Pharmacology & Therapeutics 111 (2006) 567 – 583

www.elsevier.com/locate/pharmthera

Associate editor: A.G. Ramage

Zinc and copper: Pharmacological probes and endogenous modulators of

neuronal excitability
Alistair Mathie a,*, Gemma L. Sutton a, Catherine E. Clarke b, Emma L. Veale a
a
Biophysics Section, Blackett Laboratory, Division of Cell and Molecular Biology, Imperial College London, Exhibition Road, London SW7 2AZ, United Kingdom
b
St. Vincent’s Clinical School, University of New South Wales, and Victor Chang Cardiac Research Institute, Sydney, NSW 2010, Australia

Abstract

As well as being key structural components of many proteins, increasing evidence suggests that zinc and copper ions function as signaling
molecules in the nervous system and are released from the synaptic terminals of certain neurons. In this review, we consider the actions of these
two ions on proteins that regulate neuronal excitability.
In addition to the established actions of zinc, and to a lesser degree copper, on excitatory and inhibitory ligand-gated ion channels, we show
that both ions have a number of actions on selected members of the voltage-gated-like ion channel superfamily. For example, zinc is a much more
effective blocker of one subtype of tetrodotoxin (TTX)-insensitive sodium (Na+) channel (NaV1.5) than other Na+ channels, whereas a certain T-
type calcium (Ca2+) channel subunit (CaV3.2) is particularly sensitive to zinc. For potassium (K+) channels, zinc can have profound effects on the
gating of certain KV channels whereas zinc and copper have distinct actions on closely related members of the 2 pore domain potassium channel
(K2P) channel family. In addition to direct actions on these proteins, zinc is able to permeate a number of membrane proteins such as (S)-alpha-
amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate receptors, Ca2+ channels and some transient receptor potential (trp)
channels.
There are a number of important physiological and pathophysiological consequences of these many actions of zinc and copper on membrane
proteins, in terms of regulation of neuronal excitability and neurotoxicity. Furthermore, the concentration of free zinc and copper either in the
synaptic cleft or neuronal cytoplasm may contribute to the etiology of certain disease states such as Alzheimer’s disease (AD) and epilepsy.
D 2005 Elsevier Inc. All rights reserved.

Keywords: Zinc; Copper; Voltage-gated-like ion channels; Neuronal excitability; Epilepsy; Alzheimer’s disease

Abbreviations: Ah, amyloid-h protein; AD, Alzheimer’s disease; AMPA, (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; Ca2+, calcium; CNS,
central nervous system; CSF, cerebrospinal fluid; GABA, g-aminobutyric acid; IA, transient potassium current; K+, potassium; K2P, 2 pore domain potassium
channel; Na+, sodium; NMDA, N-methyl-d-aspartate; SOCC, store-operated calcium channel; SOD, superoxide dismutase; trp, transient receptor potential; TTX,
tetrodotoxin; ZEN, zinc-enriched neurons.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
2. Zinc and copper in the brain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
3. Protein targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
3.1. How do zinc and copper alter the function of proteins? . . . . . . . . . . . . . . . . 570
3.2. Ligand-gated ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
3.2.1. Glutamate receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
3.2.2. g-Aminobutyric acidA receptors. . . . . . . . . . . . . . . . . . . . . . . . 571
3.2.3. P2X receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571

* Corresponding author. Tel.: +44 20 7594 7691; fax: +44 20 7589 0191.
E-mail address: a.mathie@imperial.ac.uk (A. Mathie).

0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.pharmthera.2005.11.004
568 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583

3.3. Voltage-gated-like ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571

3.3.1. Sodium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
3.3.2. Potassium channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573
3.3.3. Calcium channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575
3.3.4. Store-operated calcium channels and transient receptor potential channels . . 576
4. Physiological consequences of zinc and copper actions . . . . . . . . . . . . . . . . . . . . 577
4.1. Neuronal excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
4.2. Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
5. Clinical consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
5.1. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578
5.2. Epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

1. Introduction remains protein bound. The binding of zinc to l-histidine in

Zinc and copper ions are of key physiological importance in
mammalian tissue. Zinc is a vital nutrient and with the
exception of iron, it is the most abundant trace element in
the body (see Takeda, 2001). Copper is also a vital trace
element, the third most abundant in humans, and is present at
low levels in a variety of cells and tissues with the highest
concentrations in the liver (Gaetke & Chow, 2003). Both ions
have a range of physiologically important roles in humans.
They are a key structural component of many proteins and act
as co-factors for the activity of many enzymes that are critical
for brain function. These include enzymes involved in
antioxidant defense (superoxide dismutase; SOD) cellular
respiration (cytochrome c oxidase) and catecholamine synthe-
sis (dopamine-h-hydroxylase) and a plethora of other enzymes
involved in multiple biological processes required for growth,
development, and maintenance of the nervous system (Gaetke
& Chow, 2003; Barnes et al., 2005, see Frederickson et al.,
2005).
There are increasingly compelling arguments that both
ions can also function as signaling molecules, with evidence
of release from synaptic terminals and measured actions on a
wide range of membrane proteins (see Frederickson et al.,
2005). As such, in the nervous system, both ions might be
predicted to have modulatory roles in regulating neuronal
excitability. In this review, we will consider the roles of zinc
and copper ions in the nervous system, both in terms of their
physiological actions and in terms of their potential use as
pharmacological mediators of proteins that regulate neuronal
excitability.
2. Zinc and copper in the brain
In the plasma, zinc and copper are present at concentra-
tions of around 15 AM. For zinc, almost all of this is bound to
proteins such as albumin, hence free, ionic, zinc is in the
nanomolar range (see Takeda, 2001). The brain has the
highest zinc content compared to other organs (see Mocche-
giani et al., 2005) about 10-fold higher than that found in
plasma (around 100– 150 AM). This has been localized to
several specific brain regions; however, the vast majority
the plasma and cerebrospinal fluid (CSF) promotes transport
of zinc to target sites, from where its uptake across the
blood – brain barrier is tightly regulated. Following uptake,
passage of zinc through the CSF and brain extracellular fluid
compartments is unrestricted. Entry routes into glia and
neurons are still to be clarified, although several zinc
transporters have been identified (see below; Mocchegiani et
al., 2005). Even within the brain, 90% of total brain zinc is
bound to zinc metalloproteins, with much of the remaining
10% found in presynaptic vesicles, either loosely bound or
free (and therefore, histochemically reactive) (see Takeda,
2001). Indeed, it is known that free ionic zinc is a potent
killer of neurons and glia, with prolonged exposure to growth
media containing in excess of 100 nM leading to cell death
(see Frederickson et al., 2005).
Like zinc, copper accumulates in the brain. The average
copper concentration in the CSF has been estimated to be
around 70 AM. While, like zinc, most of this is protein bound,
loosely bound copper is estimated at around 0.1 –0.8 AM,
whereas the normal extracellular copper concentration in the
brain is of the order of 0.2 –1.7 AM (Gutteridge, 1984; Kardos
et al., 1989; Linder & Hazegh-Azam, 1996; Stuerenberg, 2000;
Schumann et al., 2002; White et al., 2004). However, these
values are frequently exceeded in the synaptic cleft and during
neurodegenerative disease where concentrations of copper may
reach 200 AM and 400 AM, respectively (White & Cappai,
2003).
Zinc is not uniformly distributed about the brain. Higher
concentrations are present in the grey than white matter, while
the highest concentrations are located in specific forebrain
regions including the hippocampus, amygdala and neocortex
(Slomianka et al., 1990, see Frederickson & Moncrieff, 1994;
Takeda, 2000). Histochemically stainable zinc (free plus
loosely bound zinc) is found in particularly high concentrations
in certain synaptic vesicles. Neurons that contain such vesicles
have been termed zinc-enriched neurons (ZEN). It is clear that
these neurons are not associated with a single ‘‘primary’’
synaptic neurotransmitter. For example, GABAergic ZEN
terminals have been found in the cerebellum (Wang et al.,
2002), whereas in the cerebral cortex, amygdalar nuclei,
olfactory bulb, and hippocampal formation, ZEN terminals
 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583 569

