Fisio

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 Ttype 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)-alphaamino-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. 2. 3.

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

* Corresponding author. Tel.: +44 20 7594 7691; fax: +44 20 7589 0191. E-mail address: [email protected] (A. Mathie). 0163-7258/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pharmthera.2005.11.004

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

. . . . . . . .

568 568 570 570 570 570 571 571

568

A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583

3.3.

Voltage-gated-like ion channels . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Sodium channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Potassium channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3. Calcium channels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.4. Store-operated calcium channels and transient receptor potential channels 4. Physiological consequences of zinc and copper actions . . . . . . . . . . . . . . . . . . 4.1. Neuronal excitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Neurotoxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Clinical consequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Alzheimer’s disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Epilepsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction 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 synthesis (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 concentrations 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 Mocchegiani 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

. . . . . . . . . . . . .

. . . . . . . . . . . . .

571 571 573 575 576 577 577 578 578 578 579 579 579

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

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 hippocampus, 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.

569

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 maintained (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

Low

147 days

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-3hydroxy-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) voltageindependent 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 welldocumented 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 compositions 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 mechanism 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 & Moczydlowski, 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 ‘‘cardiac’’ sodium channels, they are also expressed in neurons. White et al. (1993) identified both TTX-sensitive and TTXresistant 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

A 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

I (pA)

0

-500

zinc wash

-1000 control

-1500 -40

0

40

V (mV) 1

I (norm)

IC50 = 9.1 μM 0.5

zinc

control

0 0.1

1 nA 5 ms

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 & Begenisich, 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 envisaged 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 currents 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 conductances 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 neurodegenerative 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 conductances. 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 pyramidal 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 Ttype 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 (NG10815) 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 concentrations 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 measurement of current through the channels. 3.3.3.3. Permeation of zinc through voltage-gated Ca channels. 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 important 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 nonexcitable 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 recombinant 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 ligandgated ion channels including glutamate receptors, GABAA receptors, and P2X receptors, as well as affecting many members of the voltage-gated-like ion channel family including 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 particularly 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 observed 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 particularly 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 requirement. 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 neurotoxic 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 neurodegenerative 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 membrane 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 ADaffected 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 hippocampus 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 (Williamson & 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 (Papavasiliou & 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 concentration 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. References Acuna-Castillo, C., Morales, B., & Huidobro-Toro, J. P. (2000). Zinc and copper modulate differentially the P2X4 receptor. J Neurochem 74(4), 1529 – 1537. Akaike, N., Kostyuk, P. G., & Osipchuk, Y. V. (1989). Dihydropyridinesensitive low-threshold calcium channels in isolated rat hypothalamic neurones. J Physiol 412, 181 – 195. Alexander, S. P. H., Mathie, A., & Peters, J. A. (2004). Guide to receptors and channels. Br J Pharmacol 141, S1 – S126. Aller, M. I., Veale, E. L., Linden, A. -M., Sandu, C., Schwaninger, M., Evans, L. J., et al. (2005). Modifying the subunit composition of TASK channels alters the modulation of a leak conductance in cerebellar granule neurons. J Neurosci 25, 11455 – 11467. Assaf, S. Y., & Chung, S. -H. (1984). Release of endogenous Zn2+ from brain tissue during activity. Nature 308, 734 – 736. Backx, P. H., Yue, D. T., Lawrence, J. H., Marban, E., & Tomaselli, G. F. (1992). Molecular localization of an ion-binding site within the pore of mammalian sodium channels. Science 257(5067), 248 – 251. Barbuti, A., Ishii, S., Shimizu, T., Robinson, R. B., & Feinmark, S. J. (2002). Block of the background K(+) channel TASK-1 contributes to arrhythmogenic effects of platelet-activating factor. Am J Physiol Heart Circ Physiol 282(6), H2024 – H2030. Barnes, N., Tsivkovskii, R., Tsivkovskii, N., & Lutsenko, S. (2005). The copper-transporting ATPases, Menkes and Wilson disease proteins, have distinct roles in adult and developing cerebellum. J Biol Chem 280, 9640 – 9645. Barnham, K. J., Masters, C. L., & Bush, A. I. (2004). Neurodegenerative diseases and oxidative stress. Nat Rev Drug Discov 3, 205 – 214. Baumgarten, C. M., & Fozzard, H. A. (1989). Cd2+ and Zn2+ block unitary Na+ currents in Purkinje and ventricular cells. Biophys J 55, 313a. Blakemore, L. J., & Trombley, P. Q. (2004). Diverse modulation of olfactory bulb AMPA receptors by zinc. NeuroReport 15(5), 919 – 923. Bresink, I., Ebert, B., Parsons, C. G., & Mutschler, E. (1996). Zinc changes AMPA receptor properties: results of binding studies and patch clamp recordings. Neuropharmacology 35(4), 503 – 509. Buhl, E. H., Otis, T. S., & Mody, I. (1996). Zinc-induced collapse of augmented inhibition by GABA in a temporal lobe epilepsy model. Science 271, 369 – 373. Bush, A. I. (2003). The metallobiology of Alzheimer’s disease. TINS 26, 207 – 214. Busselberg, D., Michael, D., Evans, M. L., Carpenter, D. O., & Haas, H. L. (1992). Zinc (Zn2+) blocks voltage-gated calcium channels in cultured dorsal root ganglion cells. Brain Res 593, 77 – 81. Carl, G., Critchfield, J. W., Thompson, J. L., Holmes, G. L., Gallagher, B. B., & Keen, C. L. (1990). Genetically epilepsy prone rats are characterized by altered tissue trace element concentrations. Epilepsia 31, 247 – 252. Castelli, L., Tanzi, F., Taglietti, V., & Magistretti, J. (2003). Cu2+, Co2+, and Mn2+ modify the gating kinetics of high-voltage-activated Ca2+ channels in rat palaeocortical neurons. J Membr Biol 195, 121 – 136. Charton, G., Rovira, C., Ben-Ari, Y., & Leviel, V. (1985). Spontaneous and evoked release of endogenous Zn2+ in the hippocampal mossy fiber zone of the rat in situ. Exp Brain Res 58, 202 – 205. Chen, N., Moshaver, A., & Raymond, L. A. (1997). Differential sensitivity of recombinant N-methyl-d-aspartate receptor subtypes to zinc inhibition. Mol Pharmacol 51, 1015 – 1023. Choi, D. W., & Koh, J. Y. (1998). Zinc and brain injury. Annu Rev Neurosci 21, 347 – 375. Chu, Y., Mouat, M. F., Coffield, J. A., Orlando, R., & Grider, A. (2003). Expression of P2X6, a purinergic receptor subunit, is affected by dietary zinc deficiency in rat hippocampus. Biol Trace Elem Res 91(1), 77 – 87.

