Zn2+ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones

doi:10.1113/jphysiol.2002.020172

Zn²⁺ currents are mediated by calcium-permeable AMPA/kainate channels in cultured murine hippocampal neurones

Yousheng Jia, Jade-Ming Jeng†, Stefano L. Sensi§, John H. Weiss*†‡

Departments of * Neurology, † Neurobiology and Behavior, and ‡ Anatomy and Neurobiology, University of California at Irvine, CA 92697-4292, USA and § Department of Neurology, University 'G. d'Annunzio', Chieti, 66013, Italy

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

Permeation of the endogenous cation Zn²⁺ through calcium-permeable AMPA/kainate receptor-gated (Ca-A/K) channels might subserve pathological and/or physiological signalling roles. Voltage-clamp recording was used to directly assess Zn²⁺ flux through these channels on cultured murine hippocampal neurones. Ca-A/K channels were present in large numbers only on a minority of neurones (Ca-A/K(+) neurones), many of which were GABAergic. The presence of these channels was assessed in whole-cell or outside-out patch recording as the degree of inward rectification of kainate-activated currents, quantified via a rectification index (RI = G₊₄₀/G_-₆₀), which ranged from <0.4 (strongly inwardly rectifying) to >2 (outwardly rectifying). The specificity of a low RI as an indication of robust Ca-A/K channel expression was verified by two other techniques, kainate-stimulated cobalt-uptake labelling, and fluorescence imaging of kainate-induced increases in intracellular Ca²⁺. In addition, the degree of inward rectification of kainate-activated currents correlated strongly with the positive shift of the reversal potential (V_rev) upon switching to a sodium-free, 10 mM Ca²⁺ buffer. With Zn²⁺ (3 mM) as the only permeant extracellular cation, kainate-induced inward currents were only observed in neurones that had previously been identified as Ca-A/K(+). A comparison between the V_rev observed with 3 mM Zn²⁺ and that observed with Ca²⁺ as the permeant cation revealed a P_Ca/P_Zn of ~1.8. Inward currents recorded in 3 mM Ca²⁺ were unaffected by the addition of 0.3 mM Zn²⁺, while microfluorimetrically detected increases in the intracellular concentration of Zn²⁺ in Ca-A/K(+) neurones upon kainate exposure in the presence of 0.3 mM Zn²⁺ were only mildly attenuated by the addition of 1.8 mM Ca²⁺. These results provide direct evidence that Zn²⁺ can carry currents through Ca-A/K channels, and that there is little interference between Ca²⁺ and Zn²⁺ in permeating these channels.

(Received 7 March 2002; accepted after revision 29 May 2002)
Corresponding author J. H. Weiss: Department of Neurology, University of California at Irvine, 2101 Gillespie Neuroscience Research Facility, Irvine, CA 92697-4292, USA. Email: jweiss{at}uci.edu

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Zn²⁺ is present in particularly high levels in the mammalian brain where, in addition to its ubiquitous role as a trace element constituent of many enzymes, it is concentrated in the synaptic vesicles of certain excitatory neurones, and is released with neuronal activation, probably achieving peak synaptic levels of up to several hundred micromolar (Assaf & Chung, 1984; Howell et al. 1984).

While the physiological roles of synaptically released Zn²⁺ are poorly understood, extracellular Zn²⁺ does have inhibitory effects on both ligand-gated (NMDA and GABA) and voltage-gated (K⁺, Na⁺, Ca²⁺) ion channels, while potentiating AMPA currents (Peters et al. 1987; Westbrook & Mayer, 1987; Smart et al. 1994; Lin et al. 2001). Recent observations have indicated that endogenous synaptically released Zn²⁺ causes a tonic inhibition of NMDA receptors at the mossy fibre-CA3 synapse (Vogt et al. 2000). In addition to these extracellular effects, Zn²⁺ is seen to accumulate in postsynaptic neurones following strong presynaptic activation. Such 'Zn²⁺ translocation' has been implicated in the neurodegeneration associated with conditions of ischaemia or epilepsy (Fredrickson et al. 1989; Tonder et al. 1990; Koh et al. 1996), and might also play a role in synaptic plasticity (Lu et al. 2000; Li et al. 2001).