are glutamatergic (see Frederickson & Bush, 2001). This has
given rise to the term ‘‘gluzinergic’’ neurons, used by some.
The hippocampus is particularly rich in gluzinergic neurons in
regions such as the dentate gyrus, granule cell mossy fibers,
and in CA3 and CA1 neurons. Zinc-containing fibers from
hippocampal region innervate the cerebral cortex, amygdala,
striatum, or limbic regions (see Mocchegiani et al., 2005).
Again, just like zinc, copper is found to have a differential
distribution in the central nervous system (CNS) with certain
synaptic vesicles showing particularly high levels of copper.
Copper is also primarily associated with glutaminergic or
adrenergic neurons, particularly in regions such as the hippo-
campus, olfactory bulb, and locus coeruleus (Sato et al., 1994;
Ono & Cherian, 1999; see also Kardos et al., 1989). For now, at
least, we have been spared the term ‘‘glucupergic’’ neurons.
Giant boutons of hippocampal mossy fibers contain ¨ 300 –
350 AM of zinc (see Takeda, 2001). Release of zinc occurs
from presynaptic, small clear round vesicles within neurons
(see Frederickson et al., 2000). Pools of zinc and copper can be
released following membrane depolarization or neural activity
in a calcium (Ca2+)-dependent manner (Assaf & Chung, 1984;
Howell et al., 1984; Hartter & Barnea, 1988; Kardos et al.,
1989; Horning & Trombley, 2001). Atomic absorption
spectroscopy has also demonstrated the evoked release of zinc
from hippocampal mossy fibers (Charton et al., 1985).
A recent review by Frederickson et al. (2005) has illustrated
how 3 primary methods have been used to show synaptic
release of zinc. These are imaging of zinc in boutons to show
depletion following nerve stimulation; detection of zinc in the
perfusate following nerve stimulation; and direct imaging of
released zinc using fluorescent probes.
Of the 3 methods, it is clear that the third method is the most
direct, the fastest, and the most informative. For adult
preparations at least, fluorescent zinc probes reveal clear
increases in synaptic zinc following nerve stimulation and
estimates of concentration in the range 10 – 30 AM (e.g.
Thompson et al., 2002; Li et al., 2003). Not all studies,
however, have been able to demonstrate clear evidence of
synaptic release of zinc (Kay, 2003). So, at present, this issue is
not fully resolved.
In addition to synaptic release from neurons, there are a
number of other zinc-secreting cells in the CNS. Zinc can be
secreted from brain capillary endothelial cells and choroidal
epithelial cells to brain extracellular fluid and the CSF,
although the mechanisms underlying this are unclear at present
(see Takeda, 2001). Both zinc and copper are essential for
correct development and functioning of the brain. Furthermore,
both zinc and copper levels in the immature brain increase with
age until adulthood when a constant concentration is main-
tained (Tarohda et al., 2004, see Fig. 1).
When zinc concentrations rise inside either neurons or glial
cells, the ions can be expelled from the cell or else concentrated
in synaptic vesicles by ZnT transporters, taken up by
mitochondria or incorporated into zinc binding proteins. A
family of zinc transporters are involved in transporting zinc
across cell membranes (Colvin et al., 2003). In terms of the
nervous system, perhaps the most interesting of these are ZnT3
and ZnT4, which are required for the transport of zinc into
synaptic vesicles. The distribution of ZnT3, in particular, has
been widely studied and its presence is taken to be evidence in
favor of synaptic release of zinc in such neurons (see Harris,
2002).