580

A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583

Clapham, D. E. (2003). TRP channels as cellular sensors. Nature 426(6966), 517 – 524. Clapham, D. E., Runnels, L. W., & Strubing, C. (2001). The TRP ion channel family. Nat Rev Neurosci 2(6), 387 – 396. Clarke, C. E., Veale, E. L., Green, P. J., Meadows, H. J., & Mathie, A. (2004). Selective block of the human 2-P domain potassium channel, TASK-3, and the native leak potassium current, IKSO, by zinc. J Physiol 560(1), 51 – 62. Cloues, R. (1995). Properties of ATP-gated channels recorded from rat sympathetic neurons: voltage dependence and regulation by Zn2+ ions. J Neurophysiol 73, 312 – 319. Cloues, R., Jones, S., & Brown, D. A. (1993). Zn2+ potentiates ATP-activated currents in rat sympathetic neurons. Pflu¨gers Arch 424(2), 152 – 158. Coddou, C., Villalobos, C., Gonzalez, J., Acuna-Castillo, C., Loeb, B., & Huidobro-Toro, J. P. (2002). Formation of carnosine-Cu (II) complexes prevents and reverts the inhibitory action of copper in P2X4 and P2X7 receptors. J Neurochem 80(4), 626 – 633. Coddou, C., Morales, B., & Huidobro-Toro, J. P. (2003). Neuromodulator role of zinc and copper during prolonged ATP applications to P2X4 purinoceptors. Eur J Pharmacol 472(1 – 2), 49 – 56. Coetzee, W. A., Amarillo, Y., Chiu, J., Chow, A., Lau, D., McCormack, T., et al. (1999). Molecular diversity of K+ channels. Ann N Y Acad Sci 868, 233 – 285. Colvin, R. A., Fontaine, C. P., Laskowski, M., & Thomas, D. (2003). Zn2+ transporters and Zn2+ homeostasis in neurons. Eur J Pharmacol 479(1 – 3), 171 – 185. Cooper, B. Y., Johnson, R. D., & Rau, K. K. (2004). Characterization and function of TWIK-related acid sensing K+ channels in a rat nociceptive cell. Neuroscience 129, 209 – 224. Cote, A., Chiasson, M., Peralta, M. R., Lafortune, K., Pellegrini, L., & Toth, K. (2005). Cell-type specific action of seizure-induced intracellular zinc accumulation. J Physiol 566(3), 821 – 837. Coulter, K. L., Perier, F., Radeke, C. M., & Vandenberg, C. A. (1995). Identification and molecular localization of a pH-sensing domain for the inward rectifier potassium channel HIR. Neuron 15, 1157 – 1168. Craig, P. J., Beattie, R. E., Folly, E. A., Banerjee, M. D., Reeves, M. B., Preistley, J. V., et al. (1999). Distribution of the voltage-dependent calcium channel a1G subunit mRNA and protein throughout the mature rat brain. Eur J Neurosci 11(8), 2949 – 2964. Cusimano, A., D’Adamo, M. C., & Pessia, M. (2004). An episodic ataxia type1 mutation in the S1 segment sensitises the hKv1.1 potassium channel to extracellular Zn2+. FEBS Letters 576, 237 – 244. Davidson, J. L., & Kehl, S. J. (1995). Changes of activation and inactivation gating of the transient potassium current of rat pituitary melanotrophs caused by micromolar Cd2+ and Zn2+. Can J Physiol Pharmacol 73(1), 36 – 42. Donaldson, J., St Pierre, T., Minnich, J. L., & Barbeau, A. (1973). Determination of Na+, K+, Mg2+, Cu2+, Zn2+, and Mn2+ in rat brain regions. Can J Biochem 51, 87 – 92. Dudek, F. E. (2001). Zinc and epileptogenesis. Epilepsy Curr 1, 66 – 70. Easaw, J. C., Jassar, B. S., Harris, K. H., & Jhamandas, J. H. (1999). Zinc modulation of ionic currents in the horizontal limb of the diagonal band of Broca. Neuroscience 94(3), 785 – 795. Elinder, F., & Arhem, P. (2003). Metal ion effects on ion channel gating. Q Rev Biophys 36, 373 – 427. Ertel, E. A., Campbell, K. P., Harpold, M. M., Hofamnn, F., Mori, Y., PerezReyes, E., et al. (2000). Nomenclature of voltage-gated calcium channels. Neuron 25(3), 533 – 535. Foskett, J. K., & Wong, D. C. P. (1994). [Ca2+]i inhibition of Ca2+ release-activated Ca2+ influx underlies agonist- and thapsigargininduced [Ca2+]i oscillations in salivary acinar cells. J Biol Chem 269, 31525 – 31532. Frederickson, C. J., & Bush, A. I. (2001). Synaptically released zinc: physiological functions and pathological effects. BioMetals 14(3 – 4), 353 – 366. Fredrickson, C. J., & Moncrieff, D. W. (1994). Zinc-containing neurons. Biol Signals 3(3), 127 – 139. Frederickson, C. J., Hernandez, M. D., Goik, S. A., Morton, J. D., & McGinty, J. F. (1988). Loss of zinc staining from hippocampal mossy fibers during

