N-ethylmaleimide irreversibly inhibits the binding of [3H]threo-(±)-methylphenidate to the stimulant recognition site

NeuropharmacologyVol. 29, No. 10, pp. 901-908, 1990 Printed in Great Britain. All rights reserved

0028.3908/90 $3.00 + 0.00 Copyright 8 1990 Pergamon Press plc

N-ETHYLMALEIMIDE IRREVERSIBLY INHIBITS THE BINDING OF [3H]THREO-( f )-METHYLPHENIDATE TO THE STIMULANT RECOGNITION SITE M. M. SCHWERI Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia 31207, U.S.A. (Accepted 23 March 1990)

Summary-N-Ethylmaleimide, a nonspecific protein modifier which reacts selectively with the sulfhydryl group of cysteinyl residues under controlled conditions, irreversibly inhibited the binding of [3H]threo-( k)methylphenidate to a subset of stimulant binding sites in striatal tissue membranes from the rat in vitro. The inhibition was marked by a decrease in the II,,,,, of binding of the radiolabelled stimulant drug, while the K, remained unchanged. Pretreatment with excess unlabelled methylphenidate afforded complete protection from inactivation of the binding site by N-ethylmaleimide. Uptake of [3H]dopamine into striatal synaptosomes was likewise reduced after treatment with N-ethylmaleimide; pretreatment with large concentrations of methylphenidate provided partial protection from inactivation of transport. These findings suggest that the stimulant recognition site on the dopamine transport complex contains one or more cysteinyl residues. Key words-N-ethylmaleimide,

[‘Hlmethylphenidate

the dopamine transporter plays a vita] role in clearing dopamine from the synapse following its neuronal release, little is known concerning the molecular composition of this important constituent of the cytoplasmic membrane. The aim of the present study was to gain information about the structure of the stimulant recognition site, a subunit of the dopamine transport complex, by investigating its susceptibility to attack by the nonspecific protein modifier, N-ethylmaleimide. While this reagent reacts with both amino and sulfhydryl groups in a basic environment, it is relatively specific for thiol groups when the pH is maintained below 7.0 (Means and Feeney, 1971). N-Ethylmaleimide was utilized in these studies to ascertain the potential contribution of nucleophilic amino acids in general (and cysteine in particular), to the structure and function of the stimulant binding site. The region of the dopamine transporter which recognizes stimulant drugs has been characterized using a variety of radioligands; e.g. [3H]cocaine (Kennedy and Hanbauer, 1983) [3H]nomifensine (Dubocovitch and Zahniser, 1985), [3H]mazindol (Javitch, Blaustein and Snyder, 1983), [3H]GBR(I-[2-{diphenylmethoxy}ethyl]-4-(3-phenyl12935 propyl)-piperazine) (Berger, Janowsky, Vocci, Skolnick, Schweri and Paul, 1985) and [3H]methylphenidate (Schweri, Skolnick, Rafferty, Rice, Janowsky and Paul, 1985). While it is not yet clear whether these probes bind to the same or overlapping domains, they share sufficient features in common to suggest that they all identify sites associated with the Although

binding, stimulants, dopamine, striatum, rat.

dopamine transport complex. For instance, strong positive correlations have been noted between the potency of stimulant drugs in inhibiting the binding of these radioligands to striatal tissue (the region of the brain containing the greatest density of binding) and their potency in blocking the uptake of dopamine into nerve endings (Kennedy and Hanbauer, 1983; Javitch et al., 1983; Dubocovich and Zahniser, 1985; Schweri et al., 1985). Moreover, the binding of these radioligands decreases following surgical, chemical or pathological destruction of dopamine-containing nerve tracts (Dubocovich and Zahniser, 1985; Janowsky, Schweri, Berger, Long, Skolnick and Paul, 1985; Pimoule, Schoemaker, Javoy-Agid, Scatton, Agid and Langer, 1983). [3H]Methylphenidate was selected as a probe for the stimulant recognition site in this study primarily on the basis of prior experience with this radioligand (e.g. Schweri et al., 1985; Janowsky et al., 1985; Schweri, Jacobson, Lessor and Rice, 1987). The results described show that N-ethylmaleimide rapidly and irreversibly inactivated a subpopulation of [‘Hlmethylphenidate binding sites by reacting with one or more nucleophilic amino acid residues, situated directly in the vicinity of the stimulant recognition site. The compound was also found to interfere with the synaptosomal transport of [‘Hldopamine. Although further experiments are necessary, the present results suggest that cysteinyl groups at the methylphenidate binding site are the target of attack by this nonspecific protein modifying reagent. 901

M.M.