30 mm
Copper

Zinc

1 3 5 7 14 21 42 77 147 days

Low High
Fig. 1. Regional distribution map of copper and zinc in the brain of rats at various developmental stages. For each ion the maximum concentration observed is in red
and the minimum (0 ppm) in blue. For zinc the maximum level observed was 41.9 Ag g 1, whereas for copper it was 3.95 Ag g 1. The different regions of the brain
(top panel) are separated as follows: (A) visual cortex, (B) somatosensory cortex, (C) motor cortex, (D) granular insular cortex, (E) piriform cortex, (F) olfactory
bulb, (G) putamen (striatum), (H) olfactory tubercle, (I) corpus callosum, (J) external cortex of inferior colic, (K) hippocampus, (L) substantia nigra, (M) internal
capsule cerebral peduncle, (N) thalamic nucleus, (O) amygdaloid area, (P) globus pallidus, (Q) thalamic nucleus, (R) mesencephalic nucleus. Adapted from Tarohda
et al. (2004) with kind permission of Springer Science and Business Media.
570 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
While relatively little is currently known about copper
transport in the CNS, copper-transporting ATPases (ATP7A
and ATP7B) play a central role in distribution of copper (Puig
& Thiele, 2002). Genetic mutations in ATP7A and 7B lead to
severe neurodegenerative disorders, Menkes and Wilson
diseases, respectively (Strausak et al., 2001). These 2 proteins
are distributed in a cell-specific manner in accordance with
their distinct functional properties. Furthermore, a recent
study by Barnes et al. (2005) demonstrated that cerebellar
expression of the 2 copper ATPases is regulated individually
during development. ATP7B is constantly localized to
Purkinje neurons, allowing steady transport of copper to
ceruloplasmin. However, the expression profile of ATP7A is
variable such that during early development expression occurs
in Purkinje neurons, while during late development and into
adult life expression switches to Bergmann glia. Current data
indicate that ATP7A is responsible for the overall supply of
copper to the brain due to its presence in the choroid plexus
(Barnes et al., 2005). Intracellular copper homeostasis is
mediated by copper-induced trafficking to the plasma
membrane (ATP7A) or to vesicles (ATP7B) followed by
expulsion of excess copper from cells (Llanos & Mercer,
2002).
3. Protein targets
3.1. How do zinc and copper alter the function of proteins?
Zinc binds directly to proteins to alter their function. It
particularly targets histidine, cysteine, aspartate, and glutamic
acid residues. Unlike the situation for zinc, there are several
ways in which copper can modify the activity of a protein.
Copper is therefore potentially much more complicated than
zinc and perhaps much more interesting, at least from a
mechanistic perspective. Like zinc, it may bind directly to an
amino acid (again most likely cysteine, histidine or glutamic
acid residues) to alter protein function. However, because it is a
redox metal a second action may be to bind to cysteine residues
and oxidize them. This may catalyse the formation of
disulphide bonds between physically adjacent cysteine residues
thereby changing protein function. A third more indirect way
that copper can modulate protein function is through the
generation of free radicals, which can profoundly alter protein
and cell function (see Section 4).
It is beyond the scope of this review to detail all the effects
of these 2 ions on all membrane proteins. Instead we will focus
on the actions of these ions on proteins that are of particular
relevance for the control of neuronal excitability and/or where
zinc and copper may serve as useful pharmacological probes in
the absence of other more selective compounds. As such, we
will focus on the actions of these compounds on neuronal ion
channels; members of the voltage-gated-like ion channel
superfamily (see Yu & Catterall, 2004); in particular sodium
(Na+), potassium (K+) and calcium channels. Before doing so,
however, it is important to summarize the actions of these ions
on members of the ligand-gated ion channel superfamily, in
particular glutamate, GABAA, and purinergic P2X receptors
because much work has suggested that members of these
protein families are significant synaptic targets for zinc and
copper, with resultant consequences for synaptic transmission.
3.2. Ligand-gated ion channels
The most well-studied membrane proteins regulated by zinc
(and to a lesser extent copper) are the receptors for fast
excitatory (glutamate) and fast inhibitory (g-aminobutyric acid;
GABA) transmission in the CNS. Many of the details of these
regulations have been worked out over the last few years (see
Smart et al., 2004) to the extent that, for some receptor
subunits, the exact amino acids zinc binds to, have been
identified (e.g., Hosie et al., 2003). There is strong evidence
from studies in hippocampal mossy fibers that synaptically
released zinc can modulate both excitatory and inhibitory
synaptic transmission under physiological conditions (Vogt et
al., 2000; Ruiz et al., 2004).
3.2.1. Glutamate receptors
Excitatory amino acid receptors are the mediators of synaptic
transmission at many synapses that can undergo use-dependent
modifications of synaptic efficiency. Ionotropic excitatory
amino acid receptors can be divided into 2 large families, the
N-methyl-d-aspartate (NMDA) and the (S)-alpha-amino-3-
hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)/kainate
receptor family.
With regard to NMDA receptors, it is well established that
both zinc and copper are potent inhibitors (Peters et al., 1987;
Mayer et al., 1989; Trombley & Shepherd, 1996; Vlachova et
al., 1996; Trombley et al., 1998). While the copper binding site
has not yet been elucidated, the inhibition of NMDA receptor
currents by zinc has been shown to be mediated by 2 separate
mechanisms, a highly sensitive (low nanomolar range) voltage-
independent site on the NR2A subunit and a less sensitive
(AM) voltage-dependent site on the NR2B subunit (Paoletti et
al., 1997; Rachline et al., 2005).
AMPA/kainate receptors are also blocked by copper in the
low AM range, with binding again proposed to occur at 2
separate binding sites (Weiser & Wienrich, 1996). Unlike
copper, zinc elicits biphasic current responses from AMPA/
kainate receptors that are characterized by potentiation at low
concentrations (50 AM) and inhibition at high concentrations
(1 mM). However, a recent study by Blakemore and Trombley
(2004) has highlighted the existence of a zinc-insensitive
population of AMPA/kainate receptors, in addition to the well-
documented zinc-sensitive AMPA/kainate receptor population
within the rat olfactory bulb. Application of zinc to the
insensitive population of AMPA/kainate receptors produced
uniphasic, inhibitory responses or occasionally had no effect at
all. The differential effects of zinc on AMPA/kainate receptors
have primarily been attributed to the varying subunit composi-
tions expressed by individual receptors.
Copper reduces the efficacy of kainate at the AMPA
receptor (Weiser & Wienrich, 1996) whereas zinc significantly
increases the kinetics and specific binding of AMPA (Bresink
et al., 1996). This ability of both copper and zinc to change
 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
AMPA receptor properties may be relevant to neurotoxicity
associated with AMPA receptor activation.
As well as agonist and antagonist actions on excitatory
amino acid receptors, it has been demonstrated that zinc is
permeable through these receptors (Sensi et al., 1997; Marin et
al., 2000; Jia et al., 2002, see also Fig. 5). The Ca2+-permeable
subtype of AMPA/kainate receptors is a primary route of zinc
entry/uptake into neurons (Sensi et al., 1997) with maximum
translocation expected during intense neuronal activity. Related
to this, NMDA receptor activation has been proposed to
regulate copper homeostasis in hippocampal neurons through
the release of copper via translocation of the copper transporter
ATP7A to hippocampal neuronal processes (Schlief et al.,
2005).
3.2.2. c-Aminobutyric acidA receptors
After glutamate receptors, GABAA receptors have been
studied most extensively for zinc and (to a lesser extent) copper
sensitivity (Smart et al., 1994; Trombley & Shepherd, 1996;
Hosie et al., 2003). While normal synaptic GABAA receptors
are thought to be relatively zinc-insensitive, tonic GABAA
receptors have a much higher sensitivity to block. This occurs
because different GABAA receptor subunit combinations have
different zinc sensitivities. For example, the a1h3 splice
variant of the GABAA receptor is most sensitive to block by
zinc, with other a and h variants having lower sensitivity.
GABAA receptors that contain g subunits have greatly reduced
sensitivity due to the interposition of the g subunit and
structural changes at the a –h interface site (Hosie et al.,
2003). GABAA receptors containing g subunits are by far the
most prevalent subtypes found at GABAergic synapses. The
effects of endogenous zinc on GABAA receptors has also been
shown and modulation of GABAA receptors by zinc is
probably a vital factor in normal brain function (e.g., Xie &
Smart 1991), but this probably occurs through extrasynaptic
rather than synaptic GABAA receptors and/or following
changes in GABAA receptor composition in disease states
such as epilepsy (Dudek, 2001). Again, much less information
is available for copper effects. At least in some cases, copper is
thought to block GABAA receptors through the same mech-
anism as zinc (Narahashi et al., 1994; Trombley & Shepherd,
1996; Sharanova et al., 2000).
Inhibitory glycine receptors are also modulated by zinc;
however, zinc has a biphasic effect on these receptors
producing a potentiation of response at low concentrations
(Laube et al., 2000) but an inhibition at higher concentrations
(Laube et al., 1995).
3.2.3. P2X receptors
The P2X receptors are a family of ionotropic receptors that
are widely distributed in the brain, peripheral nerves, and many
other cell types, existing as both homomers and heteromers.
There are currently 7 members of the P2X family classed as
P2X1 – P2X7 (see review by North, 2002). The co-localization
of zinc with P2X receptors in the nervous system suggests a
physiological role in zinc modulation of ATP-evoked currents
(Nicke et al., 1998; Kanjhan et al., 1999).
571
Trace metals have been known to enhance the cationic
currents elicited by most extracellular excitatory ATP receptors
in native tissues, including those in rat superior cervical
ganglion (Cloues et al., 1993; Cloues, 1995) nodose and
coeliac ganglion neurons (Li et al., 1993) and PC12 cells
(Koizumi et al., 1995). However, not all P2X receptors respond
to zinc in the same manner. While the activity of the P2X2 and
P2X3 receptors is potentiated by zinc (Nakazawa & Ohno,
1997; Wildman et al., 1998; Wildman et al., 1999) as expected
from studies in native tissues, 1– 10 AM copper or zinc has
been found to inhibit the activity of homomeric P2X7 receptors
(Virginio et al., 1997; Coddou et al., 2002) or P2X1 receptors
(Wildman et al., 1999).
P2X2 receptor currents are also potentiated with equal
affinity by copper (Xiong et al., 1999), whereas zinc and
copper differentially modulate the P2X4 receptor. While zinc
potentiates P2X4 receptor ATP-gated currents, copper inhibits
them in a time- and concentration-dependent manner (Soto et
al., 1996; Xiong et al., 1999; Acuna-Castillo et al., 2000,
Coddou et al., 2003).
Although relatively little is known about the P2X6 receptor
(see North, 2002), increased expression of the P2X6 purinergic
receptor has been demonstrated in the hippocampus of zinc
diet-restricted rats (Chu et al., 2003). Although the role the
P2X6 receptor plays in the physiological response of the
hippocampus to zinc depletion remains to be determined, it is
known that the P2X4 and the P2X6 receptors co-assemble in
vitro, forming functional heteromeric channels (Le et al., 1998)
and that this heteromer is also potentiated by micromolar zinc.
3.3. Voltage-gated-like ion channels
3.3.1. Sodium channels
Voltage-gated sodium (Na+) channels are present in the
membrane of most excitable cells. There are 9 primary a
subunits (NaV1.1 – NaV1.9) which show differential expression
throughout the body (see Alexander et al., 2004). The majority
of Na+ channels are highly sensitive to block by tetrodotoxin
(TTX) but some are much less sensitive (see Goldin, 2001).
For example, NaV1.5, the so-called ‘‘cardiac’’ Na+ channel, and
NaV1.8 and NaV1.9 (expressed particularly highly in peripheral
nociceptive neurons) are much less sensitive to TTX than other
Na+ channels.
Modulation of Na+ channels by zinc has been widely
studied. Gilly and Armstrong (1982a) showed that millimolar
concentrations of external zinc modified the kinetics of squid
giant axon Na+ currents. Despite the low potency of this effect,
it has been suggested to arise from zinc binding to a specific
site within the channel rather than from a screening of negative
surface charges (e.g., Tanguy & Yeh, 1988; Hank & Sheets,
1992).
Thus, initially, zinc was envisaged as being a rather weak
modulator of Na+ channel activity. However Frelin et al. (1986)
compared the sensitivity of TTX-sensitive and TTX-resistant
(cardiac) Na+ channels to zinc. TTX-resistant channels were
potently inhibited by zinc with an IC50 of 50 AM, compared to
TTX-sensitive channels with an IC50 of 2 mM. Similar effects
572 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583

