Toxicology Letters 198 (2010) 171–176

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Toxicology Letters
journal homepage: www.elsevier.com/locate/toxlet

Action potential changes associated with impairment of functional properties of

sodium channels in hippocampal neurons induced by melamine
Jia-Jia Yang a , Zhuo Yang b , Tao Zhang a,∗
a
College of Life Science, Nankai University, 94 Weijin Rd, Nankai District, Tianjin 300071, PR China
b
College of Medicine, Nankai University, Tianjin 300071, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Since the melamine-contamination event happened in September 2008, there have been lots of stud-
Received 2 April 2010 ies about melamine toxicity, but very limited studies focused on central nervous system (CNS). In the
Received in revised form 16 June 2010 present study, we investigated the effects of melamine (5 × 10−4 , 5 × 10−5 and 5 × 10−6 g/ml) on voltage-
Accepted 18 June 2010
gated sodium channels (VGSCs) in hippocampal CA1 neurons using whole-cell patch-clamp recordings
Available online 25 June 2010
technique. The results showed that only 5 × 10−4 g/ml melamine reduced the amplitude of voltage-gated
sodium current (INa ). At the concentrations of 5 × 10−5 and 5 × 10−4 g/ml, melamine produced a hyper-
Keywords:
polarizing shift in the steady-state activation curve of INa and also enhanced the steady-state inactivate
Melamine
Hippocampus
processing of INa . Action potential properties and the pattern of repetitive firing were examined using
Whole-cell patch-clamp current-clamp recording, which indicated that peak amplitude and overshoot of the evoked single action
Voltage-gated sodium channels (VGSCs) potential were decreased. The half-width and the firing rate of repetitive firing were increased in a
Action potential concentration-dependent manner. The data suggest that melamine alters the action potential of hip-
pocampal CA1 neurons by impairing the functional properties of VGSCs, which may be the underlie
mechanisms of neurotoxicity induced by melamine.
© 2010 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
A major food safety incident in China was made public in
September 2008, and recently the melamine event was reappear-
ance in Shanghai, the largest city of China. Melamine has been
detected in many milk and milk-containing products, as well as
other food and feed products, which are also exported to many
countries worldwide. As an industrially synthesized chemical, it has
been widely used in products covering the fields of plastics, dyes,
fertilizers, fabrics and medicine etc (Hau et al., 2009). Although
a wide application for melamine exists, there are limited studies
available on toxicity of melamine for risk assessment.
Previous toxicological studies have demonstrated that
melamine is generally considered as having urinary system
toxicity to mammals (Gossner et al., 2009; Guan et al., 2009; Sun
et al., 2009). However, recent studies indicated that melamine
existed in rat brain and especially in the regions of cortex, striatum,
hippocampus, cerebellum and brain stem (Wu et al., 2009), what
was more our previous study found that melamine decreased
cell viability of PC12 in a concentration-dependent manner, and
affected excitability of hippocampal neurons (Yang et al., 2010).
∗ Corresponding author. Tel.: +86 22 23500237; fax: +81 22 23508800.
E-mail address: zhangtao@nankai.edu.cn (T. Zhang).
These results imply that melamine has adverse attributes, which
impels researchers to pay attention to the negative effects of
melamine on nervous system.
Ion channels usually underlie a broad range of most basic bio-
logical processes, from excitation and signaling to secretion and
absorption, and are targets for many toxins and drugs. Much dam-
age on the central nervous system (CNS) was caused by interrupting
the function of ion channels (Denac et al., 2000; Judge et al., 2007).
Voltage-gated sodium channels (VGSCs) are responsible for both
initiation and propagation of action potentials of the neurons in
the hippocampus and most electrically excitable cells in CNS (Hille,
1986). Dysfunction of VGSCs is harmful and forms the cause of cer-
tain nervous system diseases (Alekov et al., 2000; Amir et al., 2006;
Berta et al., 2008; Takahashi et al., 2000; Tarnawa et al., 2007). To
our knowledge, a number of diverse chemicals target VGSCs for
their primary actions, for example, two scorpion toxins, including
␣ and ␤ scorpion toxin, while it has not been well investigated
whether melamine could interact with VGSCs or not. If melamine
does interact, directly or indirectly, with VGSCs, certain physio-
logical properties of VGSCs could be interfered. Moreover, normal
neural activity and functions are expected to be altered via a mech-
anism involving interaction between melamine and VGSCs.
Action potentials are a fundamental property of excitable cells in
the mammalian CNS. VGSCs are responsible for the rising phase of
the action potential. And our previous works suggested that there

