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Molecular determinants of Pb2+ interaction with NMDA

receptor channels

Paola Gavazzo, Ilaria Zanardi, Irena Baranowska-Bosiacka 1,Carla Marchetti *

Istituto di Biofisica, Consiglio Nazionale delle Ricerche, via De Marini 6, 16149 Genova, Italy

Received 23 January 2007; received in revised form 26 June 2007; accepted 3 July 2007

Available online 10 July 2007

Abstract

Lead (Pb2+) is a potent neurotoxin that acts as a non-competitive, voltage-independent antagonist of the NMDA receptor (NR) channel. Pb2+

action partially overlaps with that of zinc (Zn2+), but precise coincidence with Zn2+ binding site is debated. We investigated the site of Pb2+

interaction in NR channels expressed in Xenopus laevis oocytes from the clones z1, e1 or e2 and mutated e1 or e2 forms. For each e subunit we chose

two mutations that have been identified as ‘strong mutations’ for Zn2+ binding and examined the effect of Pb2+ on channels that contained those

mutations. In e1-containing channels, mutations D102A and H128A caused a decrease of Pb2+ inhibition with a 10-fold (D102A) and four-fold

(H128A) shift of IC50. In e2-containing channels, the most effective mutation in removing Pb2+ inhibition was H127A, with a five-fold increase of

IC50, while D101A was virtually ineffective. Other mutations, D104A, T103A, and T233A, were less effective. The double mutation

D101AH127A, while reducing Zn2+ inhibition by nearly nine-fold, caused a minor (less than two-fold) shift in Pb2+ IC50. Competition

experiments showed that increasing doses of Zn2+ reduced the apparent affinity for Pb2+ in e1-containing receptors, but not in e2-containing

receptors. In addition the effect of Pb2+ on e2-containing channels was additive with that of ifenprodil, with no competition for the site. Although

none of the mutations that we have tested abolished the block by Pb2+, our results indicate that the action of this toxic metal on NR channels is more

dependent on the receptor composition than previously thought, because Zn2+ is able to displace Pb2+ from its binding site in e1-containing

channels, but not in e2-containing channels.

# 2007 Elsevier Ltd. All rights reserved.

Keywords: Glutamate receptors; NMDA receptor subunits; Heavy metals; Zinc; Amino terminal domain; Neurotoxicity

Lead (Pb2+) has been known since a long time as a potent

central neurotoxin that interferes with neuronal functions and

causes a wide variety of long lasting adverse effects, especially

in developing brains (Toscano and Guilarte, 2005). Similar to

other heavy metals, Pb2+ may exert its action through

mimicking physiological metals, primarily calcium (Ca2+)

and zinc (Zn2+), possibly competing for their binding sites

(Marchetti, 2003).

One putative site of interaction of Pb2+ in the central nervous

system is the NMDA subtype of glutamate receptor (NR), a

receptor channel involved in many forms of synaptic plasticity

and in high brain functions, such as memory and spatial

learning, cognition and behavior. The NR channel carries the

‘‘slow’’ component of the glutamate-activated post synaptic

current and it is endowed with some unique properties, such as

the requirement for two agonists, glutamate and glycine, a

Mg2+-mediated voltage-dependence and significantly high

calcium permeability. All functional NRs are tetrameric

complexes, containing the essential subunit NR1 (rat) or z1

(mouse), which exists in eight splice variants, and one or more

of the four different NR2 types (NR2A, B, C and D, rat) or e1

through e4 (mouse). The NR2 pattern of expression is tissue-

dependent and differs remarkably during development (Aka-

zawa et al., 1994; Farrant et al., 1994).

Divalent cations deeply and specifically affect the function

of NR channels. Ault et al. (1980) first recognized two distinct

effects of divalent cations on the NR channel: the Mg-like effect

(voltage-dependent block), mimicked by Co, Ni and Mn, and

www.elsevier.com/locate/neuint

Neurochemistry International 52 (2008) 329–337

* Corresponding author. Tel.: +39 010 6475578; fax: +39 010 6475500.

E-mail address: marchett@ge.ibf.cnr.it (C. Marchetti).1 Present address: Department of Biochemistry and Medical Chemistry,

Pomeranian Medical University, Szczecin, Poland.

0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved.

doi:10.1016/j.neuint.2007.07.003

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the Ca-like effect (permeation), which is mimicked by Sr, Ba

and Cd. Shortly after, Zn2+ was recognized to behave in a third

different way, with both voltage-dependent and voltage-

independent inhibitory effects (Westbrook and Mayer, 1987).

The voltage-independent Zn2+ inhibition is subunit-dependent

(Paoletti et al., 1997; Choi and Lipton, 1999) as the metal binds

with nanomolar affinity to a site in NR2A amino terminal

domain (ATD; Fayyazuddin et al., 2000; Low et al., 2000) and

with micromolar affinity to a site located in the same region of

NR2B (Rachline et al., 2005). Binding of Zn2+ restrains the

receptor activity and may regulate the amplitude of NMDA

synaptic currents (Erreger and Traynelis, 2005), so that free

Zn2+ has been suggested to act as an ‘‘atypical neurotransmit-

ter’’ (Baranano et al., 2001).