kainic acid-induced seizures: a histo-fluorescence study. Brain Res 446, 383 – 386. Frederickson, C. J., Suh, S. W., Silva, D., Frederickson, C. J., & Thompson, R. B. (2000). Importance of zinc in the central nervous system: the zinccontaining neuron. J Nutr 130, 1471S – 1483S. Frederickson, C. J., Koh, J. -Y., & Bush, A. I. (2005). The neurobiology of zinc in health and disease. Nat Rev Neurosci 6, 449 – 462. Frelin, C., Cognard, C., Vigne, P., & Lazdunski, M. (1986). Tetrodotoxinsensitive and tetrodotoxin-resistant Na+ channels differ in their sensitivity to Cd2+ and Zn2+. Eur J Pharmacol 122, 245 – 250. Fukahori, M., & Itoh, M. (1990). Effects of dietary zinc status on seizure susceptibility and hippocampal zinc content in the El (epilepsy) mouse. Brain Res 529, 16 – 22. Gaetke, L. M., & Chow, C. K. (2003). Copper toxicity, oxidative stress, and antioxidant nutrients. Toxicology 189, 147 – 163. Gilly, F. A. R., & Armstrong, C. M. (1982a). Slowing of sodium channel opening kinetics in squid axon by extracellular zinc. J Gen Physiol 79, 935 – 964. Gilly, F. A. R., & Armstrong, C. M. (1982b). Divalent cations and the activation kinetics of potassium channels in squid giant axons. J Gen Physiol 79, 965 – 996. Goldin, A. L. (2001). Resurgence of sodium channel research. Ann Rev Physiol 63, 871 – 894. Goldstein, S. A. N., Bockenhauer, D., O’Kelly, I., & Zilberberg, N. (2001). Potassium leak channels and the KCNK family of two-P-domain subunits. Nat Rev Neurosci 2, 175 – 184. Gore, A., Moran, A., Hershfinkel, M., & Sekler, I. (2004). Inhibitory mechanism of store-operated Ca2+ channels by zinc. J Biol Chem 279, 11106 – 11111. Gruss, M., Mathie, A., Lieb, W. R., & Franks, N. P. (2004). The two-poredomain K+ channels TREK-1 and TASK-3 are differentially modulated by copper and zinc. Mol Pharmacol 66, 530 – 537. Gurney, A. M., Osipenko, O. N., MacMillan, D., McFurlane, K. M., Tate, R. J., & Kempsill, F. E. J. (2003). Two-pore domain K channel, TASK-1 in pulmonary artery smooth muscle cells. Circ Res 83, 957 – 964. Gutteridge, J. M. (1984). Copper-phenanthroline-induced site-specific oxygenradical damage to DNA. Detection of loosely bound trace copper in biological fluids. Biochem J 218(3), 983 – 985. Hamed, S. A., & Abdellah, M. M. (2004). Trace elements and electrolytes homeostasis and their relation to antioxidant enzyme activity in brain hyperexcitability of epileptic patients. J Pharmacol Sci 96, 349 – 359. Hank, D. A., & Sheets, M. F. (1992). Extracellular divalent and trivalent cation effects on sodium current kinetics in single canine cardiac Purkinje cells. J Physiol 454, 267 – 298. Harris, E. D. (2002). Cellular transporters for zinc. Nutr Rev 60(4), 121 – 124. Harrison, N. L., & Gibbons, S. J. (1994). Zn2+: an endogenous modulator of ligand- and voltage-gated ion channels. Neuropharmacology 33(8), 935 – 952. Harrison, N. L., Radke, H. K., Tamkun, M. M., & Lovinger, D. M. (1992). Modulation of gating of cloned rat and human K+ channels by micromolar Zn2+. Mol Pharmacol 43, 482 – 486. Hartness, M. E., Lewis, A., Searle, G. J., O’Kelly, I., Peers, C., & Kemp, P. J. (2001). Combined antisense and pharmacological approaches implicate hTASK as an airway O(2) sensing K(+) channel. J Biol Chem 276(28), 26499 – 26508. Hartter, D. E., & Barnea, A. (1988). Brain tissue accumulates 67copper by two ligand-dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process. J Biol Chem 263(2), 799 – 805. Heinemann, S. H., Terlau, H., & Imoto, K. (1992). Molecular basis for pharmacological differences between brain and cardiac sodium channels. Eur J Physiol 422, 90 – 92. Hermosura, M. C., Monteilh-Zoller, M. K., Scharenberg, A. M., Penner, R., & Fleig, A. (2002). Dissociation of the store-operated calcium current I(CRAC) and the Mg-nucleotide-regulated metal ion current MagNuM. J Physiol 539(2), 445 – 458. Hille, B. (2001). Ionic channel of excitable membranes (3rd edR). USA’ Sinauer Associates, Inc.