on cardiac Na+ channels were seen by others (Baumgarten &
Fozzard, 1989; Ravindran et al., 1991; Schild & Moczy-
dlowski, 1991; Schild et al., 1991). It was suggested that a site
related to the TTX binding site may be important and that
cysteine residues played a role (Schild et al., 1991; also Kurata
et al., 1998).
Following elucidation of the sequences of voltage-gated Na+
channels, Satin et al. (1992) identified a key cysteine residue
(C374) in TTX-resistant cardiac Na+ channels (see Fig. 2A).
When this cysteine residue was mutated, TTX sensitivity was
induced. The mutation also lowered sensitivity to cadmium
(suggesting a lowered sensitivity to zinc also). The converse
experiments on TTX-sensitive channels confirmed these
observations. Mutation of the corresponding residue of the
brain Na+ channel to a cysteine (as is present in the cardiac Na+
channel) reduced the sensitivity of the brain Na+ channel to
TTX (Backx et al., 1992; Heinemann et al., 1992) and
increased its sensitivity to zinc (Heinemann et al., 1992).
Although NaV1.5 channels are often described as ‘‘cardi-
ac’’ sodium channels, they are also expressed in neurons.
White et al. (1993) identified both TTX-sensitive and TTX-
resistant Na+ channels in acutely dissociated neurons from
the medial entorhinal cortex of rat. The TTX-resistant
channel, like cardiac Na+ channels, was highly sensitive to
zinc, with an IC50 of around 9 AM (Fig. 2B). This high
sensitivity to zinc may be of physiological importance
considering that zinc is highly localized in the synaptic
terminals of the entorhinal cortex (see Harrison & Gibbons,
1994).
However, not all TTX-resistant Na+ channels are highly
sensitive to zinc. Kuo et al. (2004) have shown that zinc can
block TTX-resistant Na+ channels (NaV1.8 and NaV1.9) in
dorsal root ganglion neurons but that this occurs at relatively
high concentrations (IC50 of around 300 AM). This may reflect
the fact that these channels (NaV1.8 and NaV1.9) have a serine
residue in place of the key cysteine residue present in cardiac

Na channel Gene Region Sequence TTX Zinc

“Brain” NaV1.1 374-389 S L F R L M T Q D F W E N LY Q √ X

“Cardiac” NaV1.5 365-380 A L F R L M T Q D C W E R LY Q X √

“PNS” NaV1.8 347-362 S L F R L M T Q D S W E R LY Q X X

B
0

-500 zinc
I (pA)

-1000 wash

control
-1500
-40 0 40
V (mV)

IC50 = 9.1 μM
I (norm)

0.5 zinc

control 1 nA
0 5 ms

0.1 1 10 100
zinc (μM)