0378-4274/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.toxlet.2010.06.013
172 J.-J. Yang et al. / Toxicology Letters 198 (2010) 171–176
was a functional linkage between neuronal excitability and VGSCs
(Liu et al., 2009; Zhao et al., 2009). Hence, whether modulation of
VGSCs by melamine influences the properties of action potentials
should be tested.
To determine whether changes of VGSCs are associated with
altered neuronal excitability and function dysfunction of CNS,
the present study explored the potential for the neurotoxicity of
melamine in a rat hippocampal CA1 neuron model using standard
whole-cell patch-clamp techniques and attempted to investigate
the possible mechanisms of melamine on CNS.
2. Materials and methods
2.1. Slice preparation and solutions
We used a hippocampal CA1 pyramid neuron model in this study. Hippocampal
slices were cut as previously described (Tian et al., 2009) with some modifications.
Male Wistar rats on postnatal days 10–14 were provided from Experimental Ani-
mal Center, Chinese Academy of Medical Sciences. The brains were removed rapidly
and placed in an ice-cold, oxygenated (95% O2 and 5% CO2 ) high-sucrose solu-
tion that contained (in mM) sucrose 220, KCl 2.5, MgCl2 6, CaCl2 1, NaH2 PO4 1.23,
NaHCO3 26, and glucose 10, pH 7.4 (with an osmolarity of 300–305 mOsm). Hor-
izontal slices (350 ␮m in thickness) which included the entire hippocampus were
prepared with a vibratome (VT1000S, Leica, Germany). After a 1–2 h recovery period,
slices were moved to a recording chamber mounted on a BX51WI upright micro-
scope (Olympus) equipped with video-enhanced infrared-differential interference
contrast (DIC). Slices were perfused with a continuous flow of artificial CSF (ACSF;
95% O2 and 5% CO2 ) that contained (in mM): NaCl 124, KCl 2.5, MgCl2 2, CaCl2 2,
NaH2 PO4 1.23, NaHCO3 26, and glucose 10, pH 7.4. All experiments were performed
at room temperature (22–24 ◦ C). Neurons were visualized with an Olympus Optical
40× water-immersion lens.
The experiments were conducted in accordance with the guidelines of the Med-
ical Experimental Animal Administrative Committee of Nation, and all efforts were
made to minimize the number of animals used and their suffering.
2.2. Patch-clamp recording
Conventional whole-cell voltage- and current- clamp recordings were per-
formed in hippocampal CA1 neurons using pipettes with 4–6 M resistance after
being filled with pipette solution. The pipettes were pulled using a micropipette
puller (PIP5, HEKA, Germany). Slow and fast capacitance compensation was auto-
matically performed. Access resistance was continuously monitored during the
experiments. Cells were considered only when the seal resistance was >500 M
and the series resistance (<30 M) changed <20% throughout the experiment. The
standard pipette solution for current-clamp experiments was (in mM): KCl 130,
CaCl2 1, MgCl2 2, EGTA 10, Hepes 10, Mg-ATP 2, buffered to pH 7.2 with KOH. The
standard pipette solution for recording sodium current contained (in mM): CsCl 140,
MgCl2 2, Hepes 10, EGTA 10, TEA-Cl 10, Mg-ATP 2, buffered to pH 7.2 with CsOH,
4-aminopyridine (4-AP) (10 mM) and CdCl2 (0.2 mM).
2.3. Drug application
The safe blood concentration in mammals should be below level of 5 × 10−5 g/ml
proposed by the US FSIS (USDA, 2007). Thus, the concentrations of melamine,
employed in the present experiments, were 5 × 10−4 , 5 × 10−5 and 5 × 10−6 g/ml
according to our previous study (Yang et al., 2010). After the establishment of a
whole-cell configuration, the cells were allowed to stabilize for 3–5 min before
starting pulse protocols to record the currents as control. And then each final concen-
tration of melamine was used once currents were stable (about 5 min) to examine
the effects on the properties of VGSCs.
Melamine (1,3,5-triazine-2,4,6-triamine) was purchased from Tianjin Yingda
Sparseness and Noble Reagent Chemical Factory (China). 4-Aminopyridine (4-AP),
tetraethylammonium chloride (TEA-Cl), CdCl2 , EGTA, Hepes and ATP-Na2 were pur-
chased from Sigma (USA), and other reagents were of A.R. grade.
All drugs were given by large diameter (500 ␮m) flow pipette, directed at
the recorded cell. The currents were observed after melamine application, which
reached a relative steady-state in about 5 min.When a drug was not being admin-
istered; normal ACSF continuously flowed from the flow pipe. Drug solutions were
prepared by diluting the appropriate stock solution with ACSF.
2.4. Data collection and analysis
