Toxicology Letters 268 (2017) 58–62

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

Full Length Article

Toxic effects of zinc ions on kinesin – Potential molecular cause of

impaired intracellular transport
Konrad J. Böhm
Leibniz Institute on Aging – Fritz Lipmann Institute, Beutenbergstraße 11, D-07745 Jena, Germany

H I G H L I G H T S G R A P H I C A L A B S T R A C T

 Zinc ions are able to inhibit kinesin

ATPase and kinesin-based motility
generation.
 Zinc-induced inhibition depends on
the zinc/magnesium ion ratio.
 Zinc-induced inhibition depends on
temperature.
 Zinc-induced inhibition might be one
molecular cause of impaired intra-
cellular transport.

A R T I C L E I N F O A B S T R A C T

Article history:
Received 21 September 2016 In healthy organisms the metabolism of the trace element zinc is well balanced. If this balance becomes
Received in revised form 18 January 2017 destroyed the free zinc level might increase and cause toxic effects. The present study demonstrates that
Accepted 19 January 2017 under definite conditions zinc ions are able to inhibit the ATPase activity of neuron-specific KIF5A
Available online 22 January 2017 (kinesin-1). Correspondingly, the motility activity of KIF5A also decreased. The inhibition rates have been
found to depend on the magnesium ion concentration. Lowering the magnesium concentration weakens
Keywords: the inhibition. In addition, also decreases of temperature or increasing the ATP concentration result in
Zinc ions reduced inhibition. Zinc ion-mediated inhibition of KIF5A activity might be one molecular cause
Kinesin
contributing to impaired transport processes within brain and other organs in cases of zinc
Microtubules
dyshomeostasis.
ATPase
Motility © 2017 Elsevier B.V. All rights reserved.

1. Introduction concentration of zinc ions is well balanced. It is known that

Zinc is the second most abundant transition metal in the body
behind iron (Canzoniero et al., 1999). Being an essential trace
element for most living organisms, it is crucially involved in
various physiological events such as mitotic cell division or protein
and DNA synthesis (Kawahara et al., 2014). Zinc ions also play an
important role in the transmission of nerve impulses, in the
regulation of enzymatic activity, and in protein stabilization
(McCord and Aizenman, 2014). In healthy organisms the
E-mail address: kjboehm@gmx.de (K.J. Böhm).
stressful conditions like oxidative stress, heavy metal load or thiol-
modifying substances are able to disturb the intracellular zinc
homeostasis leading to increased concentrations of free zinc
within the cytoplasm (Kroncke, 2007; McCord and Aizenman,
2014). Increased cellular zinc levels have been found to be toxic
(Cuajungco and Lees, 1996; Frederickson et al., 2005; Sensi et al.,
2009) and can cause several diseases, including neuronal injury in
cases of cerebral ischemia, epilepsy, or brain trauma (Capasso et al.,
2005). Recently, the pathogenesis of some neuronal disorders like
Alzheimer's disease has been discussed in relation with zinc
dyshomeostasis (Kawahara et al., 2014; Li and Wang, 2016; McCord
and Aizenman, 2014; Mizuno and Kawahara, 2013).

http://dx.doi.org/10.1016/j.toxlet.2017.01.013
0378-4274/© 2017 Elsevier B.V. All rights reserved.
 K.J. Böhm / Toxicology Letters 268 (2017) 58–62 59