Imaging and neurotoxicity studies have suggested that Zn²⁺ can enter neurones through NMDA channels, voltage-sensitive Ca²⁺ channels (VSCC), calcium-permeable AMPA/kainate (Ca-A/K) channels, and via reverse operation of the Na⁺/Ca²⁺ exchanger (Weiss et al. 1993; Koh & Choi, 1994; Yin & Weiss, 1995; Sensi et al. 1997). Studies using the low-affinity Zn²⁺ probe Newport Green have found that in the presence of constant extracellular Zn²⁺ levels, the highest increases in intracellular Zn²⁺ concentration ([Zn²⁺]_i) are achieved upon activation of Ca-A/K channels (Sensi et al. 1999). In contrast, moderate increases in [Zn²⁺]_i are observed upon activation of VSCC, and consistent with the effective block of NMDA currents by Zn²⁺, far lower increases are detected upon their activation. However, since the increases in [Zn²⁺]_i that occur upon activation of any permeable channel reflect the net effect of intracellular buffering, extrusion and release of Zn²⁺ from intracellular stores in addition to Zn²⁺ entry, meaningful assessment of conductance requires direct measurement of Zn²⁺ currents.

Although Zn²⁺ permeation through Ca-A/K channels has not been directly characterized, a recent electrophysiological study examining the interactions between Zn²⁺ and VSCC in dissociated cortical neurones found that Zn²⁺ permeates these channels, while attenuating concurrent Ca²⁺ flux (Kerchner et al. 2000). In contrast to VSCC, however, Ca-A/K channels are likely to be concentrated on postsynaptic membranes adjacent to sites of presynaptic Zn²⁺ release, and thus might be particularly important conduits for trans-synaptic Zn²⁺ signalling.

Of note, these channels are present in substantial numbers only on distinct subpopulations of central neurones. One population of forebrain neurones that often possesses large numbers of Ca-A/K channels are the GABAergic neurones (McBain & Dingledine, 1993; Yin et al. 1994; Geiger et al. 1995). In addition, evidence for their presence on other populations of neurones, and observations that ischaemia or epilepsy results in increased numbers of these channels on hippocampal pyramidal neurones have contributed to hypotheses that their presence might be a critical factor in the selective neuronal vulnerability characteristically seen in certain nervous system diseases (Pellegrini-Giampietro et al. 1997; Weiss & Sensi, 2000).

The aim of this study was to apply electrophysiological techniques to hippocampal neurones in dissociated culture, in order to assess and compare Zn²⁺ and Ca²⁺ currents through Ca-A/K channels. Since these channels are only present in substantial numbers on a minority subpopulation of neurones, the study also necessitates approaches to characterize their presence.

METHODS

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Introduction
Methods
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Hippocampal cell culture

All experimental protocols and use of animals were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the University of California Irvine Institutional Animal Care and Use Committee (IACUC).

Mixed hippocampal cell cultures were prepared from fetal mice, gestational age 15-16 days, generally as previously described (Yin et al. 1999). Pregnant Swiss-Webster mice were deeply anaesthetized using 4 % halothane, killed by cervical dislocation and the fetuses were removed, rapidly decapitated and the heads were placed in chilled dissection medium. After isolating fetal hippocampi, the tissue was dissociated and plated at a density of approximately 10⁵ cells cm^-2, in media consisting of Eagle's minimal essential medium (with Earle's salts, supplied glutamine-free), supplemented with 10 % heat-inactivated horse serum, 10 % fetal bovine serum, glutamine (2 mM), and glucose (25 mM) on a previously established astrocyte monolayer in 35 mm glass-bottomed dishes (Mattek Cultureware, Ashland, MA, USA). Cultures were maintained in an incubator at 37 °C and in an atmosphere of 5 % CO₂. After 4-6 days in vitro, non-neuronal cell division was halted by exposure to 0.01 mM cytosine arabinoside for 1-3 days. The cells were then shifted into a maintenance medium identical to the plating medium but lacking fetal serum, with subsequent maintenance media replacement occurring twice a week. Cultures were studied after 11-15 days in vitro. The same procedure was used to prepare glial cultures, except that forebrain tissue was obtained from early postnatal (1-3 days) mice (killed by halothane anaesthesia followed by decapitation), media was supplemented with epidermal growth factor (10 ng ml^-1), and cell suspensions were plated directly onto poly-L-lysine + laminin-coated glass-bottomed dishes.