902 METHODS

Following decapitation, striatal tissue was obtained from the brains of male Sprague-Dawley rats (Lab Pets, Jonesboro, Georgia; Harlan Sprague-Dawley, Indianapolis, Indiana), weighing 150-250 g. Binding of [-‘H]methylphenidate Specific binding of [‘Hlmethylphenidate was measured as described previously (Schweri et al., 1987). Briefly, a P, fraction was prepared by differential centrifugation and resuspended in 50 vol (original wet weight) of assay buffer (50 mM Tris-Cl, 100 mM NaCl, pH 7.9 at 0°C). Samples containing 400~1 of the tissue suspension, 500 ~1 assay buffer, 50 p I water or 2 mM threo-(f)-methylphenidate (to define nonspecific binding) and 50 ~1 [‘Hlmethylphenidate [( &)-threo-[methyl-‘Hlmethylphenidate; DuPont/ New England Nuclear (Boston, Massachusetts); specific activity, 65 Ci/mmol] were incubated in Beckman Omni-Vials for 30 min in an ice bath, then centrifuged for 15 min at 29,OOOg. The resulting pellet was washed with assay buffer, shaken for 1 hr with scintillation fluid and counted in a liquid scintillation counter. In most experiments, the tissue was incubated in the presence of 8-l I nM [3H]methylphenidate. Scatchard analyses were conducted in the presence of 10-600 nM [3H]methylphenidate, after dilution of the radioligand with unlabelled methylphenidate to yield specific activities on the order of 17.45 Ci/mmol. The time course for the reaction with N-ethylmaleimide was determined by exposing a P, suspension to 0.1 mM N-ethylmaleimide for 5-60 min at O’C, terminating the reaction by centrifugation at 20,OOOg for 15 min, resuspending the pellets in assay buffer and comparing the resulting binding of [~H]methylphenidate in vehicle-treated tissue with reagent-treated tissue using the standard radioreceptor assay. The percentage inhibition was calculated using specific binding values corrected for protein content. Dose-response curves for the inhibition of the binding of [3H]methylphenidate by N-ethylmaleimide at pH 7.9 were obtained by reacting a P, suspension with 0.025 to 10mM N-ethylmaleimide in assay buffer at 0-C for 20min. The samples were then centrifuged at 20,OOOg for 15 min and the resulting pellets were resuspended in assay buffer. Binding in these samples was compared to similarly treated vehicle controls, after normalization based on protein content. Inhibition at pH 6.1 was determined in a similar manner, except the P2 fraction was exposed to the N-ethylmaleimide in 50mM sodium phosphate buffer (pH 6.1; adjusted to contain 100 mM Na+ with NaCl) for 60 min before the reaction was terminated and the resulting pellet was washed a second time in phosphate buffer before being resuspended in assay buffer.

SCHWERI

Protection of the stimulant recognition site from covalent modification by .~r-ethylmaleimide was determined as follows. A Pz fraction. suspended in assay buffer, was pre-incubated for 20 min at O’C with 5 ,uM methylphenidate. N-Ethylmaleimide (0.5 mM) was added and the sample was maintained at 0°C for 15 min longer. The sample was then quickly diluted approximately two-fold with assay buffer and immediateIy centrifuged at X.OOOg for 15 min. The pellet was resuspended in fresh assay buffer using a Tekmar Tissuemizer and centrifuged again. This procedure was repeated for a total of three washes. Binding of [3H]nlethylphenid~~te was determined in the final suspension, according to the standard radioreceptor assay described above and compared to samples treated only with methylphenidate, N-ethylmaleimide or vehicle, Eflbct of N-ethylmaleimide [‘Hldopamine

