions in order to study strophanthidin-sensitive pump currents in the absence of significant amounts of Na+-activated K+ currents. Our data show that Na+ loading evokes a relatively high density of sodium pump current in midbrain dopamine neurons compared with that recorded in other types of cell.

METHODS

Tissue preparation

Sprague-Dawley rats (150–200 g; Bantin & Kingman, Seattle, WA, USA) were anaesthetized with halothane and killed by severing major thoracic vessels. The brain was rapidly removed and horizontal slices (300 μm) containing the ventral midbrain were prepared as described previously (Johnson & North, 1992). Briefly, slices were cut in a vibratome in cold physiological saline and placed on a supporting net in a recording chamber (volume 500 μl). Each slice was immersed and perfused with a flowing (2 ml min−1) saline solution that contained (mm): NaCl, 126; KCl, 2.5; CaCl2, 2.4; MgCl2, 1.2; NaH2PO4, 1.2; NaHCO3, 19; glucose, 11. This solution was saturated with 95 % O2 and 5 % CO2, and had a pH of 7.35 at 35–37°C. Using a dissection microscope for visual guidance, the ventral tegmental area (VTA) was identified as the region lateral to the fasciculus retroflexus and medial to the medial terminal nucleus of the accessory optic tract. The substantia nigra compacta (SNC) was identified as a crescent-shaped semilucent region rostral and caudal to the medial terminal nucleus of the accessory optic tract.

Electrophysiological recordings

Unless stated otherwise, whole-cell tight-seal recordings were made with pipettes that contained (mm): sodium gluconate, 40; caesium gluconate, 67; tetraethylammonium (TEA), 20; MgCl2, 2; EGTA, 11; Hepes, 10; Mg3/2ATP, 1.5; Na3GTP, 0.2. In some experiments, equimolar concentrations of caesium gluconate were replaced with sodium gluconate and vice versa in order to set final intrapipette concentrations of Na+ to various values. The pH of the pipette solution was adjusted to 7.3–7.4 with CsOH. Osmolarities of pipette solutions were measured with a Wescor 500 vapour pressure osmometer (Logan, UT, USA) and adjusted to 290 mosmol kg−1. Membrane currents were recorded under voltage clamp (holding potential −60 mV) and amplified with an Axopatch-1D amplifier (Axon Instruments). Data were acquired and analysed using pCLAMP software, a Digidata analog/digital interface (Axon Instruments) and an IBM-compatible personal computer. Holding currents were recorded continuously using a MacLab analog/digital interface, Chart software (AD Instruments, Castle Hill, Australia), and a Macintosh IIVX computer. Series resistance was electronically compensated by 50–80 % to 10–30 MΩ; membrane potentials have been corrected for the liquid junction potential (10 mV).

Identification of cell type

All recordings were made from the ‘principal’ or most common type of neuron in the VTA and SNC. These neurons were identified by their spontaneous, broad (2 ms) action potentials (recorded under current clamp), the presence of outward current (50–150 pA at −60 mV) evoked by dopamine (30–100 μm) added to the perfusate, and by the presence of hyperpolarization-activated time-dependent inward current (> 200 pA) known as Ih (Johnson & North, 1992). Neurons that display these electrophysiological and pharmacological characteristics have been shown to contain tyrosine hydroxylase and are therefore presumed to be dopamine-containing neurons (Yung et al. 1991).

Current-voltage studies

Voltage-dependent currents were studied by recording currents during hyperpolarizing voltage steps (0–60 mV, 400 ms duration) from a holding potential of −60 mV. Currents were measured immediately after capacitative transients to minimize the influence of Ih. Current-voltage plots were nearly linear between −60 and −120 mV; consequently, chord conductance was calculated as the slope of a straight line in current-voltage plots. In some experiments, currents recorded during the experimental treatment were subtracted from those currents recorded before the treatment. Therefore, these subtracted currents represent ‘net’ currents that were evoked by an experimental condition or treatment.