The inhibitory effect of Pb2+ on NR channels was early

described as a highly specific, voltage-independent, non-

competitive allosteric interaction (Alkondon et al., 1990;

Guilarte and Miceli, 1992; Busselberg et al., 1994), which

resembles the effect of Zn2+. However, Pb2+ has no Mg-like

voltage-dependent effect and does not discriminate signifi-

cantly between NR2A-, NR2B- and NR2C-containing

receptors (Yamada et al., 1995; Omelchenko et al., 1996,

1997). Evidence of competition of Pb2+ and Zn2+for the same

binding site was suggested in two studies (Guilarte et al., 1995;

Schulte et al., 1995) that reported an increase in the IC50 for

Pb2+ inhibition of MK-801 binding in the presence of Zn2+. In

these early works, Pb2+ potency was determined based on

nominal concentration. As Pb2+ forms complexes and

precipitates and is present as a contaminant in laboratory

reagents, further studies employed a divalent chelating agent

and measured the free metal concentration by a Pb2+-sensitive

electrode, establishing the IC50 for Pb2+-binding on the NR

channel in the low mM range (Lasley and Gilbert, 1999) and

confirming that, different from Zn2+, Pb2+ modulation of NR

channels is largely subunit-independent (Gavazzo et al., 2001;

Marchetti and Gavazzo, 2005). Moreover, Lasley and Gilbert

(1999), using a [3H]-MK801binding assay under equilibrium

conditions, showed that the interaction between of Zn2+ and

Pb2+ is non-competitive, questioning that they share the same

binding site.

Because of lack of both conclusive electrophysiological data

and structural evidences, competition between Zn2+ and Pb2+,

as well as the location of the Pb2+ binding site, are still

undefined. Thus we decided to investigate directly the structural

determinants of Pb2+ interaction with NR and started this study

by examining the effect of point mutations at the Zn2+ binding

site in e1 and e2, the mouse forms of NR2A and NR2B subunits.

Our first aim was to determine whether Pb2+ and Zn2+ compete

for the same binding site and we show here that competition is

dependent on the subunit present.

1. Experimental procedure

1.1. In vitro transcription and functional expression in Xenopus

oocytes

NMDA receptor subunit mouse clones z1 (GenBank number D10028), e1

(GenBank number D10217) and e2 (GenBank number D10651) were gener-

ously provided by M. Mishina (University of Tokio, Japan). Mutant clones were

obtained by using the QuikChange Site-Directed Mutagenesis kit (Stratagene,

La Jolla, CA, USA). All mutants were verified by dideoxy sequencing across the

mutated region (Sanger et al., 1977). cRNAs were synthesized in vitro from

linearized templates of cDNA using SP6 RNA polymerase from Ambion

(Austin, TX, USA; Yamakura et al., 1993). cRNA was quantified by optical

density at 260 nm and visualized on an agarose gel by ethidium bromide in

order to verify the integrity of the synthesized molecules. Oocytes were

obtained from large females of Xenopus laevis (CNRS, Montpellier, France).

The ovarian lobes were surgically removed and the oocytes separated and

treated for 2 h with collagenase type IA (1 mg/ml), in a Ca-free solution. A

volume of 10–30 nl containing an equal amount of z1 and either e1 or e2 (wild

type or mutated form) cRNA (0.1–0.5 mg/ml) was injected in each oocyte by

means of a Nanoliter 2000 automatic injector (WPI, Sarasota, FL, USA). Cells

were incubated at 19 8C in Barth’s solution, containing (in mM) 88 NaCl, 1 KCl,

0.82 MgSO4, 0.33 Ca(NO3)2, 0.41 CaCl2, 2.4 NaHCO3, 5 Tris–HCl, 5 HEPES

(pH 7.4) and supplemented with 50 mg/ml gentamycin and 100 mM � 2-amino-

5-phosphopentanoic acid (D-AP5). Channel expression was obtained 24–48 h

after injection.

1.2. Electrophysiology

Oocytes were voltage-clamped by two electrodes, which were filled with

3 M KCl solution and had a resistance of 0.1–1 MV. The bath solution

contained (in mM): NaCl 100, KCl 2.8, Hepes 5, glycine 0.03 and 1.8 mM

CaCl2 and 3 mM Na-citrate, when the effect of Pb2+ alone was tested, or 2 mM

CaCl2 and 5 mM Na-citrate, when testing the effect of Zn2+ or of both Pb2+ and

Table 1

Measured free concentrations of Pb2+ in the different experimental conditions

Pb added

(mM)

Pb free (mM) in 3 mM

Na-citrate + 1.8 mM

CaCl2 (no Zn added)

Pb free (mM) in 5 mM

Na-citrate + 2 mM

CaCl2 + 0.2 mM Zn

Pb free (mM) in 5 mM

Na-citrate + 2 mM

CaCl2 + 0.5 mM Zn

Pb free (mM) in 5 mM

Na-citrate + 2 mM

CaCl2 + 1 mM Zn

1 0.07 � 0.01 – 0.04 � 0.02 –

5 0.21 � 0.06 – 0.08 � 0.02 –

10 0.30 � 0.03 0.09 � 0.04 0.12 � 0.03 0.21 � 0.11

30 0.50 � 0.01 0.25 � 0.03 0.27 � 0.09 0.29 � 0.04

50 0.69 � 0.05 – 0.38 � 0.08 –

100 1.39 � 0.13 0.56 � 0.08 0.62 � 0.08 0.88 � 0.02

150 1.86 � 0.27 – 0.90 � 0.08 –

200 2.52 � 0.37 1.21 � 0.10 1.23 � 0.26 –

300 3.40 � 0.48 1.31 � 0.37 1.69 � 0.18 2.17 � 0.06

500 5.41 � 0.60 2.35 � 0.48 2.89 � 0.23 3.44 � 0.4

Values are mean � S.E.M. in at least five measurements in control or in the presence of agonists glycine (30 mM) and glutamate (50 mM). Details are described in

Section 1.

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Zn2+ (see below). The pH was 7.4. Recordings were performed with a Turbo

TEC-03X amplifier (Npi Electronic, Tamm, Germany) and digitized by a

National Instrument BNC-2090 AD/DA converter at 1 kHz. Glutamate

(50 mM), as well as different concentration of Pb2+ and Zn2+, were applied

by gravity flow. The inhibitory effect of the metals was measured on the steady-

state current at �60 mV.

1.3. Lead and zinc solutions

Lead solutions were prepared daily from a stock solution of lead perchlorate

standard (0.1 M, Orion Research, Beverly, MA) and handled in plasticware.