A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583 Hirate, M., Takeda, A., Tamano, H., Enomoto, S., & Oku, N. (2002). Distribution of trace elements in the brain of EL (epilepsy) mice. Epilepsy Res 51, 109 – 116. Horning, M. S., & Trombley, P. Q. (2001). Zinc and copper influence excitability of rat olfactory bulb neurons by multiple mechanisms. J Neurophysiol 86, 1652 – 1660. Hosie, A. M., Dunne, E. L., Harvey, R. J., & Smart, T. G. (2003). Zincmediated inhibition of GABA(A) receptors: discrete binding sites underlie subtype specificity. Nat Neurosci 6(4), 362 – 369. Hoth, M., & Penner, R. (1993). Calcium release-activated calcium current in rat mast cells. J Physiol 465, 359 – 386. Howell, G. A., Welch, M. G., & Frederickson, C. J. (1984). Stimulationinduced uptake and release of zinc in hippocampal slices. Nature 308(5961), 736 – 738. Huang, R. -C., Peng, Y. -W., & Yau, K. -W. (1993). Zinc modulation of a transient potassium current and histochemical localization of the metal in neurons of the suprachiasmatic nucleus. Proc Natl Acad Sci 90, 11806 – 11810. Jeong, S. -W., Park, B. -G., Park, J. -Y., Lee, J. -W., & Lee, J. -H. (2003). Divalent metals differentially block cloned T-type calcium channels. NeuroReport 14, 1537 – 1540. Jia, Y., Jeng, J. M., Sensi, S. L., & Weiss, J. H. (2002). Zn2+ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones. J Physiol 543(1), 35 – 48. Johnson, R. P., O’Kelly, I. M., & Fearon, I. M. (2004). System-specific O2 sensitivity of the tandem pore domain K+ channel TASK-1. Am J Physiol Cell Physiol 286(2), C391 – C397. Kanjhan, R., Housley, G. D., Burton, L. D., Christie, D. L., Kippenberger, A., Thorne, P. R., et al. (1999). Distribution of the P2X2 receptor subunit of the ATP-gated ion channels in the rat central nervous system. J Comp Neurol 407, 11 – 32. Kardos, J., Kovacs, I., Hajos, F., Kalman, M., & Simonyi, M. (1989). Nerve endings from rat brain tissue release copper upon depolarisation. A possible role in regulating neuronal excitability. Neurosci Lett 103(2), 139 – 144. Kasai, H., & Neher, E. (1992). Dihydropyridine-sensitive and N-conotoxinsensitive calcium channels in mammalian neuroblastoma-glioma cell line. J Physiol 448, 161 – 188. Kase, M., Kakimoto, S., Sakuma, S., Houtani, T., Ohishi, H., Ueyama, T., et al. (1999). Distribution of neurons expressing a1G subunit mRNA of T-type voltage-dependent calcium channel in adult rat central nervous system. Neurosci Lett 268, 77 – 80. Kay, A. R. (2003). Evidence for chelatable zinc in the extracellular space of the hippocampus, but little evidence for synaptic release of Zn. J Neurosci 23(17), 6847 – 6855. Kehl, S. J., Eduljee, C., Kwan, D. C. H., Zhang, S., & Fedida, D. (2002). Molecular determinants of the inhibition of human Kv1.5 potassium currents by external protons and Zn2+. J Physiol 541(1), 9 – 24. Kerchner, G. A., Canzoniero, L. M. T., Yu, S. P., Ling, C., & Choi, D. W. (2000). Zn2+ current is mediated by voltage-gated Ca2+ channels and enhanced by extracellular acidity in mouse cortical neurones. J Physiol 528(1), 39 – 52. Kim, J. -S., Park, J. -Y., Kang, H. -W., Lee, E. -J., Bang, H., & Lee, J. -H. (2005). Zinc activates TREK-2 potassium channel activity. J Pharm Exp Ther 314, 618 – 625. Koizumi, S., Nakazawa, K., & Inoue, K. (1995). Inhibition by Zn2+ of uridine 5V-triphosphate-induced Ca(2+)-influx but not Ca(2+)-mobilization in rat phaeochromocytoma cells. Br J Pharmacol 115(8), 1502 – 1508. Kresse, W., Sekler, I., Hoffmann, A., Peters, O., Nolte, C., Moran, A., & Kettenmann, H. (2005). Zinc ions are endogenous modulators of neurotransmitter-stimulated capacitative Ca2+ entry in both cultures and in situ mouse astrocytes. Eur J Neurosci 21, 1626 – 1634. Kuo, C. -C., & Chen, F. -P. (1999). Zn2+ modulation of neuronal transient K+ current: fast and selective binding to the deactivated channels. Biophys J 77, 2552 – 2562. Kuo, C. -C., Chen, W. -Y., & Yang, Y. -C. (2004). Block of tetrodotoxinresistant Na+ channel pore by multivalent cations: gating modification and Na+ flow dependence. J Gen Physiol 124, 27 – 42.