Drugs

TEA, dopamine hydrochloride, tetrodotoxin (TTX), ouabain and strophanthidin were obtain from Sigma. A stock solution of dopamine was made daily and kept on ice to retard oxidation. Glibenclamide (Research Biochemicals, Inc.) was dissolved in dimethyl sulphoxide, whereas strophanthidin and ouabain were dissolved in ethanol. All drugs were diluted 1: 1000 in perfusate before use. Control solutions of 1: 1000 dimethyl sulphoxide or ethanol had no effect on membrane current. Approximately 30 s were required for the drug solution to enter the recording chamber; this delay was due to passage of the perfusate through a heat exchanger.

Data analysis

Numerical data in the text and error bars in figures are expressed as means ±s.e.m. In current-voltage plots, chord conductance (slope of the current-voltage curve) was determined by linear regression for each cell, and the mean value was calculated by averaging the results from all cells. Unless stated otherwise, differences in data were evaluated for significance using analysis of variance (ANOVA) (SigmaStat, Jandel Scientific, San Rafael, CA, USA). Concentration-response curves were constructed using the KaleidaGraph curve-fitting program (Synergy Software, Reading, PA, USA) on a Macintosh computer; data were fitted to the Hill-Langmuir equation y =ax/(x+b), where y is the magnitude of effect, a is the maximum effect (Emax), x is the concentration of drug or ion, and b is the EC50. An EC50 and Emax were calculated for each cell, and the mean (and s.e.m.) was derived by averaging all values.

RESULTS

K+ conductance increased by Na+ loading

In a previous study, we reported that intracellular Na+ loading increased a glibenclamide-sensitive K+ conductance in midbrain dopamine neurons (Seutin et al. 1996). When patch pipettes contained 40 mm Na+, glibenclamide (1 μm) reduced chord conductance by 6.34 ± 0.15 nS (n = 7). However, this same concentration of glibenclamide reduced chord conductance by only 0.94 ± 0.22 nS (n = 8) when pipettes contained the K+ channel blockers TEA (20 mm) and Cs+ (67 mm) during loading with 40 mm Na+. This suggests that about 85 % of the K+ conductance evoked by Na+ loading is blocked when pipette solutions contain TEA and Cs+. In order to study sodium pump currents in relative isolation, all subsequent recordings were made with pipettes that contained TEA and Cs+.

Effects of Na+ loading on holding current

Figure 1 shows that holding current was a function of the concentration of Na+ in patch pipettes ([Na+]pip). When pipettes contained 1.6 mm Na+, an inward current developed and reached a steady-state value (−148 ± 24 pA) 10 min after rupturing the membrane patch (Fig. 1A). In contrast, when pipettes contained 80 mm Na+, an outward current developed over time and reached a steady-state value of +105 ± 43 pA 10 min after membrane rupture (n = 5). When patch pipettes contained an intermediate concentration of Na+ (40 mm), a transient outward current lasting 2–3 min was followed by an inward current that measured −20 ± 12 pA when it reached steady state 10 min after beginning recording (n = 40). A summary of these data is presented in Fig. 1B. As seen in Fig. 1C, holding currents recorded with 1.6, 40 and 80 mm Na+ in pipettes were significantly different when measured 10 min after beginning recordings (P < 0.001, one-way ANOVA). Transient shifts in holding current that occur soon after beginning whole-cell recording (Fig. 1B) are most likely to be due to the net influences of progressive block of K+ channels by intracellular TEA and Cs+, and to the generation of outward currents by Na+ loading. Because about 10 min was required before holding currents reached steady state, all subsequent data were acquired at least 15 min after beginning recording.

Figure 1
Membrane currents vary over time according to [Na+]pip

Effects of strophanthidin on membrane properties

In the present studies, sodium pump currents were measured as those currents blocked by the cardiac glycoside strophanthidin. Using pipettes containing 40 mm Na+, 10 μm strophanthidin evoked an average net inward current of 411 ± 79 pA (n = 11) at −60 mV (Fig. 2A). Effects of this concentration of strophanthidin often did not resolve completely even after prolonged washout. Consequently, 10 μm strophanthidin was applied only once to each brain slice, and higher concentrations were not perfused. Currents evoked by 1–3 μm strophanthidin were seen within 2 min, they reached a peak about 5 min after application, and they were gone 30 min after washout. Ouabain at 1 and 3 μm produced currents of similar magnitude, but times needed for washout of drug effects were much longer than for strophanthidin. Consequently, all further studies utilized strophanthidin.