So far, the molecular mechanisms underlying the zinc-induced
development of diseases and the impairment of cellular processes
have not been elucidated sufficiently.
Numerous intracellular transport events are based on the
kinesin-based motility-generating system. So, it seems to be well
established that KIF5 motors are causally involved in the transport
of lysosomes, synaptic vesicles precursors, synaptic membrane
precursors, diverse other vesicles and of mitochondria (Hirokawa
et al., 2009). Interestingly, zinc ions have been reported to inhibit
the mitochondrial transport within neurons, whereby this effect
was related to phosphatidylinositol 3-kinase activation
(Malaiyandi et al., 2005). It has been well established that
mitochondria in neurons are driven along microtubule rails by
KIF5. Within the past decade, we reported that aluminum (Bohm
et al., 2015), lead (Bonacker et al., 2005) and some transition metal
ions like, mercury (Bonacker et al., 2004), cadmium (Böhm, 2014),
and copper (Böhm, 2015) are able to inhibit the KIF5A ATPase and
motility activity, suggesting that these cations are powerful to
impair kinesin-dependent cellular transport events. The question
arises whether zinc ions are also potent to affect KIF5A and
whether direct attacks of zinc ions on the kinesin might be a
further molecular cause of disturbances of the transport of
mitochondria and/or other organelles. To answer this question
the ATPase activity of human neuron-specific KIF5A, which
belongs to the kinesin-1 group, was measured. The results, which
were complemented by motility data, might indicate that the
direct interaction of zinc ions with kinesin could be another
potential molecular cause of failed intracellular transport
processes, which might underlie neuronal diseases.
2. Material and methods
The KIF5A (Niclas et al., 1994) was expressed in E. coli as
truncated tag-free construct including the N-terminal amino acids
1–560 and purified as described formerly (Kalchishkova and Böhm,
2008). Like the complete KIF5A (consisting of 1032 amino acids),
this construct forms dimers and has been proved to be functionally
intact concerning its ATPase activity and its ability to bind to and to
transport cargoes in cell-free environment (Dreblow et al., 2010;
Kalchishkova and Böhm, 2008).
The ATPase activity has been determined by measuring the
amount of inorganic phosphate released from ATP by the kinesin
(Bohm et al., 2015) at the following standard conditions: 55 nM
KIF5A560, 1.5 mM tubulin (added as fragmented paclitaxel-stabi-
lised microtubules), 1.25 mM ATP, 2.5 mM MgCl2, 50 mM Pipes, pH
6.8, 37  C. Corresponding experimental variations from these
conditions are indicated in the legends to the figures. Motility
activity has been determined using the conventional so-called
gliding assay, in which the velocity of microtubules moving across
a kinesin-coated glass surface is measured under microscopic
control (Bohm et al., 2015).
The zinc chloride (Sigma-Aldrich (99.999% trace metals basis)
used in this study was added from a 10 mM stock solution in
50 mM Pipes buffer (pH 6.8).
3. Results and discussion
Zinc ions were found to inhibit the ATPase activity in
concentration-dependent manner with an IC50 of 42.7 mM  3.1
mM at standard condition (Fig. 1a). Remarkably, the inhibition
strength was observed to depend strongly on the magnesium ion

Fig. 1. Zinc ion-caused inhibition of the kinesin activity.

(a) Dependence of ATPase activity on zinc chloride concentration at standard conditions. Mean values with SD, calculated from six independent measurements. (b) ATPase
activities (given as concentration of inorganic phosphate released within 30 min incubation in a constant volume) at 0.3 and 2.1 mM MgCl2. Insert: Dependence of the IC50
values on magnesium ions. (c) Rate of zinc ion-induced inhibition dependent on magnesium ion concentration. (d) Kinesin-mediated motility in the presence of zinc ions. The
data are based on the measurement of the velocity of microtubules gliding across a glass surface covered by KIF5A560. 1.14 mM KIF5A560, 0.4 mM tubulin. Further experimental
details are described formerly (Bohm et al., 2015). Insert: Percent inhibition. The velocities determined in the absence of zinc ions (1132 nm/s  149 nm/s at 4.0 mM MgCl2 and
1178 nm/s  141 nm/s at 0.5 mM MgCl2, respectively) were set as 0% inhibition. Mean values with SD, calculated from two independent measurements.
60 K.J. Böhm / Toxicology Letters 268 (2017) 58–62