581

Kurata, Y., Hisatome, I., Tsuboi, M., Uenishi, H., Zhang, G., Oyaizu, M., et al. (1998). Effect of sulfhydryl oxidoreduction on permeability of cardiac tetrodotoxin-insensitive sodium channel. Life Sci 63(12), 1023 – 1035. Llanos, R. M., & Mercer, J. F. B. (2002). The molecular basis of copper homeostasis and copper-related disorders. DNA Cell Biol 21(4), 259 – 270. Laube, B., Kuhse, J., & Betz, H. (2000). Kinetic and mutational analysis of Zn2+ modulation of recombinant human inhibitory glycine receptors. J Physiol 522(2), 215 – 230. Laube, B., Kuhse, J., Rundstrom, N., Kirsch, J., Schmieden, V., & Betz, H. (1995). Modulation by zinc ions of native rat and recombinant human inhibitory glycine receptors. J Physiol 483(3), 613 – 619. Lauritzen, I., Zanzouri, M., Honore, E., Duprat, F., Ehrengruber, M. U., Lazdunski, M., & Patel, A. J. (2003). K+-dependent cerebellar granule neuron apoptosis. Role of TASK leak K+ channels. J Biol Chem 278(34), 32068 – 32076. Le, K. T., Babinski, K., & Seguela, P. (1998). Central P2X4 and P2X6 channel subunits co-assemble into a novel heteromeric ATP receptor. J Neurosci 18(18), 7152 – 7159. Leonoudakis, D., Gray, A. T., Winegar, B. D., Kindler, C. H., Harada, M., Taylor, D. M., et al. (1998). An open rectifier potassium channel with two pore domains in tandem cloned rat cerebellum. J Neurosci 18(3), 868 – 877. Lesage, F. (2003). Pharmacology of neuronal background potassium channels. Neuropharmacology 44, 1 – 7. Li, C., Peoples, R. W., Li, Z., & Weight, F. F. (1993). Zn2+ potentiates excitatory action of ATP on mammalian neurons. Proc Natl Acad Sci 90(17), 8264 – 8267. Li, Y. V., Hough, C. J., & Sarvey, J. M. (2003). Do we need zinc to think? Sci Signal Transduct Knowl Environ 182, 19. Linder, M. C., & Hazegh-Azam, M. (1996). Copper biochemistry and molecular biology. Am J Clin Nutr 63(5), 797S – 811S. Lovell, M. A., Robertson, J. D., Teesdale, W. J., Campbell, J. L., & Markesbery, W. R. (1998). Copper, iron and zinc in Alzheimer’s disease senile plaques. J Neurol Sci 158, 47 – 52. Magistretti, J., Castelli, L., Taglietti, V., & Tanzi, F. (2003). Dual effect of Zn2+ on multiple types of voltage-dependent Ca2+ currents in rat palaeocortical neurons. Neuroscience 117, 249 – 264. Marin, P., Israel, M., Glowinski, J., & Premont, J. (2000). Routes of zinc entry in mouse cortical neurons: role in zinc-induced neurotoxicity. Eur J Neurosci 12(1), 8 – 18. Mathie, A., Clarke, C. E., Ranatunga, K. M., & Veale, E. (2003). What are the roles of the many different types of potassium channel expressed in cerebellar granule cells? Cerebellum 2(1), 11 – 25. Mayer, M. L., & Sugiyama, K. (1988). A modulatory action of divalent cations on transient outward current in cultured rat sensory neurons. J Physiol 396, 417 – 433. Mayer, M. L., Vyklicky Jr, L., & Westbrook, G. L. (1989). Modulation of excitatory amino acid receptors by group IIB metal cations in cultured mouse hippocampal neurones. J Physiol 415(1), 329 – 350. Maynard, C. J., Bush, A. I., Masters, C. L., Cappai, R., & Li, Q. (2005). Metals and amyloid-h in Alzheimer’s disease. Int J Exp Path 86, 147 – 159. Mocchegiani, E., Bertoni-Freddari, C., Marcellini, F., & Malavolta, M. (2005). Brain, aging and neurodegeneration: role of zinc ion availability. Prog Neurobiol 75, 367 – 390. Monteilh-Zoller, M. K., Hermosura, M. C., Nadler, M. J. S., Scharenberg, A. M., Penner, R., & Fleig, A. (2003). TRPM7 provides an ion channel mechanism for cellular entry of trace metal ions. J Gen Physiol 121, 49 – 60. Montell, C., Birnbaumer, L., & Flockerzi, V. (2002). The TRP channels, a remarkably functional family. Cell 108(5), 595 – 598. Montell, C., & Rubin, G. M. (1989). Molecular characterization of the Drosophila trp locus: a putative integral membrane protein required for phototransduction. Neuron 2(4), 1313 – 1323. Morera, F. J., Wolff, D., & Vergara, C. (2003). External copper inhibits the activity of the large-conductance calcium- and voltage-sensitive potassium channel from skeletal muscle. J Membr Biol 192, 65 – 72. Nachshen, D. A. (1984). Selectivity of the Ca binding site in synaptosome Ca Channels. J Gen Physiol 83, 941 – 967.