on

ucc.umzdation

of

A Pz fraction, prepared from the striatal tissue of five rats by differential centrifugation, was suspended in 3.0 ml of 0.32 M sucrose and 24 ml of a modified Krebs-phosphate buffer ( 120 mM NaCl. 4.9 mM KCI, 1.2 MgSO,, 11 mM glucose. 0.16 mM Na, EDTA (ethylene diamine tetraacetic acid), 1.1 mM ascorbic acid, 0.01 mM pargyline and 15.5 mM Na,HPO,, equilibrated with 95% Oz-5% CO, and adjusted to pH 7.4 with NaOH). The synaptosomal preparation was divided into four equal aliquots, which were pretreated with 5 FM methylphenidate or vehicle (0.32 M sucrose) for 20 min at O”C, before exposure to 0.2 mM N-ethylmaleimide or vehicle for 15 min longer, resulting in the following samples (listed as first treatmentisecond treatment): (1) control (vehicle/vehicle); (2) methylphenidate; vehicle; (3) vehicle/‘~-ethylmaleimide and (4) methylphenidate~~-ethylmaleimide. After the reaction with ~-ethylmaleimide was completed, a portion of the sample was set aside for measurement of uptake of [‘Hldopamine (see below) and the remainder was centrifuged at 20,OOOg for 1Omin. The resulting pellet was resuspended in 5 ml Krebs’--phosphate buffer and centrifuged a second time as described in order to remove the free drug. Accumulation of [3H]dopamine was determined, as described below, in the tissue preparation obtained by resuspending the resulting pellet in a mixture of one part 0.32 M sucrose and four parts Krebs’-phosphate buffer. Uptake of [3H]dopamine into synaptosomes was determined using a modification of the method of Harris and Baldessarini (1973j. Briefly, 250 brl of the samples described in the preceding paragraph were preincubated with 1100 ~tl Krebs’.-phosphate buffer and 100~1 water for 10 min at 37 C, prior to the addition of 50 ~1 [‘Hldopamine (3,4-[ring 2,5,6-3H]-dihydroxyphenethylamine HCI: Dupontl NEN, Boston, Massachusetts; the original specific activity [44.6-52.6 Ci/mmol] was reduced approximately 66% by addition of unlabelled dopamine).

N-Ethylmaleimide

blocks stimulant binding

Uptake was terminated exactly two minutes after the radiolabelled neurotransmitter was added by filtration through Whatman GF/C filters under vacuum. The filters were washed with two 5 ml aliquots of chilled Krebs’-phosphate buffer, shaken with 8 ml of Beckman Ready-Protein scintillation fluid and counted for radioacti~ty in a liquid scintillation counter. A~umulation of [3HJdopamine at 0°C was used to determine uptake, independent of active transport.

aQ

903

1

0

6

7

8 Buffer

Analysis of data

Data are reported as means + SEM. Scatchard analysis was conducted using the LIGAND program (Munson and Rodbard, 1980). Student’s paired t-test, two-tailed, was used in the evaluation of the parameters from the Scatchard analysis. All other data were analyzed by one-way analysis of variance (ANOVA), followed by multiple comparisons, using either the Scheffe or the Newma~Keuls test with the significance level set at P < 0.05.

RESULTS Binding Studies with [-‘H]Methylphenidate Effect of pH on reactivity of N-ethylmaleimide

The influence of pH on the reactivity of N-ethylmaleimide with the t3H]methylphenidate binding site was examined. Separate experiments were conducted using 50 mM sodium phosphate and T&-Cl buffers in order to span the pH range from 6.1 to 9.5. Individual P2 fractions, prepared from a common homogenate, were resuspended either in pH 6.1 or 7.0 phosphate buffers or in one of six Tris-Cl buffers, ranging in 0.5 unit increments from pH 7.0 to 9.5 (measured at OC). Because sodium affects binding to the stimulant recognition site (Schweri et al., 1985) the content of sodium of all buffers was adjusted to 100mM by the addition of sodium chloride. After reacting the samples with 0.5 mM N-ethylmaleimide for 20 min at 0°C the buffers were removed by centrifugation, samples were resuspended in the standard assay buffer (see Methods) and the binding of [~Hlmethylphenidate in treated samples was compared to controls exposed to vehicle under the same pH conditions. As shown in Fig. 1, samples reacted with N-ethylmaleimide at pH 6.1 underwent significantly less inactivation of the stimulant recognition site (14.8 &-5.5%) than did samples exposed to the sulfhydryl reagent in pH 7.0 phosphate buffer (38.4 k 4.1%). Fractions reacted at pH 7.0-9.5 in 50mM Tris-Cl buffers exhibited a complex pattern of inhibition (Fig. 1). Inhibition at pH 7.0 was 63.6 & 2.1% and remained constant through pH 9.0, with one exception: inhibition decreased markedly to 31.9$0.8% at pH7.5. Only at pH9.5 did inactivation of the binding site increase significantly over that seen at pH 7.0, to 79.3 i: 0.6%. This