Zinc was prepared as a stock solution of 100 mM ZnCl2 and diluted daily. The

chelating agent sodium citrate (3 mM) was used to set the free Pb2+ concentra-

tion in the bath, as in our previous work (Gavazzo et al., 2001). This chelator

appeared the more suitable to set Pb2+ concentration in an appropriate range at

pH 7.4. When both Pb2+ and Zn2+ were present, the concentration of the

chelating agent was elevated to 5 mM to increase the buffer potency, as in

Lasley and Gilbert (1999). Free ionic Pb2+ concentration in the solutions was

measured by a Pb-sensitive electrode (Orion 96-82), calibrated by the method of

Kivalo et al. (1976) against nitrilotriacetate (NTA)-buffered solutions at dif-

ferent pH, as described in Simons (1985) and in our previous work (Gavazzo

et al., 2001). The concentration of free Pb2+ in the calibrating buffers, as well as

concentrations of free Zn2+ and Ca2+ in the different solutions (see below), were

calculated by Maxchelator 2.5 software (www.stanford.edu/�cpatton/

maxc.html), assuming a temperature of 20 8C, and an ionic strenght

120 mM. The metal-ligand stability constants for citrate and NTAwith different

metals are those provided with the above quoted freeware software and are

listed at http://www.stanford.edu/�cpatton/xlsconstants.htm. The concentra-

tions of free Pb2+ measured by the Pb-sensitive electrode in the different

conditions are showed in Table 1. The values represent the mean � S.E.M.

of 5–12 measurements for each dose and were independent of the presence of

the agonists in the solution. The measured values were considered more reliable

than those obtained by software calculation. Free Zn2+ and Ca2+ were calculated

by the above software at pH 7.4. In the solution containing 3 mM citrate and

1.8 mM CaCl2, free Ca2+ ranged from 0.34 to 0.43 mM depending on the

concentration of Pb2+ added (0–500 mM). This value of free Ca2+ was in

agreement with that we measured by Fura-2 fluorescence in a previous study

(Mazzolini et al., 2001). In 5 mM citrate and 2 mM CaCl2, free Ca2+ ranged

from 0.20 to 0.23 mM with 0–500 mM Pb2+ added, and from 0.2 to 0.27 mM in

0 Pb2+ and 0–1 mM Zn2+ added. The calculated concentrations of free Zn2+ in

the different conditions are listed in Table 2. Note that free Zn2+ is dependent on

added Pb2+; however, we verified that these variations do not alter significantly

the results described in Figs. 4 and 5, where approximated values of free Zn2+

are indicated for simplicity.

1.4. Data analysis

The software for the acquisition and analysis of data was developed by

Michael Pusch and it is freely available at http://www.ge.cnr.it/ICB/conti_mor-

an_pusch/programs-pusch/programs-mik.htm. Curve fitting was performed by

Sigma Plot (SPSS Science, Chicago, IL, USA) software. Data are shown as

mean � S.E.M.

Inhibition curves were generated by fitting experimental points to a single

binding site isotherm with equation:

I

Icontrol

¼ 1

½1þ ðPb2þ=IC50ÞnH �

(1)

where I/Icontrol is the average value of the fraction of current resistant to Pb2+

inhibition, [Pb2+] the free Pb2+ concentration, IC50 the free Pb2+ dose which

provokes a 50% block of the NR current and nH is the Hill coefficient.

Competition of Zn2+ and Pb2+ for the same binding site was evaluated by the

equation

KðPbÞ�d ¼ K

ðPbÞd

�1þ ½Zn2þ�

KdðZnÞ

�(2)

where KðPbÞ�d is the apparent dissociation constant, K

ðPbÞd the dissociation

constant in the absence of Zn2+, KðZnÞd the Zn2+ dissociation constant in the

absence of Pb2+ and [Zn2+] is the free Zn2+ concentration.

2. Results

The residues important for the voltage-independent Zn2+

interaction have been previously identified in the amino

terminal domain (ATD) region of both e1 (NR2A) and e2

(NR2B) subunit. We chose two mutations for each subunit that

have been identified as ‘strong mutations’ for Zn2+ binding and

examined the effect of Pb2+ on channels that contained those

mutations. In e1 subunit, we investigated the mutants D102A

and H128A, which have been reported to exhibit a strongly

reduced zinc sensitivity (Fayyazuddin et al., 2000; Low et al.,

2000; Paoletti et al., 2000). Both these mutations partially

reduced the block exerted by Pb2+, as shown in Fig. 1. Currents

were normalized to the plateau value of the current before

(control) or after (wash) the treatment with Pb2+. The effect of

the metal was always entirely reversible. In channels containing

the wild type form of e1 subunit, the largest dose of Pb2+ (7 mM)

blocked the NR current by 85 � 2% (n = 22). Higher doses of

Pb2+ were not tested because of the limited solubility of the

metal at physiological pH (Simons, 1985; Lasley and Gilbert,

1999). Experimental points were well fitted by Eq. (1) and the

best fit gave IC50 = 1.3 mM (nH = 1.0) for channels containing

the wild type form of e1. Mutation D102A reduced the block

caused by 7 mM Pb2+ to 40 � 5% (n = 6) and caused also a 10-

fold shift of IC50 (to 11.3 mM, nH = 1.0). Mutation H128A

caused a less prominent reduction of the block and a three-fold

shift of IC50 (to 3.3 mM, nH = 0.85). Fig. 1(C) represents the

Table 2

Calculated free concentrations of Zn2+ in the different experimental conditions,

as a function of added Zn2+ in the absence of Pb2+ (top) and of added Pb2+ at

three different concentrations of added Zn2+ (bottom)

Zn added (mM) (0 Pb added) Zn free (mM)

1 0.004

10 0.043

100 0.43

150 0.67

200 0.80

300 1.38

500 2.50

1000 6.03

Pb added (mM) Zn free (mM)

+200 mM Zn +500 mM Zn +1 mM Zn

0 0.80 2.50 6.03

5 0.91 2.50 6.05

10 0.91 2.50 6.06

30 0.92 2.54 6.11

50 0.93 2.56 6.15

100 0.94 2.60 6.28

150 0.96 2.65 6.41

200 0.97 2.69 6.54

300 1.01 2.79 6.82

500 1.08 3.01 7.45

The solution contained 5 mM Na-citrate and 2 mM CaCl2. Details of the

calculation are described in Section 1.