Fig. 2. (A) Potent zinc block of NaV channels depends on a critical cysteine residue found exclusively in the ‘‘cardiac’’ NaV1.5 channel. This residue is not present in
‘‘brain’’ Na+ channels such as NaV1.1 or peripheral nervous system ‘‘PNS’’ Na+ channels such as NaV1.8. (B) These ‘‘cardiac’’ channels are present in certain neurons
and the currents are potently blocked by zinc (at a concentration of 100 AM in the examples shown). (B) Adapted from Neuron, volume 11, White, J.A., Alonso, A.,
and Kay, A.R., A heart-like Na+ current in the medial entorhinal cortex, pp. 1037 – 1047, 1993, with permission from Elsevier.
 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
NaV1.5 channels (or phenylalanine in TTX-sensitive channels)
giving reduced sensitivity to both TTX and zinc (Fig. 2A).
Taken together, it seems that only NaV1.5 channels are highly
sensitive to block by zinc.
3.3.2. Potassium channels
Potassium (K+) channels play a key role in a number of
different aspects of the electrical responses of the nervous
system. K+ channel activity determines neuronal action
potential frequency and waveform and regulates the excitability
of individual neurons (see Hille, 2001). This physiological
importance coupled with their diversity (with well over 70
different a subunits expressed in the mammalian nervous
system) (see Coetzee et al., 1999; Alexander et al., 2004)
makes them fundamental regulators of neuronal excitability. It
is important then to understand the nature and consequences of
actions of compounds such as zinc and copper on these
channels.
3.3.2.1. Voltage-gated potassium channels. As for Na+
channels, modulatory effects of divalent metal ions such as
zinc and copper on the gating of K+ channels are well
characterized (Gilly & Armstrong, 1982b; Spires & Begen-
isich, 1990; Davidson & Kehl, 1995). In a number of studies,
divalent cation effects have been shown to cause equal shifts in
the voltage-dependent kinetics of K+ channels, explained by
surface charge effects (see Hille, 2001; Elinder & Arhem,
2003). Again, as for Na+ channels, these effects were seen to
occur at millimolar concentrations and were originally envis-
aged as having little physiological relevance.
More recent studies, however, on subtypes of voltage-gated
K+ channels have revealed responses that occur at much lower
ion concentrations. Furthermore, there are examples of
particular K+ channel subtypes that are expressed in neurons,
showing notable sensitivity to these ions (e.g., Harrison et al.,
1992; Poling et al., 1996; Horning & Trombley, 2001). For
example, Harrison et al. (1992) looked at the effect of zinc on
mouse fibroblasts, transfected with cloned rat KV1.1, human
KV1.5, and human KV1.4. They found shifts in the inactivation
and activation curves of each current, at concentrations of zinc
less than 200 AM. At higher concentration of zinc (> 200 AM),
a block of these channels was seen. Related to these
observations, Huang et al. (1993) looked at the effect of zinc
on the voltage-dependent transient potassium current (IA) in
acutely dissociated neurons from the suprachiasmatic nucleus.
They found that zinc caused a shift in the steady-state
inactivation of IA, with 30 AM zinc causing a shift in the half
inactivation by 20 mV. In contrast, copper at 200 AM had little
effect on IA.
Subsequent studies support these observations (e.g., Easaw
et al., 1999; Kuo & Chen, 1999). For example, Kuo and Chen
(1999) found that zinc caused a shift in the inactivation curve
of IA currents in rat hippocampal neurons by 40 mV.
Depending on the experimental protocol they employed, zinc
was seen to apparently inhibit the channel at hyperpolarized
potentials yet enhance current at depolarized potentials. Thus, a
differential effect on the voltage dependence of inactivation
573
and activation as seen in these studies can lead to either an
apparent increase or decrease in IA, depending on the holding
potential of the cell. Similar effects have been seen on IA with
other metal ions (e.g., Mayer & Sugiyama, 1988; Watkins &
Mathie, 1994). Physiologically, IA modulates action potentials
by increasing both the rate of action potential repolarization
and accommodation. An apparent increase in IA as a result of
zinc action would likely decrease action potential frequency.
Horning and Trombley (2001) considered the excitatory
effect of both zinc and copper on rat olfactory bulb neurons.
They found that zinc (100 AM) produced modest but biphasic
effects on voltage-gated K+ currents, potentiating peak current
amplitudes of an IA current by 17% (presumably by shifting the
voltage dependence of inactivation as above) but inhibiting
steady-state current amplitudes of delayed rectifier-type cur-
rents by 15%. Copper (30 AM), however, inhibited both peak
and steady-state amplitudes of delayed rectifier-type currents
by an average of 20% and 17%, respectively. Thus, copper and
zinc can differentially influence neuronal excitability and
synaptic transmission in the rat olfactory bulb.
For many K+ channels, the site(s) of action of zinc and
copper remains to be determined. However, Kehl et al. (2002)
have shown that inhibition of human KV1.5 channels expressed
in HEK293 cells by both hydrogen ions and zinc was
substantially reduced by a mutation of histidine 463 (H) or
arginine 487 (R), amino acids located either in the channel
turret (H) or near the mouth of the pore (R) of the channel.
A particularly interesting set of experiments was carried out
recently by Cusimano et al. (2004) who found that human
KV1.1 channels were inhibited by zinc with an IC50 of around 3
mM. In other words, zinc was a rather poor blocker of these
channels. However, on mutating amino acid F184 to a cysteine,
inhibition by zinc increased, with an IC50 for the mutated
channel of 0.75 mM. Furthermore, a slowing of the activation
kinetics and a substantial shift in the voltage dependence of
activation was seen in the presence of 30 AM zinc, which was
much larger for the mutant channel compared to wild type.
Because this mutation of KV1.1 is associated with a rare
autosomal dominant neurological disorder known as episodic
ataxia type-1 (EA1), this increased sensitivity to zinc in the
mutated channel could have a profound effect on KV1.1
function and contribute to the pathogenesis of the disease.
3.3.2.2. Background or ‘‘leak’’ potassium conductances.
Background K+ currents regulate the resting membrane
potential of neurons and are fundamental regulators of neuronal
excitability (Mathie et al., 2003). As such, compounds that
regulate the activity of these channels will have a huge
influence on neuronal excitability. Two pore domain potassium
channel (K2P) is thought to underlie such leak conductances in
many neurons (see Goldstein et al., 2001; Lesage, 2003).
Leonoudakis et al. (1998) found that the K2P channel
TASK-1 was sensitive to zinc with an IC50 of 175 AM. While
this is not a particularly potent effect, initial experiments
suggested that the related K2P channel, TASK-3, was
insensitive to zinc. Because very few pharmacological agents
are available to distinguish TASK-1 from TASK-3 channels,
574 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583

zinc was seen as a useful diagnostic tool to distinguish the two.
A number of subsequent studies identified TASK-like con-
ductances in native cells and attributed the current to TASK-1
channels at least in part on the basis of zinc sensitivity
(Hartness et al., 2001; Barbuti et al., 2002; Gurney et al., 2003;
Cooper et al., 2004; Johnson et al., 2004).
In fact more recent experiments from our laboratory and
elsewhere suggest that TASK-3 channels are much more
sensitive to block by zinc than TASK-1 with an IC50 of
around 10 – 20 AM depending on the recording conditions
used (see Fig. 3; Clarke et al., 2004; Gruss et al., 2004; Kim
et al., 2005).
We have found that mutation of a glutamate at position 70
and a histidine at position 98, of TASK-3, left the channel with
a reduced sensitivity to zinc block. Conversely, mutation of a
lysine at position 70 in TASK-1 to a glutamate (as is present in
TASK-3) induced zinc sensitivity on the TASK-1 channel
(Clarke et al., 2004). TASK-3 is highly expressed in the brain
and has been implicated in neuronal apoptosis (Lauritzen et al.,
2003). Thus, modulation by a compound such as zinc could be
a useful pharmacological tool in the treatment of neurodegen-
erative and proliferative diseases.
In contrast, Kim et al. (2005) found that a different K2P
channel, TREK-2 was enhanced rather than inhibited by zinc
with an EC50 of around 90 AM. Related to this, Gruss et al.
(2004) had previously found that the K2P channel, TREK-1,
was activated by copper with an EC50 of 3 AM. As for
TASK-3, zinc inhibited TREK-1, with an IC50 of 3 AM. K2P
channels are widely distributed throughout brain and are
involved in setting the resting membrane potential and
modulating neuronal excitability. The differential effects of
zinc and copper on different K2P channels are useful
diagnostically. Furthermore, the influence of these ions on
neuronal excitability could be either increased or decreased
dependent upon the K2P channel expressed in the neuron of
interest.