Figure 2
Strophanthidin evokes inward currents

Figure 2B and C shows that currents induced by strophanthidin (10 μm) were not associated with significant changes in chord conductance. (Strophanthidin increased conductance by 0.44 ± 0.40 nS, n = 8.) Pump currents in other tissues are also associated with little change in conductance (Gadsby & Nakao, 1989; Hermans et al. 1994). Figure 2B also shows very little hyperpolarization-activated inward current (Ih) compared with that which is normally recorded in dopamine neurons. Although the initial amplitude of Ih ranged from 200 to 600 pA (when evoked by hyperpolarization to −120 mV), Ih became progressively smaller over time when recorded using pipettes that contained elevated concentrations of Na+. This reduction in Ih might have been due to the reduced transmembrane Na+ concentration gradient that is expected to occur following Na+ loading.

Lack of dependence of currents on K+ channel blockers

Because experiments with glibenclamide suggested that Na+-activated K+ currents were not completely blocked by intracellular TEA and Cs+, we wanted to ascertain whether or not our ability to measure pump current was affected by this residual K+ conductance. As seen in Fig. 3, strophanthidin (10 μm) produced a net inward current of 514 ± 147 pA (n = 5) in the presence of BaCl2 (1 mm), CsCl (3 mm), TEA (10 mm) and glibenclamide (1 μm). Because these K+ channel blockers did not significantly alter strophanthidin-induced currents compared with the control value of 411 ± 79 pA (n = 11) (P = 0.6, one-way ANOVA), these data suggest that the residual increase in K+ conductance evoked by Na+ loading does not interfere significantly with our ability to measure pump currents.

Figure 3
Lack of effect of K+ blockers, low Ca2+ or TTX on strophanthidin currents

Effect of TTX and extracellular Ca2+

Several studies have shown that cardiac glycosides cause Ca2+-dependent and TTX-sensitive release of dopamine in the brain in vivo (Westerink et al. 1989; Fairbrother et al. 1990). Therefore, we were interested to see if inward currents evoked by strophanthidin might be mediated in part by the release of neurotransmitters in the brain slice. As seen in Fig. 3, strophanthidin (10 μm) evoked an inward current of 331 ± 52 pA (at −60 mV, n = 7) in the presence of perfusate containing no added Ca2+ (equimolar substitution of Mg2+ for Ca2+); this was not significantly different from currents recorded in control perfusate (P = 0.6, one-way ANOVA). Furthermore, TTX (0.6 μm) had no effect on the amplitude of strophanthidin-evoked inward current (352 ± 61 pA, n = 5) (Fig. 3). The lack of effect of TTX or perfusate containing no added Ca2+ suggests that currents produced by strophanthidin are not mediated indirectly by the release of excitatory neurotransmitters.

Na+ dependence of strophanthidin-induced currents

Although Fig. 4 shows that effects of strophanthidin are concentration dependent (1–10 μm), this figure also shows that the magnitude of strophanthidin-induced currents is larger when the concentration of Na+ in the pipette is higher. Strophanthidin-induced currents recorded with a pipette containing 0.6 mm Na+ (Fig. 4A) were much smaller than those recorded when the pipette contained 80 mm Na+ (Fig. 4B). Note also the relatively large outward holding current when [Na+]pip was 80 mm (Fig. 4B compared with A). The greater effect of strophanthidin when [Na+]pip was 80 mm is consistent with the assumption that the increase in outward holding current with 80 mm Na+ is mostly due to an increase in sodium pump current.