9

10

pH

Fig. 1. Effect of pH on the inhibition of the binding of [‘Hlmethylphenidate by N-ethylmaleimide. Individual P, fractions of striatal tissue were resuspended in 50mM TrisCl (0) .-, or sodium phosnhate (IZI) buffers. adiusted to 100 mM Na+ with NaCi and*reacted%ith 0.5 rnM_N-ethylmaleimide for 20min. The samples were centrifuged to remove the buffers and unreacted drug, and resuspended in standard assay buffer (see Methods for details). The binding of [3H]methylphenidate was determined by the standard method. Percentage inhibition was calculated by comparison with control specific binding measured in similarly treated samples exposed to vehicle, rather than N-ethylmaleimide. In the experiment using Tris buffer, control binding ranged from 60.1 f 0.5 x lo3 cpm/mg protein in samples treated at pH 7.0, to 82.3 + 0.9 x lo3 cpm/mg protein in samples exposed to pH 8.0. Control binding in samples exposed to phosphate buffer did not vary as a function of the pH; the mean value was 67.6 f 1.2 x IO3 cpm/mg protein. Data shown are means+SEM of triplicates from separate experiments, using phosphate or Tris buffer; in some cases, the error bar is so small that it has merged with the data symbol. Similar results (with minor differences described in Results) were obtained when the experiments were repeated *Significantly different from all other samples prepared in the same buffer (one-way ANOVA followed by Scheffe multiple comparison test, P < 0.05).

experiment was repeated a second time with similar results, except maximum inhibition (59.9 f 1.5%) was observed at pH 9.0, with no further increase in inhibition at pH 9.5. When a synaptosomal preparation was reacted with 0.025-10 mM N-ethylmaleimide at pH 7.9 for 20 min (see Methods), only a subset of methylphenidate binding sites appeared to be susceptible to inactivation (Fig. 2): in three experiments, maximum inhibition never exceeded 78.5 + 2.6%. Under the experimental conditions employed, the IC,, was 0.33 2 0.12 mM (x f SEM from 3 experiments). Above pH 7.0, N-ethylmaleimide reacts rapidly with several different nucleophilic amino acid residues (Means and Feeney, 1971). Below this pH, N-ethylmaleimide reacts much more slowly, but specifically, only with cysteinyl residues (Means and Feeney, 1971; Gregory, 1955). To determine whether the binding site for methylphenidate was susceptible to attack by N-ethylmaleimide in an acidic environment, a dose-response curve was constructed for the inactivation of the stimulant binding site by the reagent at pH 6.1 (phosphate buffer), using a procedure similar to that described above, except the reaction was allowed to proceed for 60 min before

904

M. M.

001

01

1

[N-ethylmateimldel

10

100

mM

Fig. 2. Inhibition of binding of [‘Hlmethylphenidate by N-ethylmaleimide at pH 6.1 and 7.9. The P2 fractions, suspended in 50 mM sodium phosphate buffer, pH 6.1 (n) or 50mM Tris-Cl buffer, pH 7.9 (0) were reacted with N-ethylmaleimide. The reaction was terminated by centrifugation and the resulting pellets were resuspended in the standard assay buffer (pH 7.9) prior to determination of the binding of [‘Hlmethylphenidate (see Methods for experimental details). Inhibition was calculated by comparison with similarly-treated controls, exposed to vehicle instead of N-ethylmaleimide. Data shown are means + SEM of triplicates from separate experiments conducted at each pH. Points without error bars had SEM smaller than the size of the symbol on the graph.

being terminated by centrifugation and a second wash with phosphate buffer was incorporated to ensure that all unreacted drug was removed prior to reconstitution of the samples in the standard assay buffer, preparatory to measurement of the binding of [3H]methylphenidate. Under these conditions, N-ethylmaleimide was considerably less potent in inactivating the binding site, compared to the results obtained at pH 7.9 (Fig. 2). Moreover, a triphasic dose-response curve was obtained. As at pH 7.9, only a subset of receptors appeared to be affected by the reagent; as shown in Figure 2, only 84% of the available sites were inhibited at 200mM N-ethylmaleimide, the largest concentration examined. (The effect of concentrations greater than 200mM could not be evaluated, unfortunately, due to the limited solubility of the N-ethylmaleimide.) Having demonstrated inactivation of the stimulant recognition site under conditions selective for cysteine residues, all subsequent experiments were conducted in standard assay buffer (pH 7.9 at OC) because of the shorter reaction times required in the more basic environment.

Time course for inactiaation

by N-ethylmaleimide

The time course for reaction of N-ethylmaleimide with the binding site for [3H]methylphenidate was quite rapid (Fig. 3). Despite the fact that the reaction was conducted at O’C to protect the receptor from enzymatic degradation, reaction with 0.1 mM N-ethylmaleimide inactivated the site by 21% at 5 min, the earliest measured time and reached a maximum within 15 min. Extending the reaction time up to 60 min resulted in no further inhibition of [3H]methylphenidate binding.