582

A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583

Nakazawa, K., & Ohno, Y. (1997). Effects of neuroamines and divalent cations on cloned and mutated ATP-gated channels. Eur J Pharmacol 325(1), 101 – 108. Narahashi, T., Ma, J. Y., Arakawa, O., Reuveny, E., & Nakahiro, M. (1994). GABA receptor-channel complex as a target site of mercury, copper, zinc, and lanthanides. Cell Mol Neurobiol 14(6), 599 – 621. Nicke, A., Baumert, H. G., Rettinger, J., Eichele, A., Lambrecht, G., Mutschler, E., et al. (1998). P2X1 and P2X3 receptors form stable trimers: a novel structural motif of ligand-gated ion channels. EMBO J 17, 3016 – 3028. Nikonenko, I., Bancila, M., Bloc, A., Muller, D., & Bijlenga, P. (2005). Inhibition of T-type calcium channels protects neurons from delayed ischemia-induced damage. Mol Pharmacol 68, 84 – 89. Nilius, B. (2003). From TRPs to SOCs, CCEs, and CRACs: consensus and controversies. Cell Calcium 33(5 – 6), 293 – 298. North, R. A. (2002). Molecular physiology of P2X receptors. Physiol Rev 82, 1013 – 1067. Ono, S., & Cherian, M. G. (1999). Regional distribution of metallothionein, zinc and copper in the brain of different strains of rats. Biol Trace Elem Res 69(2), 151 – 159. Oyama, Y., Nishi, K., Yatani, A., & Akaike, N. (1982). Zinc current in Helix soma membrane. Comp Biochem Physiol 72C, 403 – 410. Paoletti, P., Ascher, P., & Neyton, J. (1997). High-affinity zinc inhibition of NMDA NR1-NR2A receptors. J Neurosci 17(15), 5711 – 5725. Papavasiliou, P. S., & Miller, T. (1983). Generalized seizures alter the cerebral and peripheral metabolism of essential metals in mice. Exp Neurol 82, 223 – 226. Parekh, A. B., & Penner, R. (1993). Store depletion and calcium influx. Physiol Rev 77, 901 – 930. Pei, Y., Zhao, D., Huang, J., & Cao, L. (1983). Zinc-induced seizures: a new experimental model of epilepsy. Epilepsy 24, 169 – 176. Peluffo, H., Acarin, L., Faiz, M., Castellano, B., & Gonzalez, B. (2005). Cu/Zn superoxide dismutase expression in the postnatal rat brain following an excitotoxic injury. J Neuroinflammation 2(1), 12 – 25. Peters, S., Koh, J., & Choi, D. W. (1987). Zinc selectively blocks the action of N-methyl-d-aspartate on cortical neurons. Science 236(4801), 589 – 593. Petersen, C. C., Berridge, M. J., Borgese, M. F., & Bennett, D. L. (1995). Putative capacitative calcium entry channels: expression of Drosophila trp and evidence for the existence of vertebrate homologues. Biochem J 311, 41 – 44. Poling, J. S., Vicini, S., Rogawski, M. A., & Salem, N. (1996). Docosahexaenoic acid block of neuronal voltage-gated K+ channels: subunit selective antagonism by zinc. Neuropharmacology 35, 969 – 982. Prothero, L. S., Richards, C. D., & Mathie, A. (1998). Inhibition by inorganic ions of a sustained calcium signal evoked by activation of mGlu5 receptors in rat cortical neurons and glia. Br J Pharmacol 125, 1551 – 1561. Prothero, L. S., Mathie, A., & Richards, C. D. (2000). Purinergic and muscarinic receptor activation activates a common calcium entry pathway in rat neocortical neurons and glial cells. Neuropharmacology 39, 1768 – 1778. Puig, S., & Thiele, D. J. (2002). Molecular mechanisms of copper uptake and distribution. Curr Opinion Chem Biol 6(2), 171 – 180. Rachline, J., Perin-Dureau, F., Le Goff, A., Neyton, J., & Paoletti, P. (2005). The micromolar zinc-binding domain on the NMDA receptor subunit NR2B. J Neurosci 25(2), 308 – 317. Ravindran, A., Schild, L., & Moczydlowski, E. (1991). Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin: Zn2+ induces discrete substates in cardiac Na+ channels. J Gen Physiol 97, 89 – 115. Ruiz, A., Walker, M. C., Fabian-Fine, R., & Kullmann, D. M. (2004). Endogenous zinc inhibits GABA(A) receptors in a hippocampal pathway. J Neurophysiol 91(2), 1091 – 1096. Satin, J., Kyle, J. W., Chen, M., Bell, P., Cribbs, L. L., Fozzard, H. A., & Rogart, R. B. (1992). A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties. Science 256(5060), 1202 – 1205. Sato, M., Ohtomo, K., Daimon, T., Sugiyama, T., & Iijima, K. (1994). Localization of copper to afferent terminals in rat locus ceruleus, in contrast