Fig. 3. Zinc sensitivity of TASK K2P K+ channels. (A – C) Zinc is a comparatively selective blocker of TASK-3 potassium channels (at pH 7.4) with little effect on
TASK-2 and TASK-1 at comparable concentrations. (D – F) Block of TASK-3 channels by zinc shows little voltage dependence. From Clarke et al. (2004) with
permission.
 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
3.3.2.3. Other potassium currents. There is much less
information available about the effects of zinc and copper on
other K+ channels, such as inward rectifier K+ channels or
Ca2+-activated K+ channels. Coulter et al. (1995) showed that
the inward rectifier K+ channel HIR (Kir2.3) was weakly
sensitive to block by external zinc, having an IC50 between 100
and 200 AM at physiological pH. Another strongly rectifying
channel IRK1 (Kir2.1), however, was completely insensitive to
zinc at similar concentrations. Morera et al. (2003) looked at
the effect of copper on the activity of the large conductance
calcium- and voltage-sensitive potassium channels (BKCa).
They found that copper concentrations of 20 AM and above
induced a concentration- and time-dependent decrease in the
channel open probability. Zinc, at concentrations up to 100 AM
had no effect on these channels. Copper was shown to act via
oxidation of extracellular cysteine residues, involved in gating
of the channel (Morera et al., 2003).
3.3.3. Calcium channels
Voltage-gated calcium (Ca2+) channels can be divided into 3
families based on their structural and functional characteristics
(Ertel et al., 2000; Alexander et al., 2004) and within each
family there are several different subunits. Functionally, these
differences are reflected in distinct inactivation kinetics;
activation and inactivation gating; and single channel con-
ductances. This diversity allows each type to play a different
but critical role in aspects of neuronal function.
3.3.3.1. T-type Ca2+ channels. In terms of block by zinc and
copper ions, T-type or low voltage-activated Ca2+ channels
(LVAs) are arguably the most important group. They constitute
a family of 3 channel isoforms, CaV3.1, CaV3.2, and CaV3.3.
Most brain regions express more than 1 isoform and some
neurons, such as olfactory granule cells and hippocampal
pyramidal neurons, express all 3 genes (Craig et al., 1999; Kase
et al., 1999; Talley et al., 1999). The activation and inactivation
curves of T-type Ca2+ channels overlap and cross at ¨ 60 mV,
allowing T-type Ca2+ channels to sustain a continuous Ca2+
influx in neurons and glia due to a small number of channels
being continuously open. T-type Ca2+ channels are generally
thought of as a generator of pacemaker activity and/or
regulators of hormone and neurotransmitter secretion. They
are also known to contribute to pathophysiological conditions
such as cardiac hypertrophy and absence epilepsy.
A number of papers have been published on the LVA T-type
Ca2+ channel, demonstrating inhibition by micromolar zinc.
Akaike et al. (1989) showed that the T-type Ca2+ channel
current in rat aorta smooth muscle cells in primary culture
was reversibly inhibited by zinc with an IC50 of 30 AM,
while Takahashi and Akaike (1990) showed in CA1 pyrami-
dal cells from acutely isolated rat hippocampal neurons that
zinc inhibited the T-type Ca2+ channel with an IC50 of around
20 AM. Similarly, Busselberg et al. (1992) showed that zinc
was highly specific for T-type Ca2+ channels in rat dorsal root
ganglion cells, with 20 AM zinc producing >80% block. N- and
L-type channels were less potently inhibited by zinc, with an
IC50 of 69 AM. More recently, Jeong et al. (2003) looked at the
575
effect of copper and zinc on recombinant T-type Ca2+ channels
(CaV3.1, CaV3.2, CaV3.3). Copper and zinc blocked all 3 T-
type channels but showed a high affinity for CaV3.2 channels
(IC50 = 0.9 AM and 2.3 AM for copper and zinc, respectively).
Much higher concentrations were required to block CaV3.1
and CaV3.3 channels (IC50  200 AM, see Fig. 4).
Utilizing this relatively selective block of CaV3.2 channels
by zinc, Nikonenko et al. (2005) looked at the neuroprotective
effect of various LVA Ca2+ channel antagonists in a model of in
vitro ischemia on rat organotypic hippocampal cultures. They
found 1 AM zinc was sufficient to inhibit more than 80% of the
T-type current in these neurons, which in turn prevented the
increase in intracellular calcium associated with ischemia and
delayed neuronal death.
3.3.3.2. L-, N-, P-, and Q-type Ca2+ channels. The L-, N-, P-,
and Q-type (high voltage activated, HVA) voltage-gated Ca2+
channels are, for the most part, as sensitive as T-type Ca2+
channels to inhibition by copper but are generally less sensitive
to zinc inhibition (Nachshen, 1984; Busselberg et al., 1992;
Vega et al., 1994; Easaw et al., 1999), at least compared to
recombinant CaV3.2 channels (Jeong et al., 2003). Kasai and
Neher (1992) showed that N- and L-type Ca2+ channels in
mouse neuroblastoma and rat glioma hybrid cell line (NG108-
15) were particularly sensitive to block by copper with IC50s of
7 AM and 14 AM, respectively.
Horning and Trombley (2001) looked at the effect of zinc
and copper on the rat olfactory bulb neurons in primary
cultures. These studies are of particular interest because the
mammalian olfactory bulb has one of the highest concentra-
tions of zinc and copper in the CNS (Donaldson et al., 1973;
Ono & Cherian, 1999). In these neurons, inhibition by zinc
(100 AM) of 63% and by copper (30 AM) of 52% was
observed.
Similarly, in pyramidal neurons from rat piriform cortex,
20 AM zinc inhibited each of the 4 components of HVA
current (corresponding to L-, N-, P-, and Q-type currents) by
around 35– 57% whereas copper had an IC50 of less than
1 AM for each component of the HVA current (Castelli et al.,
2003; Magistretti et al., 2003). In the study with zinc, a higher
degree of block was observed when the concentration of the
permeant ion (either Ca2+ or barium) was lowered. Thus, the
action of zinc is consistent with competitive binding of zinc
and the permeant ion to an extracellular binding site, the
occupancy of which by zinc results in Ca2+ channel block. It
is worth noting that many studies on zinc block of Ca2+
channels may underestimate the effectiveness of the ion
because they are done in artificially high concentrations of
permeating ion (barium or Ca2+) to allow reliable measure-
ment of current through the channels.
3.3.3.3. Permeation of zinc through voltage-gated Ca chan-
nels. As well as being blocked by zinc, Ca2+ channels can
mediate the entry of zinc into neurons, at least under certain
recording conditions. For example, Oyama et al. (1982) looked
at the effect of zinc on the neurones isolated from the
subesophageal ganglia of Helix aspersa. In magnesium and
576 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
Fig. 4. Differential block by copper and zinc of cloned T-type Ca2+ channels. Panel A shows block of individual current traces by copper. Both ions are much more
effective at blocking CaV3.2 channels compared to CaV3.1 or CaV3.3 channels (panels A – C). From Jeong et al. (2003) with permission from Lippincott, Williams &
Wilkins.
Ca2+-free media, 25 mM zinc generated all-or-none action
potentials. Ca2+ antagonists such as verapamil and cobalt
reduced these action potentials. Thus, the voltage-gated Ca2+
channel identified in Helix nerve cell bodies is permeable to
zinc. Similar zinc permeation through Ca2+ channels has been
observed for mammalian neurons (Sensi et al., 1997; Kerchner
et al., 2000; Sheline et al., 2002).
The fact that zinc both blocks Ca2+ channel current and
carries charge through the channel suggests that its interaction
with the channel pore may be similar to that of Ca2+, which
also blocks current through the channel carried by divalent or
monovalent cations. Furthermore, this carrying of zinc by
voltage-gated Ca2+ channels supports the notion that blockade
of these channels may have therapeutic utility in pathological
conditions, such as cardiac arrest or sustained seizures, where
excessive zinc influx may contribute to neuronal death (Choi &
Koh, 1998; see Fig. 5).
3.3.4. Store-operated calcium channels
and transient receptor potential channels
In addition to voltage-gated Ca2+ channels, another major
route for Ca2+ entry into cells is through store-operated calcium
channels (SOCCs). Ca2+ entry and the subsequent refilling of
intracellular stores in many cells, particularly cells that lack or
express small numbers of voltage-gated Ca2+ channels, has
now been established to occur through such SOCCs. This may
be of particular relevance in the CNS when considering the role
of glial cells.
This so-called ‘‘capacitive calcium influx’’ plays an impor-
tant role in shaping the Ca2+ response of various tissues and
cell types. Inhibition by heavy metals is a hallmark of SOCC
activity. The first demonstration of a Ca2+ current that
corresponded with SOCC activity was recorded in rat
peritoneal mast cells and was termed ICRAC (Hoth & Penner,
1993). One characteristic of ICRAC was inhibition by 1 mM
zinc and this has been borne out by subsequent studies (e.g.,
Parekh & Penner, 1993; Foskett & Wong, 1994; Koizumi et al.,
1995; Gore et al., 2004). Prothero et al. (1998, 2000) showed
that a rise in intracellular Ca2+ via SOCCs following activation
of metabotropic receptors in rat cortical glial cells was
powerfully inhibited by 100 AM zinc. More recently, Kresse
et al. (2005) showed a similar zinc inhibition of capacitive Ca2+
entry in mouse hippocampal astrocytes, following activation of
the metabotropic receptors, and suggested that the site of action
of zinc may involve a change in the redox potential, possibly
through an action on cysteine residues in the SOCCs (see also
Gore et al., 2004).
Transient receptor potential (trp) channels have been
identified as major pathways for cation movement in non-
excitable cells (Clapham et al., 2001; Montell et al., 2002;
Vennekens et al., 2002). Cloning of the Drosophila trp channel
(Montell & Rubin, 1989) revealed homology to voltage-gated
Ca2+ channels and it was suggested that this gene encodes a
SOCC. Interestingly, Petersen et al. (1995) showed that the
putative capacitative Ca2+ influx evoked in Xenopus oocytes
by the expression of a trp homologue was blocked by 1 mM
 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583 577