Data were acquired with EPC10 patch-clamp amplifier (HEKA, Germany) which
connected to a computer. Signals were digitized at 10 kHz, low-pass filtered at
2 kHz, stored on a computer hard disk using pulse 8.74 software (HEKA, Germany),
and analyzed with Clampfit9.0, Origin 7.5 and SPSS11.5. For activation, currents at
each test potential were converted to conductance (G) using the following formula
G = I/(Vm − Vr ), where Vr is reversal potential. The peak conductance value for each
test potential was normalized to Gmax and plotted against the test potential to pro-
duce a voltage-conductance relationship curves, which were fitted using Boltzmann
functions G/Gmax = 1/{1 + exp[(Vm − V1/2 )/k]}, where V1/2 is the voltage at which con-
ductance being half-maximal, and k is a slope factor. Steady-state inactivation curves
were fitted with the Boltzmann equations: I/Imax = 1/{1 + exp[(Vm − V1/2 )/k]}, where
I/Imax is normalized current, V1/2 the potential for half-maximal inactivation, and
k the slope factor. The time course of recovery of the IA currents from inactiva-
tion was fitted with a monoexponential function: I/Imax = A{1 − exp[−t/]}, where
Imax is the maximal current amplitude, I is the current after a recovery period of
t,  is the time constant and A is the amplitude coefficient. Statistical analysis of
the data was provided as mean ± SEM. Group statistical significance was assessed
using Student’s t-test for comparison of two groups, and one-way ANOVA followed
by a Bonferroni post hoc test for three or more groups. P < 0.05 was considered
statistically significant.
3. Results
3.1. Effects of melamine on VGSCs in pyramid neurons
The pyramid neurons were patch-clamped in the whole-cell
configuration. The sodium currents were measured during depo-
larizing voltage steps from a holding potential of −70 mV. At 10 mV
steps, 20 ms depolarizing potentials from −90 to +50 mV activated
inward currents, which were completely and reversibly blocked
by bath application of 1 ␮M TTX. Therefore, these inward currents
were thus considered as voltage-gated sodium currents (INa ).
Melamine was tested at increasing concentrations (5 × 10−4 ,
5 × 10−5 , 5 × 10−6 g/ml), and the results showed that melamine
slightly decreased the amplitude of INa in a concentration-
dependent manner (Fig. 1A). ANOVA analysis revealed that
significant inhibition of peak amplitude was only at 5 × 10−4 g/ml,
in which melamine reduced the peak amplitude to 90.9 ± 1.1%
of control (*p < 0.05 vs. control; n = 7, Fig. 1B). It was found that
the inhibition of melamine on sodium currents was not voltage-
dependent (Fig. 1C). However, melamine at the concentrations of
5 × 10−5 and 5 × 10−4 g/ml induced hyperpolarizing shifts of peak
voltage, at which the current amplitudes reached the maximum,
to approximately −50 mV. The peak voltage in 5 × 10−6 g/ml was
−40 mV, showed that there were no evident differences compared
with the peak voltage in the control group (−40 mV).
3.2. Effects of melamine on activation kinetics of INa
The steady-state activation curves were successfully fitted with
a Boltzmann equation. Representative traces of INa were pre-
sented in Fig. 2A. The curves were shifted to the left in 5 × 10−5 ,
5 × 10−4 g/ml melamine (Fig. 2B). The values of V1/2 , at which the
conductance of INa reached half of its maximum, were shifted from
−56.32 ± 1.35 mV in control to −61.78 ± 1.26 mV in 5 × 10−5 g/ml
melamine (p < 0.05 vs. control; n = 7) and −64.13 ± 1.38 mV in
5 × 10−4 g/ml melamine (p < 0.05 vs. control; n = 7). It was found
that there was no significant difference between the values of V1/2
in 5 × 10−6 g/ml melamine (−56.04 ± 1.41 mV) and those in control
(p > 0.05; n = 7). The parameters were summarized in Table 1. There
were no effects of slope factors vs. control (all n = 7, p > 0.05).
3.3. Effects of melamine on the inactivation kinetics of INa
Effects of melamine on steady-state inactivation of INa were
examined by using a double-pulse protocol. Neurons were held
at −70 mV. Currents were elicited with a 20 ms test pulse to
−10 mV, preceded by 30 ms prepulses to potentials between
−100 and −30 mV in 5 mV increments. Representative traces
were presented in Fig. 3A. As shown in Fig. 3B, peak currents
recorded in response to the test pulse were normalized to the
maximal peak current and then fitted by the Boltzmann equa-
tion to generate inactivation curves. Melamine, at the levels
of 5 × 10−5 and 5 × 10−4 g/ml respectively, shifted the steady-
 J.-J. Yang et al. / Toxicology Letters 198 (2010) 171–176 173