Electrophysiology

Whole-cell and patch recordings from cultured neurones were made using an Axopatch 200A amplifier, filtered at 1 kHz, and digitized on-line at 2 kHz with a Digidata 1320A and pCLAMP 8 acquisition software (Axon Instruments, Union City, CA, USA). Electrodes were pulled using a Narishige PP-83 (Narishige International USA, Inc., East Meadow, NY, USA), and typically had a resistance of 2-4 M $Omega$ when filled with intracellular solution containing (mM): 145 CsCl, 10 Hepes, 5 EGTA, 2 Na2ATP; pH 7.3 (adjusted with CsOH), 290-300 mosmol l^-1. Spermine (100 µM) was routinely included in the internal solution to prevent washout of rectification properties during whole-cell recording (Bowie & Mayer, 1995; Donevan & Rogawski, 1995; Isa et al. 1995; Kamboj et al. 1995; Koh et al. 1995). In some experiments, conventional outside-out patches were pulled after achieving whole-cell configuration. Except where noted, all recordings were made at room temperature at a holding potential of -70 mV in sodium-containing extracellular buffer (Na-EB) containing (mM): 140 NaCl, 3 KCl, 1 CaCl₂, 4 MgCl₂, 5 Hepes, 10 glucose; pH 7.4 (adjusted with NaOH), 300-310 mosmol l^-1. All extracellular buffers were supplemented with MK-801 (10 µM), TTX (0.5 µM), bicuculline (20 µM), and Gd³⁺ (20 µM) to block NMDA receptors, voltage-gated Na⁺ channels, GABA_A receptors and VSCC, respectively. Chemicals were applied locally using a computer-controlled focal superfusion system with a 100 µm o.d. outflow tip (DAD-12, ALA Scientific Instruments, Westbury, NY, USA).

Ca²⁺ and Zn²⁺ permeability measurements. Current-voltage (I-V) relationships were generated by applying a voltage-ramp protocol (-100 to +100 mV, 50 ms pause, then back to -100 mV, rate of ramp = 1 mV ms^-1) in the presence and absence of kainate. Agonist was applied in normal extracellular buffer as described above (Na-EB), or in identical buffer in which all cations (Na⁺, Ca²⁺, K⁺, Mg²⁺) were replaced with an equimolar concentration of N-methyl-D-glucamine (NMDG), with or without the addition of Ca²⁺ (Ca-EB) or Zn²⁺ (Zn-EB), as indicated. Zn-EB and Ca-EB were prepared immediately before use by the addition of concentrated ZnCl₂ or CaCl₂ to cation-free EB stock (containing only NMDG, Hepes and glucose, pH 7.4).

Although Ca-A/K channels may be minimally permeable to NMDG (P_NMDG/P_Na estimated at ~0.02; Jatzke et al. 2002), this should have little effect on estimated Ca²⁺ and Zn²⁺ permeability ratios.

Current responses were recorded during five consecutive voltage-ramp applications, and the kainate I-V relationship was derived by subtracting the averaged current measured in the absence of kainate from that measured in its presence (Waters & Allen, 1998). Since the I-V relationship exhibited weak hysteresis, probably due to the activation of voltage-dependent conductances during the depolarizing phase of the voltage ramp, the descending limb was used for display and subsequent analyses. Reversal potentials (V_rev), which were obtained directly from the acquired data, were used to calculate the permeability ratios of Ca²⁺ or Zn²⁺ to Cs⁺ using the constant field equation (Lewis, 1979; Iino et al. 1990):

where V_rev is the reversal potential, R, F and T are the gas and Faraday constants and the absolute temperature, respectively, and P_x represents the permeability coefficient of ion x through Ca-A/K channels.The degree of rectification of I-V relationships elicited in normal buffer (Na-EB) was quantified using a rectification index (RI), defined as the ratio of slope conductances at +40 and -60 mV:

Rectification of miniature EPSCs (mEPSCs) was estimated by comparing the mean amplitudes of mEPSCs recorded at +40 and -60 mV. To adjust for varying noise across different recordings, the detection threshold for mEPSCs was set at an amplitude of three times the current root mean square (I_RMS) of the recording; I_RMS measurement, event detection, and analysis were carried out using MiniAnalysis software (Synaptosoft, Decatur, GA, USA). Individual events were aligned along the rise time for trace averaging.