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ratio of the Pb2+ IC50 for mutant receptors over that of e1 wild

type receptors.

In wild type e2-containing channels, the current was blocked

by 91 � 2% (n = 17) in the presence of the largest concentra-

tion of Pb2+, and the best fit of the inhibition curve yielded a

value of IC50 = 1.2 mM (nH = 1.0). For this channel, we tested

the corresponding mutations H127A and D101A, which have

been recognized among those capable to shift the Zn2+

inhibition dose-dependence significantly (Rachline et al.,

2005). Mutation D101A did not change Pb2+ sensitivity,

whereas mutation H127A reduced the maximum current block

to one half with respect to the wild type current and shifted the

IC50 to 6.9 mM (Fig. 2).

We tried to find other mutations that affected Pb2+ block in

e2-containing channel more efficiently and tested T103A and

T233A, which shift up to five-fold the IC50 for Zn2+ (Rachline

et al., 2005) and mutation D104A, which does not alter Zn2+

interaction, but profoundly modifies the inhibition by the

phenolethanolamine ifenprodil or its analog RO 25-6981

(Perin-Dureau et al., 2002; Malherbe et al., 2003). All these

mutations modified the dose-dependence of Pb2+ inhibition

slightly, but less effectively than H127A (Fig. 2(B and C)).

Then we tested the effect of Pb2+ on the NR current with e2-

containing the double mutation D101AH127A. The maximum

dose of Zn2+ (6.5 mM) blocked the current of the wild type

channel by 89 � 3% (n = 8) and the current of the double

mutated channel by 54 � 3% (n = 12), with a shift of the IC50

for Zn2+ from 0.6 to 5.2 mM (Fig. 3(A)). The double mutation

was less effective on Pb2+ block, with a reduction of the block

caused by 3.4 mM free Pb2+ from 78 � 3% (n = 12) to 62 � 2%

Fig. 1. Effect of Pb2+ on NR containing z1 and wild type (wt) or D102A and H128A mutated forms of e1. Currents were elicited by 50 mM glutamate in the presence

of 30 mM glycine at a membrane potential of �60 mV. The bath solution contained 3 mM Na-citrate. Free Pb2+ concentrations were measured by a Pb-sensitive

electrode as described in the text. (A) Traces show the current block caused by increasing concentrations of Pb2+ in e1-containing channels. Vertical arrows indicate

application of 0.3, 1.4, 3.4, 5.4 and 7 mM free Pb2+, respectively. The control steady state currents were 22 and 18 mA for wild type and D102A mutation, respectively.

(B) Dose-dependence curve of the effect of Pb2+ for channels containing wild type (wt) or mutated e1. Current values were normalized to Icontrol, the value of the

current in the absence of Pb2+. Points represent averages of at least six measurements and are fitted to Eq. (1). In the wt channel, IC50 was of 1.3 mM, nH = 1.0; in the

H128A mutant, IC50 was 3.3 mM, nH = 0.85 and in D102A mutant, 11.3 mM, nH = 1.0. (C) Ratio of the Pb2+ IC50 for mutant receptors over that of e1 wild type (wt)

receptors.

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(n = 4) and a shift of IC50 for Pb2+ from 1.2 to 1.9 mM

(Fig. 3(B)).

In order to elucidate a possible competition between Zn2+

and Pb2+ directly, we measured the effect of Pb2+ in wild type

channels in the presence of fixed concentrations of Zn2+ (1, 2.6

or 6.5 mM). Results are showed in Fig. 4, for e1-containing

receptors and in Fig. 5 for e2-containing receptors. In the

presence of 3 mM Na-citrate solution, the basal Zn2+ present in

the solution is sequestered by the chelator and therefore the free

Zn2+ is negligible. This is shown in Fig. 4(A) where the current

through e1-containing channels is significantly bigger in 3 mM

Na-citrate than in a basal solution, containing no chelators, in

agreement with previous observations (Paoletti et al., 1997). A

further increase in the citrate concentration (up to 5 mM) did

not determine any further increase in the current amplitude.

Therefore the KðPbÞd equal to 1.3 mM in e1-containing receptors

and 1.2 mM in e2-containing receptors reflect the interaction of

Pb2+ with the channel in the absence of Zn2+. In e1-containing

receptors, the presence of Zn2+ modifies the block by Pb2+. In

the presence of Zn2+ and 5 mM citrate, the maximum amount of

Pb2+ added was 500 mM in all solutions. The concentration of

free Pb2+ was measured and found equal to 2.4 � 0.5 mM in

1 mM Zn2+, 2.9 � 0.2 mM in 2.6 mM Zn2+ and 3.4 � 0.4 mM in

6.5 mM Zn2+ (see Table 1). Those doses of Pb2+ determined a

block of less than 50% in each case (38 � 3%, 31 � 1%, and

20 � 3%, respectively; Fig. 4(B)). Best fit with Eq. (1) yielded

an estimate for the IC50, which was shifted from 1.3 mM (in

control conditions, 3 mM citrate and 0 Zn2+ added) to 3.8 in

1 mM free Zn2+, 7.1 mM in 2.6 mM free Zn2+ and to 19.4 mM in

6.5 mM free Zn2+. Under the assumption that Zn2+ and Pb2+

compete for the same binding site, we best fitted these values to

Eq. (2) and obtained a value of 0.48 mM for KðZnÞd (Fig. 4(B),

inset).