SCHWERI

04 0

10

20

30

40

Time

(min)

50

60

Fig. 3. Time course for inhibition of the binding of [3H]methylphenidate by 0.1 mM N-ethylmaleimide. Conditions are described in Methods; time values do not include 15 min centrifugation step at 0°C used to terminate the reaction. Data points are mean k SEM of triplicates from one experiment, which was repeated with similar results. Differences in inhibition at 15, 30 and 60 min are not statistically significant. The point without an error bar had an SEM smaller than the size of the symbol on the graph.

Characteristics qf the inhibition of the binding [‘Hlmethylphenidate by N-ethylmaleimide

of

The irreversibility of the modification of the stimulant recognition site by N-ethylmaleimide was studied by comparing the binding of [3H]methylphenidate in tissue preparations incubated with 0.5 mM N-ethylmaleimide at 0°C for 20 min, washed three times by centrifugation at 20,OOOg for 15 min and resuspended in fresh assay buffer, to similarly treated control samples exposed to vehicle rather than sulfhydryl reagent. Inhibition decreased slightly but not significantly in samples washed once (from 52.2 + 2.2% to 48 k 1.0%) then remained constant in samples washed twice more (46.9 k 2.8% inhibition after the third wash). The results described are based on triplicate analyses of binding from one experiment, which was repeated with similar results (data not shown). Scatchard analysis revealed that the decrease in binding, caused by N-ethylmaleimide was attributable to a reduction in the number of stimulant binding sites, while the affinity of the sites for [‘Hlmethylphenidate remained unchanged (Fig. 4). In these studies, a P, fraction was suspended in assay buffer and allowed to react with 0.5 mM N-ethylmaleimide at 0°C for 20 min; the reaction was terminated by centrifuging the sample at 20,OOOg for 15 min. The pellet was resuspended in assay buffer and the binding of [‘Hlmethylphenidate was measured as described in the legend to Figure 4. Control samples were treated identically, except that they were exposed to vehicle instead of N-ethylmaleimide. Based on results from three separate experiments, treatment with N-ethylmaleimide reduced the B,,, by 61.5 k 1.1%. Controls averaged 15.7 f 3.0 pmol/mg protein, compared to 6.0 k 1.l pmol/mg protein in treated samples (P < 0.04, paired f-test, 2-tailed). No significant alteration was observed in the apparent KD values: control samples averaged 167 + 85 nM, compared to 171 _t 109 nM for treated samples.

N-Ethylmaleimide

0

1

2

3

blocks stimulant binding

905

cl

MP atone

q

NEM

alone

4

Bound (pmol)

Fig. 4. Scatchard analysis of the effect of N-ethylmaleimide on the binding of [“Hlmethylphenidate. A P, fraction was suspended in assay buffer and allowed to react with 0.5 mM N-ethylmaleimide at 0°C for 20min; the reaction was terminated by centrifuging the sample at 20,OOOg for 15 min. The pellet was resuspended in assay buffer and binding to samples was measured in the presence of l&600 nM [SH]methylphenidate. Graph shown is from one experiment in which triplicate samples, containing 0.27 mg protein, were analyzed at each concentration. Data from three such experiments showed the I?,,,, differed significantly (P < 0.04, paired t-test, 2-tailed) between controls (0) [ 15.7 + 3 .Opmol/mg protein] and treated samples (0) [6.0 _t 1.1 pmol/mg protein], while the KD was unchanged (167 k 85 nM for controls and 171 k 109 nM for treated samples).

Protection from inhibition by N-ethylmaleimide Inhibition of the binding of [3H]methylphenidate by N-ethylmaleimide was prevented if the samples were first equilibrated with saturating concentrations of unlabelled methylphenidate (Fig. 5; see Methods for experimental procedure), suggesting that the sulfhydryl reagent attacked the stimulant recognition site at the same region where stimulant agents, such as methylphenidate, bind, rather than allosterically. As shown in Figure 5, 5 PM methylphenidate occupied the majority of the stimulant recognition sites, inhibiting the binding of 10 nM [3H]methylphenidate by approximately 90% in unwashed samples. After three washes, however, the unlabelled methylphenidate was completely removed, as indicated by the absence of inhibition in the washed samples. (Binding actually increased slightly in these samples, compared to untreated

controls, resulting in a small but statistically significant negative inhibition [- 10.1 & 1.9%].) In contrast, inhibition in samples treated with 0.5 mM N-ethylmaleimide remained elevated after three washes (54.6 + 0.6%, compared to 62.8 k 1.7% in unwashed samples). Samples pretreated with 5 PM methylphenidate prior to exposure to the sulfhydryl reagent, however, were completely protected from inactivation by the compound; after washing, inhibition in these samples averaged -7.7 f 1.3%, not significantly different from either the untreated controls or samples treated only with methylphenidate. This experiment was repeated with similar results.