Imaging studies

Cultures were mounted on the stage of a Nikon Diaphot inverted microscope (Nikon Instruments, Melville, NY, USA) equipped with a 75 W xenon lamp, a computer-controlled filter wheel, and a times 40, 1.3 NA epifluorescence oil-immersion objective. Emitted signals were acquired with a 12 bit cooled digital CCD camera (Hamamatsu, Bridgewater, NJ, USA), using neutral-density filters to minimize photobleaching, and background signals were subtracted before analysis. All experiments were carried out at room temperature (25 °C) in a static 1.5 ml bath.

Intracellular Ca²⁺ ([Ca²⁺]_i) imaging for pre-identification of Ca-A/K(+) neurones. Cultures were loaded in the dark with 2.5 µM of Fura-2 AM in Na-EB containing 0.1 % pluronic acid and 0.75 % DMSO, for 20 min at 25 °C, then washed in Na-EB and kept in the dark for an additional 20 min. Cells were alternately illuminated at 340 and 380 nm, and fluorescence was monitored at 510 nm. To pre-identify Ca-A/K(+) neurones, a microscope field was focally perfused for 5 s via a pneumatic pressure-ejection system with kainate (50 µM) in sodium-free, 10 mM Ca²⁺ buffer (10Ca-EB), along with MK-801 (10 µM) and Gd³⁺ (20 µM) to block Ca²⁺ influx through NMDA channels and VSCC. Cultures were washed for 60 s after termination of the kainate exposure, and allowed to recover to baseline fluorescence levels before further experiments were performed. [Ca²⁺]_i responses to this exposure were highly segregated between neurones; while most showed relatively little response, neurones that responded abruptly (340/380 fluorescence ratio increase of more than two times baseline within 6 s) were provisionally identified as Ca-A/K(+) (see Fig. 2B; Sensi et al. 1999).

Intracellular Zn²⁺ ([Zn²⁺]_i) imaging. To monitor [Zn²⁺]_i, cells were loaded in the dark at 25 °C with 5 µM of the diacetate ester of the zinc-selective probe, Newport Green (K_d = 1 µM; Haugland, 1996), in a Hepes-buffered salt solution (HSS, consisting of (mM): 120 NaCl, 5.4 KCl, 0.8 MgCl₂, 20 Hepes, 15 glucose, 1.8 CaCl₂, 10 NaOH, pH 7.4) containing 0.2 % pluronic acid and 1.5 % DMSO. After 30 min, cultures were washed with HSS and kept in the dark for an additional 30 min before imaging. Cells were illuminated at 490 nm and fluorescence monitored at 530 nm. Agonist exposures were by bath exchange with either normal or calcium-free HSS containing kainate (100 µM) and Zn²⁺ (300 µM), with MK-801 (10 µM) and Gd³⁺ (20 µM), and were terminated after 5 min by washing with calcium-free HSS. Due to the slow recovery of the kainate-induced increases in [Zn²⁺]_i, the two experimental conditions were performed in separate age-matched culture dishes. As with [Ca²⁺]_i imaging, these exposures resulted in a distinct segregation of [Zn²⁺]_i responses (see Fig. 7A) corresponding to the level of Ca-A/K channel expression (Sensi et al. 1999); neurones that responded abruptly (>5 % fluorescence increase within 1 min) were provisionally identified as Ca-A/K(+). Using these criteria, the proportion of neurones considered to be Ca-A/K(+) in each condition was closely matched.

Histochemistry

Kainate-stimulated cobalt-uptake labelling ('Co²⁺ staining') to identify Ca-A/K(+) neurones (Pruss et al. 1991) was performed generally as described previously. Briefly, cultures were loaded with Co²⁺ by exposure to 150 µM kainate with 3.75 mM Co²⁺, and intracellular Co²⁺ precipitated with (NH₄)₂S. Cultures were then fixed and developed using a modified Timm's stain procedure (Yin et al. 1998). After staining, dishes were reinserted onto the microscope stage and fields re-matched.

For glutamic acid decarboxylase (GAD) immunohistochemistry, paraformaldehyde-fixed cultures were washed with phosphate-buffered saline, and blocked in 5 % horse serum. Primary antibody exposures were at 1:1000 (1 h, 37 °C). Biotinylated horse anti-mouse antibody, avidin-biotin complex (ABC) solution, and 3-amino-9-ethyl-carbazole were used to visualize stained cells. For GAD and Co²⁺ stain co-labelling, cultures were first subjected to kainate-stimulated Co²⁺ loading, precipitation and fixation as described. Cultures were then GAD immunostained and multiple fields photographed before and again after development of the Co²⁺ stain.