concentration (Fig. 1b): In the absence of zinc the ATP hydrolysis

rate at 0.3 mM MgCl2 was by a factor of 2 lower than that one at
2.1 mM. But, at zinc concentrations above 100 mM the ATP
hydrolysis rate at 0.3 mM MgCl2 exceeded that one measured at
2.1 mM (Fig. 1b) and with increasing the zinc concentration up to
300 mM there was been practically no inhibition. Only above
300 mM zinc caused a relative slight decrease of the ATP hydrolysis
rate (Fig. 1b). Correspondingly, the IC50 values were found to vary
from 26 mM (at 4 mM MgCl2) to about 515 mM (at 0.01 mM MgCl2)
(Fig. 1b inset). Moreover, ATPase measurements at constant 50 mM
zinc chloride showed a linear dependence of inhibition on the
magnesium ion concentration (Fig. 1c).
The motility assay supported the results obtained from the
ATPase measurements. In the presence of zinc ions the velocity of
gliding microtubules decreased in concentration-dependent
manner, whereby the effect of zinc was also dependent on
magnesium (Fig. 1d). At 0.5 mM and at 4.0 mM MgCl2 IC50 values of
310 mM  50 mM and 25.9 mM  1.2 mM, respectively, were calcu-
lated (Fig. 1d, insert).
The zinc ion-induced inhibition of ATPase activity of kinesin
was significantly weakened by increasing the ATP concentration in
the assay (Fig. 2a). Elevation of the ATP concentration by factor 2
was found to result in a twofold IC50 increase. Consistently, the
percent inhibition (measured at 50 mM ZnCl2) decreased expo-
nentially with increasing the ATP concentration (Fig. 2b). ATP
concentration-dependent measurements of the ATPase activity in
the presence of 0 and 50 mM ZnCl2 the revealed nearly equal vmax
values, but different Km values of 0.3 and 1.0 mM, respectively
(Fig. 2c). It is known that ATP binding to kinesin is accompanied by
conformational changes in the motor domain of kinesin (Chang
et al., 2013). Considering this, it might be suggested that nucleoside
triphosphate binding causes structural alterations protecting the
motor domain against metal ions. However, it has to be also taken
into account that zinc ions are able to form complexes with ATP
(Krezel and Maret, 2016). As a result, the effective zinc
concentration in the assay mixture and, consequently, the
inhibition strength should be lowered. This explanation is
consistent with the IC50 shift towards higher values (Fig. 2a).
Furthermore, it is commonly accepted that the substrate for
kinesin is a magnesium-ATP complex (Cohn et al., 1989). But,
kinesin can also use some other divalent cations as substrate
cofactor to generate activity (Böhm et al., 1997). The higher Km
values measured at 2.5 mM ATP might result from increasing
formation of zinc-ATP and its binding to kinesin with lower affinity.
The usage of zinc-ATP as substrate could also explain the more or
less insensibility of kinesin to zinc ions under conditions of
magnesium ion lack (see Fig. 1b, curve for 0.3 mM Mg2+). Fig. 2. Dependence of zinc-induced inhibition on the ATP concentration.
(a) Inhibition curves at 1.25 mM and 2.50 mM ATP with IC50 values 26.1 mM  1.9
Cysteine residues play a central role in zinc binding to proteins mM and 53.5 mM  4.4 mM, respectively. (b) ATP dependence of inhibition
(Pace and Weerapana, 2014). The human KIF5A (NCBI Reference (calculated by dividing the ATPase activity measured in the presence of 50 mM
Sequence NP_004975.2), used in this study, comprises seven ZnCl2 at a given ATP concentration by activity measured in the absence of ZnCl2 at
cysteine residues in its motor domain, which is responsible for the corresponding concentration) at 2.1 mM MgCl2. (c) ATP dependence of the
ATPase activity at 0 and 50 mM ZnCl2.
both hydrolysing the ATP and microtubule binding. It is commonly
known that dithiothreitol (DTT) protects the thiol groups of
proteins from oxidation. But DTT itself represents an effective zinc-
binding agent, which might reduce zinc-caused inhibition effects Beside this, the experiments with DTT and cysteine might also
(Maret et al., 1999). The kinesin inhibition curves and correspond- indicate that the sulfhydryl groups in the motor domain are not
ingly the IC50 values recorded in the presence of DTT at standard accessible for zinc ions or they do not necessarily participate in ATP
condition were very close to those of the control without additive hydrolysis.
(Fig. 3a). This might be interpreted as meaning that oxidation of Microtubule binding to kinesin is known to stimulate its ATPase
the thiol groups does not play a significant role in kinesin ATPase activity (Hackney, 1994). Therefore, the zinc ion-induced inhibition
inhibition and/or under the experimental conditions of the present of KIF5A ATPase activity might be due to microtubule destruction.
study DTT does not lower the effective zinc ion concentration. But, transmission electron microscopy revealed that zinc ions had
Unlike DTT, the addition of L-cysteine significantly weakened the no visible effect on the microtubules in the standard assay
inhibitory effect (Fig. 3a). This might be due to complexation of mixtures at least up to 500 mM (result not shown). Within this
zinc ions with the free amino acid, as a result of which the effective context, it has to be emphasized that for both the ATPase and the
zinc ion concentration exerting ATPase inhibition is lowered. gliding assay the microtubules were stabilised by paclitaxel, which
 K.J. Böhm / Toxicology Letters 268 (2017) 58–62
Fig. 3. (a) Zinc-induced inhibition in the presence of DTT and L-cysteine. Standard
conditions with corresponding additives. IC50 values: no additive 34.5  2.3 mM;