z s ._ r D r E

s

20

0

I

-201

Unwashed

*

I

Washed

Fig. 5. Protection of the binding of [3H]methylphenidate from inactivation by N-ethylmaleimide, by pretreatment with excess methylphenidate. Samples of tissue were preincubated in the presence of 5 PM unlabelled methylphenidate, prior to reaction with 0.5 mM N-ethylmaleimide. The tissue was washed three times to remove unreacted and/or reversibly bound drugs. See Methods for detailed description of procedure. The percentage inhibition is expressed as mean f SEM (n = 3), calculated by comparing the binding in treated samples to vehicle-treated controls before (814 fmol/mg protein) and after (910 fmol/mg protein) washing; concentration of [3H]methylphenidate was 9.9 nM. Samples pretreated with excess methylphenidate (MP) 0; samples exposed to N-ethylmaleimide (NEM) q; samples pretreated with excess methylphenidate, prior to exposure to N-ethylmaleimide q. *Significantly different from washed vehicle-treated controls. **Significantly different from all other washed treatment groups. Data evaluated by Scheffe analysis (P < 0.05) following one way ANOVA. The experiment was repeated with similar results.

Studies of the accumulation of [3H]dopamine

In order to determine whether inactivation of the stimulant recognition site by N-ethylmaleimide was accompanied by an irreversible reduction in the transport of dopamine, accumulation of [‘Hldopamine by striatal synaptosomes was measured after treatment with the sulfhydryl reagent. As shown in Table 1, the uptake of [3H]dopamine was reduced by 79.2 + 0.2% in synaptosomes treated with 0.2mM N-ethylmaleimide for 15min. After two washes, the inhibition of accumulation of [3H]dopamine remained high (75.0 + 0.3%). In contrast, the marked inhibition of the uptake of [‘Hldopamine (79.2 f 0.3%) observed in samples exposed to 5 PM unlabelled methylphenidate was completely removed by washing. Indeed, similar to the results obtained with the binding of [‘HImethylphenidate, uptake of [‘Hldopamine in samples exposed to large concentrations of methylphenidate and then washed, actually surpassed that seen in vehicle-treated controls by 9.6%. Contrary to the results obtained with the binding of [3H]methyl-

M. M. SCHWERI

906 Table I Protection

bl methylphenldate from ~“activatm” by n;-ethylmaleimide somal accumulation of [‘H]dopaml”e Percentage

Tissue status

Control

of accumulation

of [‘HJdopamine

Y-Ethylmalamide

Methylphenidate

0 5 0.6 (3) 0 * I.5 (2)

Unwashed Washed

inhibition

Protected

79.2 + 0.2 (3) 75.0 * 0.3 (3)

79.2 * 0.3 (3) -9.6 + I.4 (3Y

of synapto-

91.6 + 0.1 (3) 70.8 k 1.0 (3)d.h

Inhibition of accumulation of [‘H]dopamine by samples subjected to I of 4 treatments. was measured before and after washing to remove reversibly bound agents (see Methods for exper”nenral detads). Treatment groups are defined as follows: controlpretreated ~lth xhwle (0.32 M sucrose). before exposure to vehicle: methylphenidatepretreated v+ith 5 11M methylphenidate, before exposure to vehicle; N-ethylmaleimide --pretreated with vehicle. before exposure to 0.2 mM N-ethylmaleimide; protectedpretreated wth 5 PM methylphcnidate. before exposure to 0 2 mM N-ethylmaleimide. Uptake of [‘Hldopamine in controls before washing was 533 & 3 x IO’cpm:mg protem. compared to 250 t 4 x IO’cpm mg protein after washing. The percentage Inhibition I” the protected samples after eashmg wab calculated by comparison to the methylphenidate-treated samples. m order to correct for the s~nall but significant enhancement of accutnulation of [‘H]dopamine, observed after wsshlng. m samples treated with methylphenidate. Each \aIue is the mean & SEM: number of samples is I” parentheses D&i from washed samples were analyzed by one-way ANOVA, followed different from bq Student Ncwnan Keuls multiple comparison test. *Significantly control.