Data analysis

Electrophysiological data were analysed using Clampfit 8.0 (Axon Instruments, Union City, CA, USA) and SigmaPlot 2000 software (SPSS Science, Chicago, IL, USA). Fluorescence imaging experiments were analysed by outlining neuronal somata and gathering data with Meta Imaging Series 4.0 software (Universal Imaging, West Chester, PA, USA). Student's paired t-test, one-way ANOVA, and Tukey's Multiple Comparison Test were carried out using Prism 3.0 (GraphPad Software, San Diego, CA, USA). All averaged values are given as means ± S.E.M.

Chemicals and reagents

Pregnant Swiss-Webster mice were obtained from Simonsen Laboratories (Gilroy, CA, USA). All tissue culture media and sera were obtained from Gibco Life Technologies (Grand Island, NY, USA). 1-Naphthyl acetyl spermine (NAS) was kindly provided by Daicel Chemical (Tokyo, Japan). Newport Green was obtained from Molecular Probes (Eugene, OR, USA) and Fura-2 AM from TefLabs (Austin, TX, USA). MK-801 was obtained from Research Biochemicals (Natick, MA, USA); GYKI 53655 was a gift of Eli Lilly (Indianapolis, IN, USA). Anti-GAD (GAD-6) was obtained from the Developmental Studies Hybridoma Bank at the University of Iowa, Iowa City; secondary antibody and ABC solution were obtained from Vector Laboratories (Burlingame, CA, USA). All other chemicals and reagents were obtained from Sigma Aldrich (St Louis, MO, USA).

RESULTS

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Methods
Results
Discussion
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Electrophysiological detection of Ca-A/K channels in subpopulations of hippocampal neurones

Prior studies have indicated that Ca-A/K channels are present in large numbers only on a minority (<30 %) of neurones (Ca-A/K(+) neurones) in cortical (Yin et al. 1994) or hippocampal (Iino et al. 1990; Yin et al. 1999) cultures. In the absence of electrophysiology, Ca-A/K(+) neurones can be identified by a histochemical stain ('Co²⁺ stain') based on selective kainate-induced uptake of Co²⁺ ions through these channels (and the relative Co²⁺ impermeability of VSCC and NMDA channels; Pruss et al. 1991; Yin et al. 1994, 1998). Using this technique combined with GAD immunocytochemistry we have found that most (70-90 %), but not all, Ca-A/K(+) neurones in both cortical (Yin et al. 1994) and hippocampal cultures are GABAergic (of 179 Co²⁺ stained neurones in our hippocampal cultures, 143 were GAD(+)).

Because of the selective expression of Ca-A/K channels, their presence must be evaluated in each neurone studied before attempting to elicit Ca²⁺ or Zn²⁺ currents. Most Ca-A/K channels on these neurones are comprised of AMPA subunits, for which kainate is an effective and relatively non-desensitizing agonist. The Ca²⁺ permeability of AMPA channels is determined by the presence of the GluR2 subunit (Burnashev et al. 1992). Calcium-impermeable channels (which contain GluR2) yield linear (or mildly outwardly rectifying) I-V curves, whereas in the presence of intracellular polyamines (like spermine), calcium-permeable AMPA channels show inward rectification (Bowie & Mayer, 1995; Donevan & Rogawski, 1995; Isa et al. 1995; Kamboj et al. 1995; Koh et al. 1995).

Initial electrophysiological experiments to assess the presence of Ca-A/K channels in the cultures were carried out in outside-out patches. This recording configuration was employed as it provided excellent stability and minimized space clamp artefact. Neurones were chosen for recording in a quasi-random fashion (generally >20 µm diameter, soma not contacting adjacent neurones), and were patched with electrodes containing spermine (100 µM); the control extracellular buffer (Na-EB) was supplemented with MK-801, TTX, bicuculline and Gd³⁺. Outside-out patches were pulled and left for 5 min to achieve maximal dialysis and inhibition of contaminating K⁺ currents, before eliciting I-V relationships of kainate-induced currents using a voltage-ramp protocol (Fig. 1A). The degree of inward rectification was quantified by calculating the RI, defined as the ratio of slope conductances at +40 and -60 mV. Among 101 patches from different neurones, the degree of rectification ranged widely (Fig. 1B), from strongly inward to outward (0.002 < RI < 2.50; mean RI = 0.93 ± 0.06). However, in general agreement with prior studies (Iino et al. 1990, 1994), only a minority (27 %) displayed strong inward rectification (RI < 0.4).