1 mM DTT 36.2  0.2 mM; 1 mM L-cysteine 77.6  9.9 mM, respectively. (b) Depen-
dence of the inhibition, caused by 50 mM ZnCl2, on the amount of microtubules.
Standard conditions with varying microtubule concentration.
counteracts disassembly processes caused by numerous noxae, e.g.
calcium ions.
Another reason of decreased ATPase activity might be
disturbances in kinesin-microtubule binding. The critical micro-
tubule-interacting residues of kinesin are primarily positively
charged, which is consistent with a primarily electrostatic
interaction of kinesin with the negatively charged microtubule
surface (Tucker and Goldstein, 1997; Woehlke et al., 1997). So, it
might be possible that zinc ions prevent kinesin binding to the
microtubule surface by neutralising the negative charges. It was
observed that elevated microtubule concentrations in the standard
ATPase assay weakened the zinc ion-induced inhibition, which
seems to support such mechanism (Fig. 3b). In addition, the
motility assays performed under microscopic control (AVEC DIC
mode) displayed a trend towards a decreased kinesin-microtubule
binding caused by zinc ions. On the other hand, it should be taken
into account that porcine brain tubulin also contains cysteine
residues (alpha tubulin 12 and beta tubulin 8, respectively) (Krauhs
et al., 1981; Ponstingl et al., 1981) that might sequester zinc ions.
The temperature dependence of the ATPase activity of kinesin-
1, purified from porcine brain, had an optimum around 37  C
(Böhm et al., 2000). A very similar temperature curve revealed the
human KIF5A, used in this study (Fig. 4a). Strikingly, in the
presence of zinc ions the activity optimum was shifted to about
32  C (Fig. 4a). When the data points of the corresponding
inhibition-temperature plot were fitted by linear functions within
the ranges 22–30  C and 31–39  C, two distinct slopes with an
intersection point at 29.6  C became evident (Fig. 4b). The different
sensitivity of kinesin to zinc might be due to a temperature-caused
conformational change. Such a change has been already suggested
61
Fig. 4. Dependence of zinc-induced inhibition on temperature.
(a) Temperature dependence of the ATPase activity (concentration of inorganic
phosphate released within 30 min incubation in a given volume) at 0 and 250 mM
ZnCl2. (b) Inhibition (calculated by dividing the ATPase activity measured in the
presence of 250 mM ZnCl2 at a given temperature by the ATPase activity measured
in the absence of ZnCl2 at the corresponding temperature) plotted against
temperature.
on the basis of Arrhenius plots determined from temperature-
dependent ATPase activity and motility measurements performed
with purified kinesin-1 (Böhm et al., 2000).
3.1. Concluding discussion
Zinc is an essential trace element required to maintain quite
different life processes. In healthy organisms, the cellular amount
of zinc ions is well balanced. However, recently an increasing
number of reports have been published in which especially
neuronal diseases are casually related to elevated levels of metal
ions, including zinc (McCord and Aizenman, 2014; Sensi et al.,
2011).
Using a cell-free experimental approach, including neuronal
KIF5A, isolated microtubules, magnesium ions and ATP, zinc ions
were shown to inhibit both the kinesin ATPase and motility
activity. Dependent on concrete conditions, IC50 values down to
26 mM (Fig. 2a) were determined. Within this context, the
physiological relevance of such relatively high values has to be
discussed. Human cells contain 200–300 mM zinc in total (Maret,
2015). For brain, a value of about 150 mM was given (Sensi et al.,
2011). Most of the cation is bound to proteins or concentrated
within subcellular compartments, e.g., synaptic vesicles. The
concentration of free zinc ions in neuronal cells is in the picomolar
range (Maret, 2015). However, e.g., in cases of oxidative stress,
bound intracellular zinc can be released into the cytosol (McCord
and Aizenman, 2014). On the one hand, it seems to be questionable
62 K.J. Böhm / Toxicology Letters 268 (2017) 58–62
if under such circumstances the cytosolic zinc ion concentration
can reach micromolar values on average. On the other hand, it
cannot be excluded that in certain cytoplasmic space, around the
kinesin-based transport machinery, the free zinc level is spatially
and/or temporarily raised.
Moreover, the data presented in this study were obtained from
a simplified molecular two-protein model in artificial environment
with ATP and magnesium ions, which themselves were shown to
modulate zinc-induced inhibition. Beside this, all measurements
were performed in 50 mM Pipes buffer. As Pipes is known to form
complexes with zinc ions (Wyrzykowski et al., 2014), the effective
zinc concentration causing kinesin inhibition should be signifi-
cantly lower than determined. Accordingly, a single experiment
with reduced Pipes (15 mM) and 5 mM MgCl2, revealed an
inhibition with an IC50 of about 13 mM zinc.
Regardless the uncertainty concerning the physiological
parameters (among them the concentrations of free zinc, kinesin,
microtubules, magnesium) within cells, the present study provides
first evidence that zinc ions are able to inhibit kinesin ATPase and
kinesin-driven motility, which might cause a breakdown of the
kinesin-dependent transport system and finally initiate disease.
Acknowledgement
The author is very grateful to Mrs Marina Wollmann for her
skilful excellent assistance in technical performing the experi-
ments described in this study.
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