Fig. 1. Effect of 5 × 10−4 , 5 × 10−5 and 5 × 10−6 g/ml melamine on the amplitude of INa current. Neurons were held at −70 mV, and current traces evoked by +30 mV. Averaged
current traces obtained from neurons in each group (A); a bar graph showing concentration–response after 5 min treatment with melamine at 5 × 10−4 , 5 × 10−5 and
5 × 10−6 g/ml of INa (n = 7, *p < 0.05 vs. control) (B); effects of different concentrations on the current–voltage relationship of INa (C).

Fig. 2. Effect of melamine of different concentrations on activation kinetics of INa . Neurons were held at −70 mV, and current of INa was generated by applying pulses from
−90 to +50 mV at 10 mV steps for 20 ms (A). The steady-state activation curves of INa . Peak amplitudes were converted into conductance by using the equation G = I/(Vm − Vr ),
normalized conductance of sodium channels was plotted against the voltages of conditioning pulses, and fitted with a Boltzmann function. (B) Each point represents
mean ± SEM (n = 7).

Table 1
Effects of melamine on the kinetic properties of VGSCs (n = 7).

Groups Parameters of activation Parameters of inactivation The time constant for the recovery  (ms)

Vh (mV) k Vh (mV) k

Control −56.32 ± 1.35 3.78 ± 1.00 –35.46 ± 1.34 8.63 ± 0.39 3.19 ± 0.17
5 × 10−6 g/ml −56.04 ± 1.41 4.03 ± 1.06 –37.07 ± 1.51 8.87 ± 0.58 3.19 ± 0.16
5 × 10−5 g/ml −61.78 ± 1.26* 3.23 ± 1.33 –38.14 ± 1.41* 8.92 ± 0.40 3.26 ± 0.19
5 × 10−4 g/ml −64.13 ± 1.38* 3.43 ± 0.92 –41.05 ± 1.53* 8.98 ± 0.41 3.21 ± 0.20

Note: Vh , the membrane potential at half-activation or half-inactivation; k, slope factor; , the time constant for the recovery from inactivation.
*
P < 0.05 vs. control.