Figure 4
Concentration-dependent effects of strophanthidin recorded with different concentrations of Na+ in pipettes ([Na+]pip)

Figure 4C summarizes the concentration-dependent effects of strophanthidin recorded with three different values for [Na+]pip. (A complete set of data for six different [Na+]pip is shown in Table 1.) When pipettes contained 80 mm Na+, 1, 3 and 10 μm strophanthidin evoked net inward currents of 129 ± 30, 336 ± 47 and 513 ± 77 pA, respectively (n = 7 each). These currents were significantly greater than those recorded with pipettes that contained 0.6 mm Na+ (36 ± 7, 116 ± 18 and 235 ± 43 pA, respectively, n = 9, P < 0.05, two-way ANOVA with repeated measures). By fitting these data to the Hill-Langmuir equation, we estimated that the strophanthidin EC50 was 8.4 ± 3.9 μm when [Na+]pip was 80 mm (n = 7) and 8.9 ± 2.0 μm when [Na+]pip was 0.6 mm (n = 9). As seen in Table 1, none of the strophanthidin EC50 values that were calculated for each of six different [Na+]pip were significantly different (P = 0.9, one-way ANOVA). These data suggest that the potency of strophanthidin (as opposed to its efficacy) does not depend upon [Na+]pip. The pooled average EC50 for strophanthidin was 8.2 ± 1.2 μm (n = 41), and the concentration of strophanthidin that would inhibit 90 % of pump current (EC90) was estimated at about 50 μm.

Table 1
Strophanthidin-evoked currents with different [Na+]pip

Data in Fig. 5A, which are also presented in Table 1, show the effects of six different [Na+]pip on magnitudes of currents evoked by 1, 3 and 10 μm strophanthidin. This figure shows that currents evoked by strophanthidin progressively increase as a function of increasing values of [Na+]pip; currents appear to approach maximum values when the [Na+]pip is 50–100 mm. By fitting these data to the Hill-Langmuir equation, we estimated that the concentrations of Na+ needed for half-maximal activation of strophanthidin-sensitive currents (EC50) were 2.4, 0.8 and 0.6 mm Na+ for strophanthidin concentrations of 1, 3 and 10 μm, respectively. The finding that a substantial amount of strophanthidin-sensitive current can be demonstrated when [Na+]pip is as low as 0.6 mm suggests that the sodium pump has a relatively low threshold for activation in dopamine neurons.

Figure 5
Strophanthidin currents are dependent upon [Na+]pip

In order to estimate the maximum current capable of being generated by the sodium pump, we used the Hill equation to calculate the theoretical maximum current (Emax) that could be evoked by strophanthidin at each [Na+]pip (see Table 1). The relationship between Emax and [Na+]pip is shown graphically in Fig. 5B. These data suggest that the sodium pump can generate a maximum current of 830 pA, and the pump is half-maximally activated at an internal Na+ concentration of 1.3 mm. Given an estimate of 4 or 5 mm for a basal intracellular concentration of Na+ (Partridge & Thomas, 1976; Alvarez-Leefmans et al. 1994), these data suggest that the basal level of sodium pump activity is relatively high in dopamine neurons.

K+ dependence of strophanthidin-induced currents

Because the sodium pump requires extracellular K+ in order to generate currents, we next sought to determine the dependence of strophanthidin-induced currents on the extracellular concentration of K+ ([K+]o). When recording with [Na+]pip= 40 mm and perfusate that contained a relatively high concentration of K+ (7.5 mm), strophanthidin (10 μm) evoked a net inward current of 358 ± 43 pA (n = 6). This was not significantly different from the magnitude of current evoked by strophanthidin recorded in control (2.5 mm K+) perfusate (411 ± 79 pA, n = 11). However, strophanthidin-induced currents were significantly smaller (231 ± 7 pA, n = 7, P = 0.01, one-way ANOVA) when recorded in perfusate that contained a low (0.2 mm) concentration of K+ (Fig. 6). Assuming a maximum current value of 411 pA (the average current evoked by 10 μm strophanthidin when [Na+]pip= 40 mm and [K+]o= 2.5 mm), we estimate that strophanthidin-induced currents are half-maximal when [K+]o is about 0.2 mm. Thus, it appears that a normal extracellular concentration of K+ is sufficient for maximum activation of the sodium pump in dopamine neurons.