In contrast, in e2-containing receptors, the dose-dependence

of Pb2+ inhibition was not significantly modified in the presence

of increasing doses of Zn2+. The largest dose of Pb2+ added

caused a > 80% block, with IC50 close to 1.2 mM, in all cases

(Fig. 5).

Fig. 2. Effect of Pb2+ on NMDA receptors containing z1 and wild type (wt) or mutated forms of e2. Currents were elicited by 50 mM glutamate in the presence of

30 mM glycine at a membrane potential of �60 mV. The bath solution contained 3 mM Na-citrate. Free Pb2+ concentrations were measured by a Pb-sensitive

electrode as described in the text. (A) Traces show the current block caused by increasing concentrations of Pb2+ in e2-containing channels. Vertical arrows indicate

application of 0.3, 1.4, 3.4, 5.4 and 7 mM free Pb2+, respectively. The control steady state currents were 2.5, 2.2 and 2.8 mA for wild type, D101A and H127A

mutation, respectively. (B) Dose-dependence curve of the effect of Pb2+ for channels containing wild type (wt) or mutated e2. Current values were normalized to

Icontrol, the value of the current in the absence of Pb2+. Points are averages of at least six measurements and are fitted to Eq. (1). In the wild type channel, IC50 had a

value of 1.2 mM, nH = 1.1; in D101A mutant the same parameters had values 1.0 mM and 1.0; in T233A, 1.7 mM and 1.2; in T103A, 2.7 mM and 1.3; in D104A,

3.2 mM and 1.3, and finally in H127A, 6.9 mM and 0.8. (C) Ratio of the Pb2+ IC50 for mutant receptors over that of e2 wild type (wt) receptors.

P. Gavazzo et al. / Neurochemistry International 52 (2008) 329–337 333

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Finally, and because the D104A mutation caused a shift of

IC50 for Pb2+, we tested if Pb2+ competes for the same site as

ifenprodil, as it has been shown for Zn2+. These experiments

were designed as in Rachline et al. (2005). Binding and

unbinding of ifenprodil to its site is significantly slower than

that of divalent cations. Similar to Zn2+, unbinding of Pb2+

developed with a time constant of 2 s (Fig. 6(A)), while

ifenprodil unbound from its site with a time constant of

approximately 100 s (Fig. 6(B)). Then the two blockers were

applied together in sequence. First, ifenprodil was applied at a

concentration (200 nM) to occupy half of its binding sites and

subsequently a high (7 mM) concentration of Pb2+ was applied

for several minutes. This dose of Pb2+ should be able to displace

ifenprodil in the case the two binding sites are coincident and, if

this is the case, a single, fast off-relaxation time-course should

result in wash. However, the off-relaxation curve showed a

double exponential time-course, with a fast (2 s time constant)

and a slow (100 s time constant) component of equal weight

(Fig. 6(C and D)), suggesting that the effect of Pb2+ on e2-

containing channels is additive with that of ifenprodil, with no

competition for the binding site.

3. Discussion

In this work, we have presented new data on the putative

Pb2+ binding site on the NR channel. Because of previous

(Guilarte et al., 1995; Schulte et al., 1995), although

controversial (Lasley and Gilbert, 1999), evidences, we started

our investigation from the assumption that Pb2+ binds to or

close to the Zn2+ binding site. We took advantage of the most

Fig. 3. Dose-dependence of the inhibitory effects of Zn2+ and Pb2+ on NMDA

receptors containing z1 and wild type (circles) or double-mutated

D101AH127A (triangles) e2. Currents were elicited by 50 mM glutamate in

the presence of 30 mM glycine at a membrane potential of �60 mV. Current

values were normalized to Icontrol, the value of the current in the absence of Zn2+

or Pb2+. Points represent averages of at least six measurements and were fitted to

Eq. (1). (A) Dose-dependence curve of Zn2+ inhibition. The bath solution

contained 5 mM Na-citrate and free Zn2+ concentrations were calculated as

described in the text. The IC50 was 0.6 mM (nH = 0.95) in the wild type (circles)

and 5.2 mM (nH = 0.73) in the double mutant (triangles). (B) Dose-dependence

curve of Pb2+ inhibition. The bath solution contained 3 mM Na-citrate. The

double mutation shifted the IC50 from 1.2 mM in control (nH = 1.1, circles) to

1.95 mM (nH = 0.9, triangles).

Fig. 4. Effect of citrate and of Pb2+ in the presence of Zn2+ in NR channels

containing z1 and wild type e1. Currents were elicited by 50 mM glutamate in

the presence of 30 mM glycine at a membrane potential of�60 mV. (A) Current

traces showing the response to 50 mM glutamate in the presence of 30 mM

glycine at a membrane potential of �60 mV in a solution without divalent

chelators (left) and in 3 mM Na-citrate (right). Further elevation of citrate

concentration to 5 mM did not produce any further change in the current. (B)

Dose-dependence curve of Pb2+ inhibition in the presence of the indicated

concentration of Zn2+ for channels containing wild type e1. The bath solution

contained 5 mM Na-citrate. Current values were normalized to Icontrol, the value

of the current in the absence of Pb2+. Points represent averages of at least four

measurements and were fitted to Eq. (1). The IC50 was shifted from 1.3 mM

(nH = 1.0) in Pb2+ alone, to 3.8 mM (nH = 0.9) in 1 mM, to 7.1 mM (nH = 0.8) in

2.6 mM and to 19.4 mM (nH = 0.8) in 6.5 mM free Zn2+. The inset shows the

dependence of the apparent dissociation constant KðPbÞ�d on the free Zn2+

concentration and the straight line is the best fit with Eq. (2). See text for

explanation.