to mitochondrial copper in cerebellum. J Histochem Cytochem 42(12), 1585 – 1591. Schild, L., & Moczydlowski, E. (1991). Competitive binding interaction between Zn2+ and saxitoxin in cardiac Na+ channels. Biophys J 59, 523 – 537. Schild, L., Ravindran, A., & Moczydlowski, E. (1991). Zn2+-induced subconductance events in cardiac Na+ channels prolonged by batrachotoxin. J Gen Physiol 97, 117 – 142. Schlief, M. L., Craig, A. M., & Gitlin, J. D. (2005). NMDA receptor activation mediates copper homeostasis in hippocampal neurons. J Neurosci 25(1), 239 – 246. Schmitz, C., Perraud, A., Johnson, C., Inabe, K., Smith, M., Penner, R., et al. (2003). Regulation of vertebrate cellular Mg2+ homeostasis by TRPM7. Cell 114, 191 – 200. Schumann, K., Classen, H. G., Dieter, H. H., Konig, J., Multhaup, G., Rukgauer, M., Summer, K. H., Bernhardt, J., & Biesalski, H. K. (2002). Hohenheim consensus workshop: copper. Eur J Clin Nutr 56, 469 – 483. Sensi, S. L., Canzoniero, L. M., Yu, S. P., Ying, H. S., Koh, J. Y., Kerchner, G. A., et al. (1997). Measurement of intracellular free zinc in living cortical neurons: routes of entry. J Neurosci 17(24), 9554 – 9564. Sharanova, I. N., Vorobjev, V. S., & Haas, H. L. (2000). Interactions between copper and zinc at GABAA receptors in acutely isolated cerebellar Purkinje cells of the rat. Br J Pharmacol 130, 851 – 856. Sheline, C. T., Ying, H. S., Ling, C. S., Canzoniero, L. M. T., & Choi, D. W. (2002). Depolarization-induced zinc influx into cultured cortical neurons. Neurobiol Dis 10, 41 – 53. Slomianka, L., Danscher, G., & Frederickson, C. J. (1990). Labelling of the neurons of origin of zinc-containing pathways by intraperitoneal injections of sodium selenite. Neuroscience 38(3), 843 – 854. Sloviter, R. (1985). A selective loss of hippocampal mossy fiber Timm stain accompanies granule cell seizure activity induced by perforant path stimulation. Brain Res 330, 150 – 153. Smart, T. G., Xie, X., & Krishek, B. J. (1994). Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc. Prog Neurobiol 42(3), 341 – 393. Smart, T. G., Hosie, A. M., & Miller, P. S. (2004). Zn2+ ions: modulators of excitatory and inhibitory synaptic activity. Neuroscientist 10(5), 432 – 442. Soto, F., Garcia-Guzman, M., Gomez-Hernandez, J. M., Hollmann, M., Karschin, C., & Stuhmer, W. (1996). P2X4: an ATP-activated ionotropic receptor cloned from rat brain. Proc Natl Acad Sci 93(8), 3684 – 3688. Spires, S., & Begenisich, T. (1990). Modification of potassium channel kinetics by histidine-specific reagents. J Gen Physiol 96(4), 757 – 775. Strausak, D., Mercer, J. F. B., Dieter, H. H., Stremmel, W., & Multhaup, G. (2001). Copper in disorders with neurological symptoms: Alzheimer’s, Menkes, and Wilson diseases. Brain Res Bull 55, 175 – 185. Stuerenberg, H. J. (2000). CSF copper concentrations, blood – brain barrier function, and ceruloplasmin synthesis during the treatment of Wilson’s disease. J Neural Transm 107, 321 – 329. Takahashi, K., & Akaike, N. (1990). Calcium antagonist effects on lowthreshold (T-type) calcium current in rat isolated hippocampal CA1 pyramidal neurons. J Pharmacol Exp Ther 256, 169 – 175. Takeda, A. (2000). Movement of zinc and its functional significance in the brain. Brain Res Rev 34, 137 – 148. Takeda, A. (2001). Zinc homeostasis and functions of zinc in the brain. BioMetals 14, 343 – 351. Takeda, A., Hirate, M., Tamano, H., & Oku, N. (2003). Zinc movement in the brain under kainate-induced seizures. Epilepsy Res 54, 123 – 129. Talley, E. M., Cribbs, L. L., Lee, J. H., Daud, A., Perez-Reyes, E., & Bayliss, D. A. (1999). Differential distribution of three members of a gene family encoding low voltage-activated (T-type) calcium channels. J Neurosci 19(6), 1895 – 1911. Tanguy, J., & Yeh, J. Z. (1988). Divalent cation block of normal and BTXmodified sodium channels in squid axons. Biophys J 53, 229a. Tarohda, T., Yamamoto, M., & Amano, R. (2004). Regional distribution of manganese, iron, copper, and zinc in the rat brain during development. Anal Bioanal Chem 380, 240 – 246.