P < 0.05:

experiment

wo\

hs~gndicantly

repeated

ditkent

H second

tme

phenidate. however, pretreatment with saturating amounts of methylphenidate did not afford complete protection from attack by N-ethylmaleimide. Nevertheless, a small, but statistically significant degree of protection was observed: inhibition was reduced from 75.0 + 0.3% in samples treated with N-ethylmaleimide alone, to 70.8 & I .O% in samples pretreated with methylphenidate (P < 0.05. Student Newman-Keuls multiple comparisons test). This experiment was repeated with similar results.

DISCUSSION

This work demonstrates that IV-ethylmaleimide irreversibly inhibited binding to a subset of stimulant recognition sites in striatal tissue. The data reported suggest that the inactivation of these sites was due to the covalent modification of one or more cysteinyl residues located in a region of the molecular complex essential to stimulant binding. although further experiments will be required before this conclusion can be accepted without reservation. This conclusion is supported by several lines of evidence. The ability of N-ethylmaleimide to inactivate the binding of [3H]methylphenidate following reaction with striatal tissue preparations at pH 6.1 strongly implicates cysteinyl groups as the reactive amino acid residues at the stimulant recognition site. Above pH 7.0. this reagent can form adducts with free amino groups. as well as sulfhydryl moieties (Means and Feeney, 1971). Below pH 7.0, however, N-ethylmaleimide reacts slowly, but specifically. only with thiol groups (Means and Feeney, 1971). The multiphasic nature of the doseeresponse curve at the lower pH suggests that (1) the stimulant recognition site may exist in several different conformations or (2) the site may contain multiple cysteine groups. having diRerent reactivities towards the sulfhydryl reagent. While N-ethylmaleimide was much less potent at this

from wth

N-ethylmalelmide smilar

group,

P i

0.05. Ttus

results.

acidic pH, maximum inhibition of approximately 80% was observed under both conditions. The increase in inhibition, seen at pH 9.0-9.5, suggests that a lysine residue may also be present in this region of the dopamine transporter, although it is unlikely that the unprotonated c-amino group (pK, = 10.5; Lehninger, 1970) of this amino acid was available for reaction at the pH of 7.9 employed in these experiments. The transient, but marked, decrease in reactivity occurring at pH 7.5 may perhaps be due to a conformational change in the macromolecule induced by deprotonation of the imidazole group of histidine, thereby preventing access of N-ethylmaleimide to its target nucleophile. Due to the experimental design, it cannot be determined from the present data whether the inhibition caused by N-ethylmaleimide at pH 7 in phosphate buffer, differed significantly from that measured in Tris-Cl buffer at the same pH (Fig. 1). The experiments employing the two buffer systems were conducted separately; therefore, experiment-toexperiment variation dictates that comparisons be made only within an individual buffering system. not between different experiments. The combined results of the Scatchard analysis and the washout studies further support the conclusion that N-ethylmaleimide covalently modified the stimulant recognition site. Scatchard analysis of the binding of [3H]methylphenidate in tissue treated with the sulfhydryl reagent, revealed a significant decrease in B,,,, but no change in K,; such results would be expected after treatment with an agent which effectively reduces the number of receptors available for binding by irreversibly inactivating them, but does not alter the affinity of the remaining binding sites. The failure of the inhibition to dissipate after repeated washings further supports the interpretation that N-ethylmaleimide covalently. and therefore irreversibly, modified the recognition site.