P. Gavazzo et al. / Neurochemistry International 52 (2008) 329–337334

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recent findings concerning the location of the Zn2+ binding

domain on e (NR2) subunits, selected some residues that have

been shown to be involved in Zn2+ interaction and investigated

how the same residues may influence Pb2+ interaction.

In NR2A, or e1, subunit, residues D102 and H128 are both

located in a region lining the putative Zn2+ binding cleft and it

has been shown that a substitution by either A or S strongly

reduces the Zn2+ binding affinity (Fayyazuddin et al., 2000;

Paoletti et al., 2000; Low et al., 2000). The two point mutations

D102A and H128A affected Pb2+ inhibition, with D102A being

more effective. In NR2B, or e2 subunit, the corresponding D101

and H127 residues have been shown to control intermediate

affinity Zn2+ inhibition (Rachline et al., 2005), as well as

inhibition by the NR2B-selective antagonist ifenprodil (Perin-

Dureau et al., 2002; Rachline et al., 2005) and its analog RO 25-

6981 (Malherbe et al., 2003). Only mutation H127A modified

the blocking properties of Pb2+. The point mutation D101A,

which was shown to affect profoundly not only Zn2+, but also

ifenprodil inhibition (Malherbe et al., 2003; Rachline et al.,

2005), had no effect on Pb2+ action (Fig. 2). We also tested other

three point mutations in the ATD of e2 subunit: T103A and

T233, mutations that affect both ifenprodil and Zn2+ inhibition,

and D104A, a mutation that decreases only ifenprodil

sensitivity (Rachline et al., 2005), but none of these mutations

was more effective than H127A on Pb2+ inhibition. Finally, the

double mutant D101AH127A caused a larger shift in Zn2+

inhibition than in Pb2+ inhibition. However both shifts were of a

lesser extent than that caused by H127A mutation alone. This

can be explained by the proximity and close interaction of the

two residues. When substituted amino acids are near to or in

direct contact with each other, the effects are often highly non-

additive, because each mutation affects the environment of the

Fig. 5. Dose-dependence curve of Pb2+ inhibition in the presence of the

indicated concentration of Zn2+ for channels containing wild type e2. Currents

were elicited by 50 mM glutamate in the presence of 30 mM glycine and 5 mM

Na-citrate at a membrane potential of�60 mV. Current values were normalized

to Icontrol, the value of the current in the absence of Pb2+. Points represent

averages of at least five measurements. The solid line shows the best fit with

Eq. (1) in the absence of Zn2+ (same as in Fig. 2). The IC50 was close to 1.2 mM

in all cases.

Fig. 6. Evidence for lack of competition for the same binding site between Pb2+ and ifenprodil in channels containing wild type e2. Currents were elicited by 50 mM

glutamate in the presence of 30 mM glycine at a membrane potential of�60 mV. (A–C) Current traces illustrating the effect on the steady-state current of 7 mM Pb2+

alone (A), 200 nM ifenprodil alone (B) and application in sequence of the two blockers, with simultaneous removal (C). The high dose of Pb2+ (7 mM) blocked the

current almost completely, while 200 nm ifenprodil caused a 50% inhibition with slower on and off rates. The time course of current recovery after the combined

application displays two components, corresponding to the sum of the two individual off-relaxation curves. (D) Off-relaxation time courses shown on an expanded

scale. Circles: 7 mM Pb2+; the best fit with a single exponential decaying function gave a time constant of 2 s. Squares: 200 nM ifenprodil only, the best fit gave a time

constant of 100 s. Triangles: time course of simultaneous removal of the two agonists, the best fit with the sum of two exponential decaying functions yielded 2 and

100 s time constants, with equal weight.

P. Gavazzo et al. / Neurochemistry International 52 (2008) 329–337 335

Author's personal copy

other (Wells, 1990; Skinner and Terwilliger, 1996). The effects

of such two mutations can be antagonistic or synergistic, with

respect to a particular function, and in this case the double

mutant pertubs the system less than the two individual mutants.

Although these data confirmed an interaction of Pb2+ in the

ATD of e1 and e2 subunit, they also suggested a difference

between the two channel types, because a residue of the e1 high-

affinity binding site for Zn2+ (D102) is important for Pb2+

inhibition, while a residue controlling the e2 intermediate-

affinity site for Zn2+ (D101) is not. This difference was

confirmed by direct competition experiments. In e1-containing

channels, Pb2+ inhibition curve was shifted in the presence of

increasing doses of Zn2+, suggesting a partial overlap or close

interaction of the two binding sites (see later). In contrast, in e2-

containing channels, we did not observe any shift in the Pb2+

dose-dependence curve in the presence of Zn2+, suggesting that

despite some involvement of the same residue (H127) in both

Pb2+ and Zn2+ interaction, the two binding sites are distinct.

This latter conclusion finds an indirect confirmation in the

observation that Pb2+ binds to a site that is distinct from that of

ifenprodil (Fig. 6). In contrast, it has been shown that this

specific NR2B antagonists binds in the ATD (Perin-Dureau

et al., 2002) to a site overlapping with that for Zn2+ (Rachline

et al., 2005).

While the absence of an apparent competition indicates that

in e2-containing channels Pb2+ binds to a site distinct from that

for Zn2+ and ifenprodil, in e1-containing channels, the

competition observed between Pb2+ and Zn2+ does not imply

necessarily that the two metal bind to the same site, but possibly

that when Pb2+ occupies its site, it modifies Zn2+ binding. From

the data shown in Fig. 4, Pb2+ appears to compete for a Zn2+

binding site whose affinity can be estimated of 0.48 mM, a value

20–30-fold larger than that reported for the high affinity Zn2+

binding measured with tricine as chelator (Paoletti et al., 1997).