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Figure 1. Some hippocampal neurones display inwardly rectifying kainate-activated currents
A, recording protocol. Current-voltage (I-V) relationships were elicited in both whole-cell and outside-out patch configurations, from a holding potential of -70 mV, by applying ascending and descending voltage ramps (top). Applying this protocol to an outside-out patch in normal extracellular buffer (Na-EB), in the presence and absence of kainate (100 µM), yielded the currents shown (middle). The I-V curve was derived by subtracting the current in the absence of kainate from that in its presence (bottom); the descending limb of the curve was used for analyses. B, kainate elicits inwardly rectifying currents in some neurones. Outside-out patches were pulled and subjected to the recording protocol illustrated in A, and rectification quantified by calculating a rectification index (RI; see Methods). While some patches yielded strongly inwardly rectifying I-V curves (left; RI = 0.22), others showed near-linear or outwardly rectifying responses (right; RI = 1.49). C, evidence for synaptic calcium-permeable AMPA/kainate receptor-gated (Ca-A/K) channels on some neurones. Rectification of synaptic AMPA/kainate channels was assessed by comparing the mean amplitude of mEPSCs at +40 and -60 mV, recorded in the presence of NMDA antagonists. Neurones with low whole-cell RI values, indicative of the presence of somatic Ca-A/K channels, generally also showed inward rectification of miniature EPSCs (mEPSCs; left), whereas other neurones did not (right). Insets depict the corresponding averaged mEPSC traces at +40 and -60 mV (scale bar = 5 pA, 5 ms).

Inward rectification of kainate currents in outside-out patches only provides evidence for the presence of Ca-A/K channels on the somatic membrane. To assess the presence of these channels at synaptic sites, some neurones in which mEPSCs were observed were left in whole-cell configuration, and rectification of mEPSCs estimated by comparing the mean amplitude of events at +40 mV with those at -60 mV. In a set of 11 cells with inwardly rectifying kainate currents (mean whole-cell RI of 0.44 ± 0.05), the I₊₄₀/I-₆₀ ratios were also low (0.43 ± 0.04; Fig. 1C). Thus, the apparent inward rectification of mEPSCs provides support for the presence of Ca-A/K channels in postsynaptic membranes of functional synapses.

In some experiments, outside-out patch recording (to assess RI) was followed by kainate-stimulated cobalt-uptake labelling. These techniques for assessing the presence of Ca-A/K channels were highly correlated; six out of six neurones with RI > 1.0 were unlabelled, whereas neurones with strong inward rectification (RI < 0.4, n= 12) were all labelled by the procedure (Fig. 2A). However, since cobalt-uptake histology requires fixation of the cells, it is not useful for pre-identification of Ca-A/K(+) neurones prior to recording. An alternative approach enabling detection of Ca-A/K channels in living neurones makes use of fluorescent Ca²⁺ imaging techniques to measure increases in [Ca²⁺]_i upon kainate exposure under conditions that minimize Ca²⁺ influx through VSCC and NMDA channels. Neurones were loaded with the calcium-sensitive fluorescent probe, Fura-2, and imaged before and during brief focal perfusion with kainate in sodium-free, high Ca²⁺ buffer (10Ca-EB), as described earlier. These exposures resulted in distinct segregation of responses between individual neurones, with a minority (<30 %) showing particularly abrupt rises (see Methods; Fig. 2B). Prior studies have revealed that assessment of the presence of Ca-A/K channels by this technique correlates closely with assessment by Co²⁺ labelling (Sensi et al. 1999). In addition, we found that 73 ± 8 % of neurones pre-identified as Ca-A/K(+) by this technique were also GAD(+) (n = 10 experiments, comprising 38 pre-identified Ca-A/K(+) neurones).