Fig. 3. Effect of melamine of different concentrations on inactivation kinetics of INa . Neurons were held at −70 mV, and currents were elicited with a 20 ms test pulse to
−10 mV proceeded by 30 ms prepulses to potentials between −100 and −30 mV in 5 mV increments (A). Peak amplitudes for INa currents were normalized and plotted vs.
command potentials and the data were fitted with Boltzmann function. (B) Each point represents mean ± SEM (n = 7).
174 J.-J. Yang et al. / Toxicology Letters 198 (2010) 171–176
state inactivation curves in the hyperpolarizing direction. The
value of V1/2 was −35.46 ± 1.34 mV in control (n = 7), but it was
significantly changed to −41.05 ± 1.53 mV and −38.14 ± 1.41 mV
in 5 × 10−4 g/ml (p < 0.05 vs. control; n = 7) and 5 × 10−5 g/ml
melamine (p < 0.05 vs. control; n = 7), respectively. There were no
significant changes in 5 × 10−6 g/ml melamine (−37.07 ± 1.51 mV;
p > 0.05; n = 7) compared with that of control level. Whereas, k of
these INa inactivation curves was not affected by its concentrations
tested (p > 0.05).
3.4. Effects of melamine on recovery from inactivation of INa
To study the time course of recovery of sodium channels from
inactivation, we applied a double-pulse protocol as following. Cells
were held at −70 mV. A 50 ms conditioning depolarizing pulse
of −40 mV was applied to inactivate the sodium channels fully,
and then a 50 ms test pulse of −40 mV was applied after a series
of −90 mV intervals varying from 2 to 36 ms in 2 ms increments.
Fig. 4A showed representative traces. The peak value of INa evoked
by the conditioning pulse was designated as I1 , while the peak value
of INa evoked by the test pulse was designated as I2 . The ratio of I2
to I1 represents the recovery of INa from inactivation. The plot of
I2 /I1 vs. the duration of the +90 mV intervals was well fitted with
a monoexponential function: I/Imax = A + B exp(−t/), where t is the
recovery interval of the conditioning prepulse and  is the time con-
stant for the recovery from inactivation of INa (Fig. 4B). In this study,
melamine had no significant effect on . The time constants of INa
were 3.19 ± 0.17, 3.21 ± 0.19, 3.26 ± 0.19, and 3.19 ± 0.16 ms in con-
trol (n = 7), and were 5 × 10−6 , 5 × 10−5 , 5 × 10−4 g/ml melamine (all
p > 0.05 vs. control; n = 7), respectively.
3.5. Effects of melamine on action potential
To determine if the electrophysiological differences in sodium
channel properties altered neuronal excitability, we measured
action potential properties and the pattern of repetitive firing
using whole-cell current-clamp recordings. Evoked action poten-
tials were generated from a holding potential of −70 mV. Single
action potential was elicited by brief depolarizing current pulses
(5 ms, 100 pA), and repetitive firing was evoked by a prolonged
depolarizing current injection (500 ms, 50 pA). The number of
action potentials was counted, and only overshooting action poten-
tials more positive than 0 mV were included. Peak Amplitude,
overshoot, spike half-width, V-threshold of single action poten-
tial and firing rate of repetitive firing were measured before and
after drug applications (Table 2). Peak amplitude of the evoked
single action potential was only decreased at a concentration of
5 × 10−4 g/ml. V-threshold and half-width were increased in the
present of the 5 × 10−4 melamine solutions, while the firing rate of
repetitive firing was also increased in a concentration-dependent
manner (n = 6, p < 0.05, Fig. 5).
4. Discussion
In the present study, we investigated the effects of melamine on
VGSCs. Our results showed that acute melamine exposure impaired
certain electrophysiological properties of VGSCs, including neg-
ative shifting of activation and inactivation curves. Furthermore,
action potential properties and the pattern of repetitive firing are
changed as well. These findings provide important new knowledge
to understand potential effects of melamine in nervous system.
Although previous toxicological studies have demonstrated that
melamine is generally considered as having a low toxicity to mam-
mals, with a large oral LD50 of 3161 mg/kg in rats (Melnick et al.,
1984), its sequestration in tissues or cells and long-term expo-
sure to the bioenvironment can not be neglected (Gossner et al.,