This discrepancy can be due to several factors. First, the

inhibition curves (Fig. 4(B)) do not reach 50% block, so that

IC50 values are undefined and the extent of the shift most likely

underestimated. Then, and more significantly, it is entirely

possible that Pb2+ is not capable to fully displace Zn2+ from its

site or that the two binding sites are not exactly overlapping. It

has been recently proposed that allosteric modulators, Zn2+ and

phenolethanolamines (such as ifenprodil), as well as Pb2+, act

through regulatory sites that are located in the ATD, but are

coupled to the agonist binding site and stabilize the non-

conducting desensitized configuration (Erreger and Traynelis,

2005; Huggins and Grant, 2005). Therefore, it is possible that

when Zn2+ occupies its site, it decouples Pb2+ binding and

channel closure, even if the two binding sites are not coincident.

The difference between e1-containing and e2-containing

channels may have some interesting outcome, because Zn2+ is

able to displace Pb2+ from it binding site in e1-containing

channels, but not in e2-containing channels. In basal

physiological conditions, when Zn2+ occupies the e1 high

affinity site, the IC50 for Pb2+ would be different from the value

we have measured in 0 free Zn2+ (1.3 mM), with IC50 increasing

by a factor of 3 in 1 mM free Zn2+; on the contrary the IC50 in

e2-containing channels is close to 1 mM irrespective of free

Zn2+ concentration, resulting in a different sensitivity to Pb2+ in

the two receptor types when free Zn2+ is not negligible.

It should be noted that, with either subunit, the shifts we

observed in the dose-dependent curve are always not very

prominent, so that additional structural elements beside the

residues we have mutated must contribute to Pb2+ binding. In

the case of Zn2+ binding to NR2B-containing receptors, the

existence of an additional low-affinity site located in a region

different from ATD, was also suggested (Rachline et al., 2005).

As for Pb2+, earlier studies provided biochemical evidence for

two sites of interaction on the NR channel (Guilarte, 1997), one

of higher affinity, with IC50 ranging from 0.3 to 4.7 mM and a

second of lower affinity (IC50 � 70 mM), and whose relative

abundance was related to development and to different

populations of NRs. However, those values were based on

nominal metal concentrations and cannot be directly compared

to the values observed in the current study. In another study,

using free metal ion concentrations, Lasley and Gilbert (1999)

observed only one allosteric binding site, with IC50 close to

0.6 mM. All these authors worked with native channels, whose

composition in subunits is not known and frequently

heterotrimeric, that is they contain two different types of e(NR2) subunit; so their results cannot be directly related to our

data, which were obtained in oocytes injected with only one

type of e subunit and expressing a channel protein of defined

composition. In addition, a role of the z1 subunit, which was

never mutated in this study, and of its different splice variants,

would also require attention, because it is likely it partecipates

to Pb2+ binding site, as it does to the Zn2+ binding site

(Traynelis et al., 1998).

In conclusion, all the mutations tested, in either subunit, fell

short of abolishing Pb2+ block, suggesting the involvement of

additional structural elements, different from those important

for Zn2+ binding. This work is the first to address the problem of

the location of Pb2+ interaction site on NR channels from the

structural point of view and many possibilities are still open and

deserve further investigation. Our study indicates that the effect

of Pb2+ on NR channels is more dependent on the receptor

composition than previously thought. In e1-containing chan-

nels, some residues that are important for high affinity binding

of Zn2+ participate in Pb2+ interaction and Zn2+ is able to

displace Pb2+ from its binding site, while this is not observed in

e2-containing channels.

References

Akazawa, C., Shigemoto, R., Bessho, Y., Nakanishi, S., Mizuno, N., 1994.

Differential expression of five N-methyl-D-aspartate receptor subunit

mRNAs in the cerebellum of developing and adult rats. J. Comp. Neurol.

347, 150–160.

Alkondon, M., Costa, A.C., Radhakrishnan, V., Aronstam, R.S., Albuquerque,

E.X., 1990. Selective blockade of NMDA-activated channel currents may be

implicated in learning deficits caused by lead. FEBS Lett. 261, 124–130.

Ault, B., Evans, R.H., Francis, A.A., Oakes, D.J., Watkins, J.C., 1980. Selective

depression of excitatory amino acid induced depolarizations by magnesium

ions in isolated spinal cord preparations. J. Physiol. 307, 413–428.

Baranano, D.E., Ferris, C.D., Snyder, S.H., 2001. Atypical neural messengers.

Trends Neurosci. 24, 99–106.

P. Gavazzo et al. / Neurochemistry International 52 (2008) 329–337336

Author's personal copy

Busselberg, D., Michael, D., Platt, B., 1994. Pb2+ reduces voltage- and N-

methyl-D-aspartate (NMDA)-activated calcium channel currents. Cell. Mol.

Neurobiol. 14, 711–722.

Choi, Y.B., Lipton, S.A., 1999. Identification and mechanism of action of two

histidine residues underlying high-affinity Zn2+ inhibition of the NMDA

receptor. Neuron 23, 171–180.

Erreger, K., Traynelis, S.F., 2005. Allosteric interaction between zinc and

glutamate binding domains on NR2A causes desensitization of NMDA

receptors. J. Physiol. 569, 381–393.

Farrant, M., Feldmeyer, D., Takahashi, T., Cull-Candy, S.G., 1994. NMDA-

receptor channel diversity in the developing cerebellum. Nature 368, 335–

339.

Fayyazuddin, A., Villarroel, A., Le Goff, A., Lerma, J., Neyton, J., 2000. Four

residues of the extracellular N-terminal domain of the NR2A subunit control

high-affinity Zn2+ binding to NMDA receptors. Neuron 25, 683–694.

Gavazzo, P., Gazzoli, A., Mazzolini, M., Marchetti, C., 2001. Lead inhibition of

NMDA channels in native and recombinant receptors. Neuroreport 12,

3121–3125.