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Figure 2. Strong correlation between inward rectification of kainate-induced currents and other methodologies in the identification of putative Ca-A/K channel-expressing (Ca-A/K(+)) neurones
A, Ca-A/K(+) neurones can be identified by a histochemical stain based on kainate-stimulated uptake of Co²⁺ ions. Cobalt-labelled (top) and unlabelled (bottom) neurones are shown along with I-V curves generated from kainate exposures to outside-out patches pulled from each neurone (insets). B, Ca-A/K(+) neurones can be identified using Ca²⁺ imaging techniques. A hippocampal culture was loaded with the fluorescent Ca²⁺ probe, Fura-2; pseudocolour images convey relative intracellular Ca²⁺ concentrations ([Ca²⁺]_i) before (i) and after (ii) brief exposure to kainate in sodium-free, high Ca²⁺ buffer (10Ca-EB). Traces (iii) depict the time course of fluorescence ratio changes in individual neurones; coloured lines correspond to specific neurones indicated by coloured dots in (ii). I-V curves (iv) are from outside-out patches pulled from the same two neurones. Note that the neurone with a rapid and high [Ca²⁺]_i rise (red) displays strong inward rectification (RI = 0.13), whereas the one with little [Ca²⁺]_i rise (blue) does not (RI = 1.65).

Neurones pre-identified by imaging as possessing Ca-A/K channels generally yielded inwardly rectifying I-V curves upon recording in either the outside-out patch (Fig. 2B) or the whole-cell configuration. Of 33 pre-identified neurones subjected to whole-cell recording, 27 exhibited -0.46 ± 0.02). A cutoff of 0.7 for strong inward rectification was chosen arbitrarily for whole-cell recordings, in which RI values of Ca-A/K(+) neurones were generally greater than those obtained in outside-out patch recording. Thus, these three techniques, based respectively on the assessment of Co²⁺ uptake, increases in [Ca²⁺]_i, or current rectification upon kainate exposure, each identify a common subset of hippocampal neurones possessing large numbers of Ca-A/K channels.

Direct measurement of Ca²⁺ currents through Ca-A/K channels

Although rectification of I-V curves from outside-out patches suggests the presence of Ca-A/K channels, it provides neither direct nor quantitative measures of ion flux. In order to determine permeability ratios for Ca²⁺ and Cs⁺ ions, we carried out further experiments examining the changes in V_rev that occurred upon removing extracellular Na⁺ and altering Ca²⁺ concentration. After generation of an I-V curve on an outside-out patch in Na-EB (Fig. 3), I-V relationships were elicited again during perfusion with buffer containing Ca²⁺ (10 mM) as the only permeant cation (10Ca-EB). Most neurones that had linear or outwardly rectifying I-V curves in Na-EB buffer showed little or no kainate-activated inward current in 10Ca-EB at potentials up to -90 mV (Fig. 3A). However, a subpopulation of neurones (with inwardly rectifying I-V curves) did demonstrate inward Ca²⁺ currents in 10Ca-EB (Fig. 3B), with V_rev changing from -90 mV in the absence of all permeant cations, to V_rev ranging from -55 mV to -19 mV in the presence of high Ca²⁺.

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Figure 3. Correlation between two indices of the contribution of Ca-A/K channels to the total kainate-activated current
A, outside-out patch recording of kainate-activated currents from a Ca-A/K(-) neurone. After pulling an outside-out patch, the I-V relationship of kainate-activated currents was elicited first in Na-EB and then in 10Ca-EB. Note the near-linear I-V curve in Na-EB, and the lack of inward current in 10Ca-EB with very negative reversal potential (V_rev, arrow). B, outside-out patch recording of kainate-activated currents from a Ca-A/K(+) neurone. After pulling an outside-out patch, the I-V relationship of kainate-activated currents was elicited in Na-EB and then in 10Ca-EB. Note the strong inward rectification in Na-EB, and the inward current at -70 mV in 10Ca-EB with positively shifted V_rev (arrow). C, scatter plot of V_rev against RI from 20 outside-out patches. Note the strong correlation between a positive shift in V_rev and decreasing RI, both indicative of an increasing fraction of current through Ca-A/K channels (r = -0.84; P < 0.0001). Arrows indicate individual neurones illustrated in A and B.

In agreement with prior reports (Iino et al. 1994; Itazawa et al. 1997), a scatter plot between the RI elicited in normal buffer and the V_rev observed upon changing to 10Ca-EB shows a close correlation between these measures (Fig. 3C; correlation coefficient r = -0.84, n = 20, P < 0.0001). As suggested previously (Iino et al. 1994), the continuous distribution of each of these measures provides compelling evidence for a mix of calcium-permeable and calcium-impermeable channels on many neurones. V_rev can be related to relative permeability (P_Ca/P_Cs) by the extended constant field equation (Lewis, 1979). Assuming that neurones with the most positively shifted values of V_rev are those with the greatest contribution of Ca-A/K channels to the total current, a V_rev in 10Ca-EB of -19 mV yields an estimated P_Ca/P_Cs of ~2.5, in general agreement with previous studies (Iino et al. 1990; Wollmuth & Sakmann, 1998).