2009). Our previous work indicated that melamine could impair
cell functions (Yang et al., 2010) and even notably decreased
the viability of PC12, a model in vitro for the neuron research
(Ishima et al., 2008; Xiao et al., 2008), which suggested that
melamine was able to induce neuron death. It also illustrated
that melamine increased spontaneous firing rate mainly through
inhibiting transient outward potassium current (IA ) and delayed
rectifier potassium current (IK ), which primarily contributed to the
repolarization of action potential (Yang et al., 2010).
It is well established that VGSCs underlie axonal and somatic
action potentials and actively propagate information within the
dendritic tree of pyramidal neurons (Stuart and Sakmann, 1994)
and also play a significant role in determining membrane excitabil-
ity in the CNS (Devor, 2006). Recent studies have revealed that
VGSCs consist of three subunits (expressed as a trimmer), namely,
one ␣-subunit (260 kDa) that forms the core protein of the channel
(possessing the TTX-binding sites) and two ␤-subunits (30–40 kDa)
that modify the channel function as an auxiliary subunit. The
␣-subunit consists of four repeat bridges domains (I–IV), each con-
taining six transmembrane segments (S1–S6) and one membrane
reentrant segment (SS1/SS2) connected by internal and external
polypeptide loops (Catterall, 1992; Cestele et al., 1998; Noda et al.,
1989).
In this study, acute exposure to melamine induced negative
shifts of steady-state activation (Fig. 2) which was consistent
with the descend threshold for activation (Table 2). Moreover,
melamine exposure caused a negative shift of steady-state inacti-
vation (Fig. 3), suggesting that sodium channels were shifted to the
inactivated state and prevented to recovery from the inactivated
state to the resting state. Such an effect would be or at least partially
responsible for the reduction of peak sodium current by melamine,
as shown in Fig. 1B. What’s more, these findings suggest possible
locations for the binding site of melamine on the sodium chan-
nels. In previous studies, ␤-scorpion toxins (a peptidic neurotoxin,
which specifically modulates sodium channels gating) binding to
neurotoxin receptor site 4 in the S3–S4 extracellular loop in domain
␣ of the sodium channel ␣ subunit was reported to shift the volt-
age dependence of activation to more negative potentials (Cestele
et al., 1998; Wang and Strichartz, 1983). Moreover, ␤-scorpion tox-
ins enhanced closed state inactivation, thus causing a left shift of
steady-state inactivation (Cestele et al., 1998). And our data were
consistent with the results. The coincidence between ␤-scorpion
toxins and melamine suggests that melamine may bind to a recep-
tor site in the S3–S4 loop at the extracellular end of the S4 segment
in domain II of ␣ subunit, by covalent or electrostatic interactions,
resulting in conformational changes of the channel. It is only at rel-
atively high concentrations at which melamine showed significant
effects on hippocampal neuron channels, suggesting that melamine
binds on sodium channels with relatively low affinity.
In the study, action potential properties and the pattern of repet-
itive firing were examined for further investigating the effects of
melamine. The results (in Table 2) showed that the peak amplitude,
overshoot and voltage threshold of evoked single action poten-
tial were significantly reduced at relatively high concentration
melamine. These properties were determined by the changes of
VGSCs since they generate the upstroke of action potential (AP).
Additionally, the low threshold value for action potential gener-
ation suggested that they were more readily excited than that in
normal neurons. This might due to a function of more negative
activation threshold of the INa , discussed before. The activation of
sodium channels suggests that the transition from a resting, closed
conformation to an open conformation is accompanied by the out-
ward translocation of several positive charges across the membrane
(Armstrong, 1981; Hodgkin and Huxley, 1952). The lower threshold
value and the negative shifts steady-state activation indicated that
the decreased difficulty for sodium channels to transit from closed
 J.-J. Yang et al. / Toxicology Letters 198 (2010) 171–176 175