A. Mathie et al. / Pharmacology & Therapeutics 111 (2006) 567 – 583 Thompson, R. B., Peterson, D., Mahoney, W., Cramer, M., Maliwal, B. P., Won Suh, S., et al. (2002). Fluorescent zinc indicators for neurobiology. J Neurosci Methods 118(1), 63 – 75. Tovar, K. R., Sprouffske, K., & Westbrook, G. L. (2000). Fast NMDA receptormediated synaptic currents in neurons from mice lacking the NR2B subunit. J Neurophysiol 83(1), 616 – 620. Trombley, P. Q., Horning, M. S., & Blakemore, L. J. (1998). Carnosine modulates zinc and copper effects on amino acid receptors and synaptic transmission. NeuroReport 9(15), 3503 – 3507. Trombley, P. Q., & Shepherd, G. M. (1996). Differential modulation by zinc and copper of amino acid receptors from rat olfactory bulb neurons. J Neurophysiol 76(4), 2536 – 2546. Valko, M., Morris, H., & Cronin, M. T. D. (2005). Metals, toxicity and oxidative stress. Curr Med Chem 12, 1161 – 1208. Vega, M. T., Villalobos, C., Garrido, B., Gandia, L., Bulbena, O., GarciaSancho, J., et al. (1994). Permeation by zinc of bovine chromaffin cell calcium channels: relevance to secretion. Pflu¨gers Arch 429, 231 – 239. Vennekens, R., Voets, T., Bindels, R. J., Droogmans, G., & Nilius, B. (2002). Current understanding of mammalian TRP homologues. Cell Calcium 31(6), 253 – 264. Virginio, C., Church, D., North, R. A., & Surprenant, A. (1997). Effects of divalent cations, protons and calmidazolium at the rat P2X7 receptor. Neuropharmacology 36(9), 1285 – 1294. Vlachova, V., Zemkova, H., & Vyklicky, L., Jr. (1996). Copper modulation of NMDA responses in mouse and rat cultured hippocampal neurons. Eur J Neurosci 8(11), 2257 – 2264. Voets, T., Nilius, B., Hoefs, S., vanderKemp, A. W., Droogmans, G., Bindels, R. J., et al. (2004). TRPM6 forms the Mg2+ influx channel involved in intestinal and renal Mg2+ absorption. J Biol Chem 279, 19 – 25. Vogt, K., Mellor, J., Tong, G., & Nicoll, R. (2000). The actions of synaptically released zinc at hippocampal mossy fiber synapses. Neuron 26(1), 187 – 196. Wang, Z., Danscher, G., Kim, Y. K., Dahlstrom, A., & Mook Jo, S. (2002). Inhibitory zinc-enriched terminals in the mouse cerebellum: double-

583

immunohistochemistry for zinc transporter 3 and glutamate decarboxylase. Neurosci Lett 321(1 – 2), 37 – 40. Watkins, C. S., & Mathie, A. (1994). Modulation of the gating of the transient outward potassium current of rat isolated cerebellar granule neurons by lanthanum. Pflu¨gers Arch 428, 209 – 216. Weiser, T., & Wienrich, M. (1996). The effects of copper ions on glutamate receptors in cultured rat cortical neurons. Brain Res 742(1 – 2), 211 – 218. White, A. R., Barnham, K. J., Huang, X., Voltakis, I., Beyreuther, K., Masters, C. L., et al. (2004). Iron inhibits neurotoxicity induced by trace copper and biological reductants. J Biol Inorg Chem 9, 269 – 280. White, A. R., & Cappai, R. (2003). Neurotoxicity from glutathione depletion is dependent on extracellular trace copper. J Neurosci Res 71, 889 – 897. White, J. A., Alonso, A., & Kay, A. R. (1993). A heart-like Na+ current in the medial entorhinal cortex. Neuron 11, 1037 – 1047. Wildman, S. S., King, B. F., & Burnstock, G. (1998). Zn2+ modulation of ATPresponses at recombinant P2X2 receptors and its dependence on extracellular pH. Br J Pharmacol 123(6), 1214 – 1220. Wildman, S. S., King, B. F., & Burnstock, G. (1999). Modulatory activity of extracellular H+ and Zn2+ on ATP-responses at rP2X1 and rP2X3 receptors. Br J Pharmacol 128, 486 – 492. Williamson, A., & Spencer, D. (1995). Zinc reduces dentate granule cell hyperexcitability in epileptic humans. NeuroReport 6, 1562 – 1564. Xie, X. M., & Smart, T. G. (1991). A physiological role for endogenous zinc in rat hippocampal synaptic neurotransmission. Nature 349(6309), 521 – 524. Xiong, K., Peoples, R. W., Montgomery, J. P., Chiang, Y., Stewart, R. R., Weight, F. F., et al. (1999). Differential modulation by copper and zinc of P2X2 and P2X4 receptor function. J Neurophysiol 81(5), 2088 – 2089. Yu, F. H., & Catterall, W. A. (2004). The VGL-chanome: a protein superfamily specialized for electrical signalling and ionic homeostasis. Sci Signal Transduct Knowl Environ 253, 15. Zecca, L., Stroppolo, A., Gatti, A., Tampellini, D., Toscani, M., Gallorini, M., et al. (2004). The role of iron and copper molecules in the neuronal vulnerability of locus caeruleus and substantia nigra during aging. Proc Natl Acad Sci 101, 9843 – 9848.