Guilarte, T.R., 1997. Pb2+ inhibits NMDA receptor function at high and low

affinity sites: developmental and regional brain expression. Neurotoxicol-

ogy 18, 43–51.

Guilarte, T.R., Miceli, R.C., 1992. Age-dependent effects of lead on [3H]MK-

801 binding to the NMDA receptor-gated ionophore: in vitro and in vivo

studies. Neurosci. Lett. 148, 27–30.

Guilarte, T.R., Miceli, R.C., Jett, D.A., 1995. Biochemical evidence of an

interaction of lead at the zinc allosteric sites of the NMDA receptor

complex: effects of neuronal development. Neurotoxicology 16, 63–

71.

Huggins, D.J., Grant, G.H., 2005. The function of the amino terminal domain in

NMDA receptor modulation. J. Mol. Graph. Model. 23, 381–388.

Kivalo, P., Virtanen, R., Wickstrom, K., Wilson, M., 1976. An evaluation of

some commercial lead(II)-selective electrodes. Anal. Chim. Acta 87, 401–

409.

Lasley, S.M., Gilbert, M.E., 1999. Lead inhibits the rat N-methyl-D-aspartate

receptor channel by binding to a site distinct from the zinc allosteric site.

Toxicol. Appl. Pharmacol. 159, 224–233.

Low, C.M., Zheng, F., Lyuboslavsky, P., Traynelis, S.F., 2000. Molecular

determinants of coordinated proton and zinc inhibition of N-methyl-D-

aspartate NR1/NR2A receptors. Proc. Natl. Acad. Sci. U.S.A. 97,

11062–11067.

Malherbe, P., Mutel, V., Broger, C., Perin-Dureau, F., Kemp, J.A., Neyton, J.,

Paoletti, P., Kew, J.N., 2003. Identification of critical residues in the amino

terminal domain of the human NR2B subunit involved in the RO 25-6981

binding pocket. J. Pharmacol. Exp. Ther. 307, 897–905.

Marchetti, C., 2003. Molecular targets of lead in brain neurotoxicity. Neuro-

toxicity Res. 5, 221–236.

Marchetti, C., Gavazzo, P., 2005. NMDA receptors as targets of heavy metal

interaction and toxicity. Neurotoxicity Res. 8, 245–258.

Mazzolini, M., Traverso, S., Marchetti, C., 2001. Multiple pathways of Pb2+

permeation in rat cerebellar granule neurones. J. Neurochem. 79, 407–416.

Omelchenko, I.A., Nelson, C.S., Marino, J.L., Allen, C.N., 1996. The sensitivity

of N-methyl-D-aspartate receptors to lead inhibition is dependent on the

receptor subunit composition. J. Pharmacol. Exp. Ther. 278, 15–20.

Omelchenko, I.A., Nelson, C.S., Allen, C.N., 1997. Lead inhibition of N-

methyl-D-aspartate receptors containing NR2A, NR2C and NR2D subunits.

J. Pharmacol. Exp. Ther. 282, 1458–1464.

Paoletti, P., Ascher, P., Neyton, J., 1997. High-affinity zinc inhibition of NMDA

NR1-NR2A receptors. J. Neurosci. 17, 5711–5725.

Paoletti, P., Perin-Dureau, F., Fayyazuddin, A., Le Goff, A., Callebaut, I.,

Neyton, J., 2000. Molecular organization of a zinc binding N-terminal

modulatory domain in a NMDA receptor subunit. Neuron 28, 911–925.

Perin-Dureau, F., Rachline, J., Neyton, J., Paoletti, P., 2002. Mapping the

binding site of the neuroprotectant ifenprodil on NMDA receptors. J.

Neurosci. 22, 5955–5965.

Rachline, J., Perin-Dureau, F., Le Goff, A., Neyton, J., Paoletti, P., 2005. The

micromolar zinc-binding domain on the NMDA receptor subunit NR2B. J.

Neurosci. 25, 308–317.

Sanger, F., Nicklens, S., Coulson, A.R., 1977. DNA sequencing with chain-

terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463–5467.

Schulte, S., Muller, W.E., Friedberg, K.D., 1995. In vitro and in vivo effects of

lead on specific 3H-MK-801 binding to NMDA-receptors in the brain of

mice. Neurotoxicology 16, 309–317.

Simons, T.J., 1985. Influence of lead ions on cation permeability in human red

cell ghosts. J. Membr. Biol. 84, 61–71.

Skinner, M.M., Terwilliger, T.C., 1996. Potential use of additivity of mutational

effects in simplifying protein engineering. Proc. Natl. Acad. Sci. U.S.A. 93,

10753–10757.

Toscano, C.D., Guilarte, T.R., 2005. Lead neurotoxicity: from exposure to

molecular effects. Brain Res. Brain Res. Rev. 49, 529–554.

Traynelis, S.F., Burgess, M.F., Zheng, F., Lyuboslavsky, P., Powers, J.L., 1998.

Control of voltage-independent zinc inhibition of NMDA receptors by the

NR1 subunit. J. Neurosci. 18, 6163–6175.

Wells, J.A., 1990. Additivity of mutational effects in proteins. Biochemistry 29,

8509–8517.

Westbrook, G.L., Mayer, M.L., 1987. Micromolar concentrations of Zn2+

antagonize NMDA and GABA responses of hippocampal neurons. Nature

328, 640–643.

Yamada, Y., Ujihara, H., Sada, H., Ban, T., 1995. Pb2+ reduces the current from

NMDA receptors expressed in Xenopus oocytes. FEBS Lett. 377, 390–392.

Yamakura, T., Mori, H., Masaki, H., Shimoji, K., Mishina, M., 1993. Different

sensitivities of NMDA receptor channel subtypes to non-competitive

antagonists. Neuroreport 4, 687–690.

P. Gavazzo et al. / Neurochemistry International 52 (2008) 329–337 337