N-Ethylmaleimide

blocks

The complete protection of the stimulant binding site from inactivation by N-ethylmaleimide afforded by pretreatment with excess unlabelled methylphenidate suggests that the protein modifier requires direct access to the domain on the dopamine transporter recognized by stimulant drugs, in order to inhibit their binding. These results argue against the likelihood that the observed decrease in the binding of [3H]methylphenidate was due to covalent modification of an allosteric site by the sulfhydryl reagent. (It is also possible, however, that occupation of the stimulant recognition site by methylphenidate may simply stabilize it in a conformation incompatible with attack by N-ethylmaleimide.) As predicted from the binding studies with [3H]methylphenidate, N-ethylmaleimide was found to also irreversibly inhibit transport of [3H]dopamine by synaptosomes (Table 1). Unlike the results obtained in the binding studies, however, pretreatment with 5 PM methylphenidate provided only a small degree of protection of the transport function from inactivation by N-ethylmaleimide. One explanation for the discrepancy in the protection afforded by large concentrations of methylphenidate in the binding vs the transport studies, is that the decrease in transport activity observed after treatment with the sulfhydryl reagent may reflect the covalent modification, not only of the stimulant recognition site on the dopamine transporter, but of other cysteine-containing proteins which contribute to uptake of dopamine, as well. Saturating concentrations of a reversibly bound stimulant would protect only the binding site for methylphenidate, leaving other proteins (essential for the uptake process itself, or to the maintenance of synaptosomal viability) open to attack. While the present study is consistent with previous work suggesting that the binding site for methylphenidate is a component of the transport complex for dopamine (Schweri et al., 1985) further elucidation of the role that cysteinyl groups play in this functional relationship awaits development of a site-directed affinity label targeted specifically to the cysteinyl residue(s) of the stimulant recognition site. The presence of a nucleophihc group at the stimulant binding site is consistent with previous reports that Metaphit (a phencyclidine derivative containing an isothiocyanate moiety at the meta-position of the aromatic ring) irreversibly inhibits the binding of [‘HIcocaine (Berger, Jacobson, Rice, Lessor and Reith, 1986) [‘Hlmethylphenidate (Schweri et al., 1987; Schweri, Jacobson, Lessor and Rice, 1989) and [‘Hlmazindol (Zimanyi, Jacobson, Rice, Lajtha and Reith, 1989) to the site, as well as synaptosomal transport of [3H]dopamine (Schweri et al., 1989). Unlike N-ethylmaleimide, derivatives containing the isothiocyanate group react readily with a broad range of nucleophiles, including primary and secondary amines, as well as mercaptans (for review, see Assony, 1961). Metaphit also differs from N-ethylmaleimide

stimulant

binding

907

in that the derivative of phencychdine is not a general protein modifier; rather, it acts as an affinity label only at sites which recognize the parent compound, phencyclidine (Rafferty et al., 1985; Schweri et al., 1987). However, binding could be inhibited by N-ethylmaleimide to only 80% of the stimulant recognition sites identified using [3H]methylphenidate (Fig. 2). These results suggest that two subpopulations of stimulant recognition sites may exist, which have similar affinities for [3H]methylphenidate but differ markedly in their reactivity towards N-ethylmaleimide. After free drug was removed by repeated washing, the binding of [3H]methylphenidate in samples which had been pre-treated with large concentrations of unlabelled methylphenidate to block attack by N-ethylmaleimide was found to have increased slightly, but significantly, over that observed in samples pre-treated with vehicle only (Fig. 5). A parallel increase in the transport of [3H]dopamine was likewise observed in synaptosomes after the removal of large concentrations of methylphenidate from the incubation medium (Table l), again illustrating the apparent interrelationship between the binding site for methylphenidate and the dopamine transporter. While the protective effect associated with the occupation of receptors by their respective ligands has been reported for several other systems (e.g. Korneyev and Factor, 1981), this phenomenon has not been described for the stimulant recognition site. In conclusion, this research suggests that the site on the dopamine transporter recognized by stimulant drugs contains one or more reactive nucleophiles. While the identity of the nucleophiles is not known for certain, cysteine residues are likely candidates. Besides advancing our knowledge of the molecular composition of the dopamine transport complex, this information should also prove valuable in the design of both site-directed affinity labels, as well as crosslinking reagents, targeted to the stimulant recognition site. Acknowledgements-The author thank MS Tami Reeves, Mm Michele Cassidy, and Mr Paul Ossi for their technical assistance. This work was supported by grant NS-22546 from the National Institutes of Health.

REFERENCES

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908

M. M. SCHWERI

Dubocovich M. L. and Zahniser N. R. (1985) Binding characteristics of the dopamine uptake inhibitor (‘H]nomifensine to striatal membranes. Biochem. Pharmac. 34: 1137-1144. Gregory .I. D. (195.5) The stability of N-ethylmaleimide and its reaction with sulfhydryl groups. J. Am. Chem. SQC.77: 3922-3923.

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Schweri M. M.. Jacobson A. E.. Lessor R. A. and Rice K. C. (1989) Metaphit inhibits dopamine transport and binding of [“Hlmethylphenidate, a proposed marker for the dopamine transport complex. Lij? Sci. 45: 1689-1698. Schweri M. M., Skolnick P., Rafferty M. F., Rice K. D., Janowsky A. J. and Paul S. M. (1985) 13H]Threo(~)-methylphenidate binding to 3,4-dihydroxyphenylethylamine uptake sites in corpus striatum: Correlation with the stimulant properties of ritalinic acid esters. J. ~eur#~hem. 45: IO62- 1070. Zimanyi I., Jacobson A, E., Rice K. C., Lajtha A. and Reith M. E. A. (1989) Long-term blockade of the dopamine uptake complex by Metaphit, an isothiocyanate derivative of phencyclidine. Synapse 3: 239-245.