Figure 6
Effects of 0.2, 2.5 and 7.5 mm extracellular K+ on currents evoked by 10 μm strophanthidin

DISCUSSION

In the present study, our findings that sodium pump currents are half-maximal when internal Na+ concentration is 1.3 mm and external K+ concentration is 0.2 mm suggest there is a high basal level of sodium pump activity in ventral midbrain dopamine neurons. Consequently, the sodium pump is likely to influence the membrane potential and/or the pattern of action potential generation in these neurons. Despite the high level of basal activity, our data also show that the sodium pump is capable of significantly increasing its activity in response to increasing intracellular concentrations of Na+. This ability to generate a relatively high density of pump current is consistent with the postulated role of the sodium pump in regulating the firing pattern of dopamine neurons (Johnson et al. 1992).

Compared with other tissues, the sodium pump in dopamine neurons appears to be relatively sensitive to inhibition by cardiac glycosides. This may at first be surprising when one considers that rat cardiac muscle has a well-known resistance to cardiac glycosides (Gold et al. 1947). However, it is now clear that Na+-K+-ATPase can be composed of at least three different types of α-subunit (Shull et al. 1986), and the subunit expressed in rat heart (α1) typically binds cardiac glycosides with low affinity (Kd≥ 100 μm) (Jewell & Lingrel, 1991). On the other hand, neurons in rat brain also express the α2- and α3-subunits (Brines & Robbins, 1993), which have much higher affinities for cardiac glycosides (Berrebi-Bertrand et al. 1990). Moreover, an in situ hybridization study showed that the ventral midbrain expresses relatively high levels of the message for the α3-subunit (Hieber et al. 1991). Furthermore, our finding that the strophanthidin EC50 is relatively low (8.2 μm) is consistent with the hypothesis that Na+-K+-ATPase contains the α3-subunit in dopamine neurons. Because the locus coeruleus has been shown to express α1-subunits (Hieber et al. 1991), it would be interesting to establish whether or not strophanthidin is less potent for inhibiting pump current in these cells compared with dopamine neurons.

Although strophanthidin is relatively potent in dopamine neurons, the most remarkable finding is the high efficacy of this drug. When recording with pipettes that contained 80 mm Na+, we showed that 10 μm strophanthidin evoked 513 pA of inward current. Given a dopamine neuron with a radius of 15 μm2 (Grace & Bunney, 1983), this suggests that the sodium pump generates current at a density of 18 μA cm−2. This is very close to the estimate of 19 μA cm−2 that we calculated previously for dopamine neurons using strophanthidin to block pump currents in the presence of NMDA (Johnson et al. 1992). Furthermore, if we take our estimate of 830 pA as the maximum amount of current capable of being generated by the pump, this suggests a maximum pump current density of 29 μA cm−2. This compares with frog skin, toad bladder and the Sachs organ of the electric eel, which generate pump current densities of 20, 44 and 86 μA cm−2, respectively (Bonting & Caravaggio, 1963). However, it is clear that other excitable cells are less able to generate sodium pump currents. For example, Senatorov et al. (1997) reported that 80 μm strophanthidin evoked about 35 pA of inward current in rat auditory thalamic neurons when recording with patch pipettes that contained 20 mm Na+. Moreover, Nakao & Gadsby (1989) showed that 2 mm strophanthidin inhibited 150 pA of pump current in guinea-pig cardiac myocytes when recording with pipettes that contained 50 mm Na+; these and other data suggest that cardiac myocytes generate pump currents at a maximum density of 1.1 μA cm−2 (De Weer et al. 1988; Glitsch et al. 1989). Therefore, it appears that dopamine neurons can generate pump current at higher density compared with cardiac myocytes and auditory thalamic neurons. It remains to be seen whether or not this large capacity to generate sodium pump current is unique to midbrain dopamine neurons.