Fig. 4. Effect of melamine of different concentrations on recovery from inactivation of INa . The currents were obtained as followed protocol: cells were held at −70 mV, a
50 ms conditioning depolarizing pulse of −40 mV was applied to inactivate the sodium channels fully, and then a 50 ms test pulse of −40 mV was applied after a series of
−90 mV intervals varying from 2 to 36 ms in 2 ms increments (A). The peak value of INa evoked by the conditioning pulse was designated as Imax , while the peak value of INa
evoked by the test pulse was designated as I. The ratio of I to Imax represented the recovery of INa from inactivation (B). Each point represents mean ± SEM (n = 7).

Table 2
Effects of melamine on properties of action potentials (n = 7).

Groups Number of APs Peak amplitude (mV) Overshoot (mV) Spike half-width (ms) V-threshold (mV)

Control 10.13 ± 0.52 106.62 ± 3.59 36.62 ± 2.58 2.28 ± 0.52 −44.72 ± 3.10
5 × 10−6 g/ml 10.66 ± 0.35 105.84 ± 3.33 35.84 ± 3.21 2.58 ± 0.72 −47.91 ± 2.34
5 × 10−5 g/ml 11.77 ± 0.47* 100.64 ± 4.21 30.60 ± 3.32* 2.69 ± 0.34* −48.93 ± 3.45
5 × 10−4 g/ml 12.88 ± 0.64** 93.47 ± 3.39* 25.47 ± 2.92** 2.94 ± 0.64* −54.91 ± 3.65**
*
P < 0.05 vs. control.
**
P < 0.01 vs. control.

to open conformation, which means that the IIS4 voltage sensors
became easier to move outward, thus enhance the sensitivity of
the voltage dependence negatively (Stuhmer et al., 1989; Yang et
al., 1997).
Meanwhile, The AP firing rate was increased significantly
(Fig. 5B), which were consistent with our previous report that
melamine increased the spontaneous firing rates through inhibit-
ing voltage-gated K+ currents (Liu et al., 2007). And the duration of
action potentials was increased as well. It is well known that the
properties of action potential are also dependent on the K+ currents
and other repolarization currents (Golod et al., 1998; Lin, 1997).
Therefore, VGSCs may be one of attacking sites of melamine, and
the effects of melamine on action potential may, at least in part, be
due to the changes of kinetic of VGSCs.
In humans, VGSCs, especially Nav1.3, Nav1.7, Nav1.8, and
Nav1.9, may play an important role in many nervous system dis-
eases, and numerous publications have attempted to elucidate the
mechanism of these channels. In addition, these channels have been

Fig. 5. Effect of melamine of different concentrations on action potential and action potential firing rate. In the current-clamp mode, neurons were held at −70 mV, and single
action potentials were elicited using a 10 ms depolarizing current pulse before and after application of melamine (A). To observe the effect of melamine on action potential
firing rate, a long-term depolarizing current (500 ms, 50 pA) was given to the neurons (B).
176 J.-J. Yang et al. / Toxicology Letters 198 (2010) 171–176
considered as possible molecular targets. In our present study, the
impairment of VGSCs by melamine was substantiated. It is well
known that voltage-gated sodium channels play a role in neu-
rotransmitter release (Sitte et al., 2004), therefore, the effects of
melamine mean that modulation of VGSCs may be able to pro-
duce functional dysfunction at message transmission in CNS. Action
potentials are a fundamental property of excitable cells in the
mammalian CNS, and modification of VGSCs could influence their
generation, the propagation to synapse terminals, or the local
depolarization of neuron. Results obtained from previous studies
supported the idea that VGSCs and action potential were involved
in Alzheimer’s disease (AD), epilepsy, Parkinson disorder and injury
in CNS neurons (Fritch et al., 2009; Janszky, 2009; Mashimo et al.,
2010; Ogiwara et al., 2009).
The modulation of VGSCs in hippocampal neurons may be one
aspects of neurotoxicity of melamine. However, the detailed mech-
anisms are still unknown, and further investigations are needed.
Although the mechanisms by which impaired the functional prop-
erties of sodium channels remain a matter of conjecture, our results
provide new insights into melamine toxicology and present the
potential risks of melamine application, for environmental usage.
Conflict of interest
None.
Acknowledgements
This work was supported by grants from the National Natu-
ral Science Foundation of China (30870827) and the “111” Project
(B08011).
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