Fig. 5. Zinc release from zinc containing neuron terminals. (A) During normal stimulation, zinc (red circles) is released from presynaptic terminals. It can act on
postsynaptic channel proteins such as GABAA receptors, NMDA receptors, or many other different ion channels to alter their activity. In addition, it may permeate
through certain channel proteins (such as voltage-gated Ca2+ channels, trp channels, or AMPA/kainate receptors as indicated in the schematic) to enter the
postsynaptic cell. (B) Following excessive excitation, zinc is depleted from presynaptic vesicles so less is available for release; however, the intracellular
concentration of zinc in the postsynaptic cell is increased, which may be protective or detrimental to the neuron depending on the particular neuron considered (see
text for further details, developed from an idea of Takeda, 2000).

zinc. More recently, however, there has been much debate over
the exact correlation between functional SOCCs and recombi-
nant trp channels (see Clapham, 2003; Nilius, 2003). It is
possible that block by zinc could provide a useful diagnostic
tool to help in this debate (see Gore et al., 2004).
Like voltage-gated Ca2+ channels, at least some trp
channels are permeable to zinc. For example, 2 channels of
the TRPM subfamily, TRPM6 and TRPM7, have been shown
to be permeable to various divalent cations, including zinc
(Hermosura et al., 2002; Monteilh-Zoller et al., 2003; Schmitz
et al., 2003; Voets et al., 2004). Indeed, Monteilh-Zoller et al.
(2003) showed that TRPM7 was 2-fold more permeant to zinc
than to Ca2+. This suggests that TRPM7, in a similar manner to
voltage-gated Ca2+ channels or zinc-permeable AMPA/kainate
receptors, could potentially play a major role in mediating the
severe neurotoxic effects associated with high levels of zinc in
the brain during ischemia.
4. Physiological consequences of zinc and copper actions
4.1. Neuronal excitability
As shown in the previous section, physiologically relevant
concentrations of zinc and copper can modulate several ligand-
gated ion channels including glutamate receptors, GABAA
receptors, and P2X receptors, as well as affecting many
members of the voltage-gated-like ion channel family includ-
ing K+, Na+, and Ca2+ channels. With such a wide range of
actions, it is difficult to predict (and indeed measure) what the
net effects on neuronal excitability of these ions might be.
Nevertheless, a number of striking features emerge from
studies of these receptors and channels, principally the
differential sensitivity of certain members of each family to
zinc and, although much less thoroughly studied, occasionally
to copper too. For ligand-gated ion channels, for example,
NMDA receptors that lack the NR2B subunit are much more
sensitive to zinc modulation than all other glutamate receptors
(see Chen et al., 1997; Tovar et al., 2000), whereas, for
GABAA receptors, it is those that lack the g subunit; that is,
those expressed primarily extra-synaptically, that are most
sensitive to zinc. Similarly for Na+ channels, zinc is a much
more effective blocker of 1 subtype of TTX-insensitive Na+
channel (NaV1.5) than other Na+ channels, whereas for Ca2+
channels a certain T-type channel subunit (CaV3.2) is partic-
ularly sensitive. For K+ channels, zinc can have profound
effects on the gating of certain K V channel subtypes
(particularly those that show fast inactivation such as KV1.4),
whereas it displays rather selective blocking actions on closely
related members of the K2P channel family (compare the
effects on TASK-3 and TASK-1).
There are 2 main consequences of such differential effects
of zinc. Firstly, zinc can provide a useful diagnostic tool to
identify channels in mammalian neurons that underlie ob-
served currents, when other pharmacological tools are lacking.
This has been exemplified recently by the use of zinc to
identify TASK-3 channels as underlying background K+
currents in cerebellar granule cells (see Clarke et al., 2004;
Aller et al., 2005). In a more physiological sense, these
differences are useful because they allow the possibility of
differential responses of neurons to either exogenously applied
zinc or synaptically released zinc (see Fig. 5), depending on
the particular ion channels that neuron expresses at a given
time and where they are localized. KV1.4, for example, has
been found highly localized in axons and terminals, with a
possible role in modulating the excitability of nerve terminals.
In the mossy fiber terminals of the hippocampus, high
concentrations of zinc are also found and thus zinc may play
a physiological role in hippocampal transmission through
regulation of KV1.4. In terms of changes with time, a good
example is the altered sensitivity of GABAA receptors to
released zinc that can occur in temporal lobe epilepsy (see
below and Dudek, 2001).
578 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
4.2. Neurotoxicity
Neurotoxicity can be observed following unwanted rises in
extracellular copper or zinc concentration. Extracellular levels
of copper are significantly elevated during aging and some
neurodegenerative disorders. The ability of copper to undertake
redox cycling to activate molecular oxygen is used by a variety
of enzymes. Although normally bound to proteins, excess
copper may be released and become free to catalyze the
formation of highly reactive hydroxyl radicals, implicated in
disorders associated with abnormal copper metabolism and
neurodegenerative changes (Barnham et al., 2004; Valko et al.,
2005). Ironically, one of the most important roles of copper,
physiologically, is in controlling free radical reactions partic-
ularly as part of the enzyme, copper/zinc superoxide dismutase
(SOD1). So, for example, a dietary deficiency of copper
increases cellular susceptibility to oxidative damage. Thus,
either too little or too much copper in the brain can lead to an
increased vulnerability to oxidative damage.
The brain is particularly susceptible to oxidative stress due
to its high energetic requirement (utilizing 20% basal oxygen
consumption), in addition to the high levels of transition metals
and reduced antioxidant defenses compared to other organs
(Maynard et al., 2005). Within the brain, neurons are the most
metabolically active cells having the greatest oxygen require-
ment. Thus, SOD expression is most prominent amongst
neuronal populations consisting of large neurons that are
continually vulnerable to oxidative damage (Peluffo et al.,
2005). In the adult CNS, SOD1 is expressed in numerous
regions, the most notable being hippocampal pyramidal
neurons, granule neurons of the dentate gyrus, cortical neurons
(especially pyramidal cells), neurons of the substantia nigra, as
well as distinctly high expression in motor neurons of the
spinal cord (Peluffo et al., 2005).
Although not a redox-active metal-like copper, zinc too is
required to allow optimal antioxidant responses and for DNA
repair. Zinc is a functional and structural component of several
enzymes and transcription factors involved in the antioxidant
response and DNA integrity. Inadequate levels of zinc in the
brain lead to abnormal functioning of SOD, which in addition
to its dismutase activity, acquires peroxidase activity resulting
in generation of peroxynitrite and neuronal death (Mocchegiani