Our finding that strophanthidin-induced currents are half-maximal when [Na+]pip is 1.3 mm suggests that the sodium pump has a high rate of basal activity in dopamine neurons. This value for the Na+ EC50 is similar to estimates of 1–3 mm Na+ for rat Na+-K+-ATPase expressed in cultured cells (Jewell & Lingrel, 1991; Jewell et al. 1992), but it is lower that the estimate of 5–19 mm in guinea-pig and rabbit cardiac myocytes (Nakao & Gadsby, 1989; Shattock & Matsuura, 1993) and in rat renal proximal tubule cells (Ibarra et al. 1993). Because the affinity for Na+ differs according to the subunit composition of Na+-K+-ATPase (Jewell & Lingrel, 1991; Therien et al. 1996), these differences could be due to tissue differences in the expression of pump subunits. Nevertheless, if one assumes that the resting concentration of Na+ in neurons is about 4–5 mm (Partridge & Thomas, 1976; Alvarez-Leefmans et al. 1994), then one would expect that the sodium pump is usually more than half-maximally activated in dopamine neurons. Despite its low threshold for activation, our data suggest that sodium pump activity does not saturate until intracellular concentrations of Na+ are 50–100 mm. This suggests that the sodium pump in dopamine neurons responds to a wide range of intracellular concentrations of Na+.

With a ‘normal’ extracellular concentration of 2.5 mm K+, we found that the sodium pump was capable of generating maximal current in dopamine neurons. Moreover, a 50 % reduction in activity did not occur until [K+]o was reduced to 0.2 mm. This EC50 for K+ is relatively low compared with 2.8 mm reported for rabbit vagus nerve (Rang & Ritchie, 1968), 1–2.5 mm in Na+-K+-ATPase expressed in HeLa cells (Therien et al. 1996) and 1–2 mm in cardiac myocytes (Nakao & Gadsby, 1989; Shattock & Matsuura, 1993). The low EC50 for K+ implies that increases in extracellular K+ that might occur during cerebral ischaemia or trauma would not potentiate sodium pump activity in dopamine neurons. It is also interesting that raised extracellular K+ (to 7.5 mm) did not reduce the potency of strophanthidin in our studies. This suggests that mildly elevated concentrations of K+ do not interfere with the binding of cardiac glycosides to Na+-K+-ATPase in dopamine neurons (Akera & Brody, 1971).

The ability of dopamine neurons to generate large amounts of sodium pump current is consistent with the role of the sodium pump in burst firing (Johnson et al. 1992). In the in vitro model of burst firing, NMDA receptor/ion channels permit an influx of Na+ which is then removed from the cell by the sodium pump. Because pump activity is electrogenic, this causes the repetitive membrane hyperpolarizations that underlie burst firing of action potentials. It seems reasonable to assume that the ability to generate pump currents at high density may be a necessary condition that enables burst firing by this mechanism. It would be interesting to know if other cells that have been shown to utilize the sodium pump during burst firing can also generate pump currents at such high density (Willis et al. 1974; Ballerini et al. 1997).

A high capacity to generate pump current also suggests that dopamine neurons are well prepared to maintain intracellular homeostasis even when Na+ influx is increased during glutamate-mediated synaptic transmission. Because accumulation of intracellular Na+ favours a rise in intracellular Ca2+ (Baker et al. 1969), a failure to maintain Na+ homeostasis can initiate processes that lead to cell death (Stys et al. 1992). Consequently, a large capacity to pump Na+ out of a cell should protect against glutamate-induced toxicity. In Parkinson's disease, however, dopamine neurons have been shown to have an impaired ability to synthesize ATP, and this defect may predispose these neurons to degenerate over time (Schapira et al. 1990; Hattori et al. 1991). A failure of dopamine neurons to synthesize enough ATP to maintain sodium pump activity would be expected to facilitate greatly the toxic effects of glutamate, even at concentrations that are normally seen during synaptic transmission (Lees, 1991).

In conclusion, our data suggest that ventral midbrain dopamine neurons are capable of generating relatively dense sodium pump currents in response to intracellular Na+ loading. The ability of dopamine neurons to generate pump currents at high density is consistent with the proposed role of the sodium pump in NMDA-mediated burst firing. Furthermore, sodium pump activity would be expected to confer protection against glutamate-induced toxicity. It remains to be seen whether other central neurons can generate pump currents at a density that is comparable to that recorded in ventral midbrain dopamine neurons.

Acknowledgments

This work was supported by a grant from the National Institute of Neurological Disease and Stroke (NS31889). We would like to thank Dr Carmen C. Canavier and Dr Martin J. Kelly for their helpful comments during the preparation of this manuscript.

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