et al., 2005).
The physiological intracellular concentration of free zinc in
eukaryotic cells lies in the low picomolar range. Increases in
free intracellular zinc (nanomolar concentrations), following
ischemia, for example, can lead to neurotoxicity (see Fig. 5;
Frederickson et al., 2005).
However, intracellularly accumulated zinc may be neuro-
toxic or neuroprotective depending on its concentration and the
particular neuron of interest (Cote et al., 2005). For example,
within the strata oriens and lucidum of the CA3 region of the
hippocampus, high intracellular concentrations of zinc resulted
in cell death, an effect that could be ameliorated by reducing
zinc levels. Conversely, moderate intracellular concentrations
of zinc in neurons of the CA3 pyramidal layer resulted in cell
survival. Surprisingly, zinc chelation led to an increase in the
mortality rate of these CA3 pyramidal cells (Cote et al., 2005).
Similarly, if the free intracellular zinc is decreased in some
neurons, for example, through the use of zinc chelators such as
TPEN, this can trigger apoptosis (Frederickson et al., 2005).
Thus, the intracellular concentration of these ions also needs to
be tightly regulated by neurons.
5. Clinical consequences
In the previous section, a number of potential physiological
and pathophysiological consequences of zinc and copper
actions were considered. It is of interest, finally, to consider
these ideas in terms of known disease states, specifically
Alzheimer’s disease (AD) and certain forms of epilepsy.
5.1. Alzheimer’s disease
Alzheimer’s disease (AD) is a progressive neurodegener-
ative disorder characterized by amyloidal plaques and
neurofibrillary tangles in conjunction with neuronal cell loss
or dysfunction. The primary constituent of amyloid deposits
associated with all cases of AD is amyloid-h protein (Ah),
derived from the proteolytic cleavage of amyloid precursor
protein. In healthy individuals, Ah is predominantly mem-
brane associated; however, in the AD brain there is a
marked augmentation in the proportion of aggregated
(diffuse and plaque amyloid) and soluble Ah peptides (see
Bush, 2003).
A number of recent studies have demonstrated that the
toxicity and aggregation of Ah during AD is promoted by
aberrant interactions with metals, in particular copper and zinc
(see Maynard et al., 2005). Ah contains selective high and low
affinity copper and zinc binding sites that facilitate its
precipitation via interactions with these metal ions. The AD-
affected brain contains increased concentrations of copper and
zinc within the core and peripheral regions of amyloid plaques
(Lovell et al., 1998; Zecca et al., 2004; Maynard et al., 2005).
Interaction between Ah and oxidized metal ions renders the Ah
peptide toxic to neurons in cell culture (see Maynard et al.,
2005), an effect that is abolished under copper-free conditions
(see Bush, 2003). If copper has a primary role in the toxicity of
Ah peptide actions, zinc seems to play a key role in
aggregation of amyloid deposits. At neutral pH, interactions
between zinc and Ah result in the formation of insoluble
aggregates, whereas binding of copper is competitive resulting
in soluble Ah complexes. In contrast, within the slightly acidic
environment of an elderly or inflamed brain, copper causes
insolubilization and aggregation of Ah (see Maynard et al.,
2005).
Neuronal death in AD occurs selectively in the hippocam-
pus and neocortex as well as in particular subcortical regions.
Because zinc-releasing neurons are also seen in these regions
high concentrations of free, ionic zinc may be released during
synaptic transmission. Under conditions of reduced metabolism
in the aged or AD-affected brain, pools of extracellular zinc
could accumulate, promoting aggregation of Ah. In support of
this idea, studies using mice that are deficient in the synaptic
 A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583
zinc transporter, ZnT3 showed an ¨ 50% reduction in amyloid
load compared to wild-type animals (see Maynard et al., 2005).
5.2. Epilepsy
Several of the ion channel proteins (such as Na+ channels,
T-type Ca2+ channels, and GABAA receptor channels), which
zinc and copper act on, have been implicated in certain forms
of epilepsy, thus it is difficult to make simple predictions about
the pro- or anti-convulsive actions of these ions. This is
compounded by experimental observations where zinc has
been documented to act as either an anticonvulsant (William-
son & Spencer, 1995) or a pro-convulsant (Pei et al., 1983).
Seizure activity in human epileptics and model animals
influences the distribution of essential trace elements, including
copper and zinc, in the brain and peripheral tissues (Papava-
siliou & Miller, 1983; Carl et al., 1990; Hirate et al., 2002). A
number of studies suggest that altered zinc homeostasis in the
brain contributes to the occurrence of epileptic seizures (e.g.,
Buhl et al., 1996; Takeda et al., 2003). Prior to seizure activity,
zinc concentrations are higher in the brain of epileptic (EL)
mice than control mice. The enhanced uptake of zinc into the
brain may reflect a compensatory mechanism for maintaining
correct brain function in EL mice (Hirate et al., 2002; Takeda et
al., 2003). Conversely, postseizure activity, the zinc concen-
tration in the brain of EL mice is significantly lower than that in
control mice (Hirate et al., 2002). The likelihood of seizure
activity in EL mice is diminished by zinc supplementation,
whereas it is augmented by zinc deprivation (Fukahori & Itoh,
1990; Hirate et al., 2002; Takeda et al., 2003). Kainate-induced
seizures in EL mice trigger a substantial, but tissue-specific
loss of zinc from the brain. A reduction of zinc occurs in the
hippocampus, amygdala, and cerebral cortex where there is an
abundance of gluzinergic neuron terminals but not in the
cerebellum (Sloviter, 1985; Frederickson et al., 1988; Takeda et
al., 2003).
Perhaps the single most interesting observation to date
concerning the importance of zinc in epilepsy arises from
observed structural and receptor distribution changes that occur
in the hippocampus of patients with temporal lobe epilepsy or
in animal models of this condition (Dudek, 2001). Two quite
distinct changes occur, but the relationship between the two has
clear functional consequences. Firstly, the composition of the
GABAA receptors in dentate granule cells changes from
relatively zinc-insensitive receptors (incorporating g subunits)
to receptors comprised, primarily, of zinc-sensitive subunits
(see Section 3.2.2). Secondly, mossy fiber terminals (which
release zinc) are reorganized to innervate the dentate granule
cells. It has been proposed that zinc released from mossy fibers
during intense stimulation will block these novel GABAA
receptors, decrease inhibition, and contribute to the generation
of seizures in the dentate gyrus (Buhl et al., 1996; Hamed &
Abdellah, 2004). This represents an excellent example of the
functional consequences that arise from the combination of an
alteration of zinc release from synaptic terminals and the
selective sensitivity of particular protein subunits to modulation
by zinc.
579
Acknowledgments
Our work is supported by the MRC and ARC. CEC is an
Australian Research Council Postdoctoral Fellow.
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