As further confirmation that strong inward rectification is indicative of Ca-A/K channel activation, we made use of NAS, a synthetic analogue of Joro spider toxin, which blocks Ca-A/K channels in a voltage-dependent fashion (Koike et al. 1997). After carrying out the usual I-V protocol in Na-EB on outside-out patches, the protocol was repeated in the presence of 300 µM NAS. In six patches with strongly rectifying currents (RI = 0.17 ± 0.04), NAS decreased the current at -70 mV by 88 ± 3.0 %, whereas non-inwardly rectifying currents (RI = 1.41 ± 0.08; n = 7) were little altered (block of 2.9 ± 5.3 %; Fig. 4). Under the present exposure paradigm, 30 µM NAS was less efficacious at blocking inwardly rectifying currents (data not shown). The relatively low potency of NAS in the present paradigm might in large part reflect the slow use dependence of the block (Koike et al. 1997).

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Figure 4. Effects of the voltage-dependent Ca-A/K channel blocker 1-naphthyl acetyl spermine (NAS) on kainate-induced currents
A, NAS blocks kainate-induced currents in Ca-A/K(+) neurones. After pulling an outside-out patch, the I-V relationship of kainate (KA)-activated currents was elicited in Na-EB alone, and in the presence of 300 µM NAS. Note that this strongly inwardly rectifying current (RI = 0.16) was blocked by NAS in a voltage-dependent manner, with greater block at negative potentials. Inset, effect of 300 µM NAS on the kainate response of a Ca-A/K(+) neurone at -70 mV. B, NAS has no effect on kainate-induced currents in Ca-A/K(-) neurones. After pulling an outside-out patch, the I-V relationship of kainate-activated currents was elicited without and with 300 µM NAS in Na-EB, as described earlier. Note the lack of effect of NAS on this outwardly rectifying current (RI = 1.57).

Characterization of Zn²⁺ currents though Ca-A/K channels

Subsequent experiments sought to directly characterize the ability of Zn²⁺ to permeate Ca-A/K channels. Initial attempts to measure Zn²⁺ currents used outside-out patches, as above, perfused with sodium-free, potassium-free and calcium-free buffer containing 3 mM Zn²⁺ (3Zn-EB), as perfusion with higher levels of Zn²⁺ (10 mM) generally resulted in instability of the recording. However, because inward currents recorded under these conditions at -70 mV were very small, studies comparing Zn²⁺ and Ca²⁺ flux through Ca-A/K channels were carried out in the whole-cell configuration, in neurones pre-identified as Ca-A/K(+) on the basis of Ca²⁺ imaging or by RI < 0.7. In pre-identified Ca-A/K(+) neurones perfused with kainate in 3Zn-EB, inward currents at -70 mV were invariably observed (Fig. 5A and C; mean -89.5 ± 7.8 pA, n = 28).

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Figure 5. Kainate induces whole-cell Zn²⁺ currents in Ca-A/K(+) neurones
A, continuous current recordings in Ca-A/K(+) and Ca-A/K(-) neurones. After establishing a stable whole-cell voltage clamp (-70 mV) in Na-EB, neurones were microperfused with 3Zn-EB (containing 3 mM Zn²⁺ as the only permeant ion), before application of kainate (also in 3Zn-EB). Note that while the 3Zn-EB alone resulted in an increased outward current in both neurones, subsequent application of kainate (KA) induced an inward current in a Ca-A/K(+) neurone (top trace), while increasing the outward current in a Ca-A/K(-) neurone (bottom trace). B, NAS blocks inward Zn²⁺ currents in Ca-A/K(+) neurones. A Ca-A/K(+) neurone was patched in whole-cell mode and a kainate-induced I-V relationship elicited in the presence of 3Zn-EB alone, or in the additional presence of NAS (300 µM). Note that NAS caused a marked negative shift in V_rev, eliminating the inward Zn²⁺ current at -70 mV. The inset shows the effect of the NAS on a kainate-induced Zn²⁺ current at -90 mV. C, increasing extracellular Zn²⁺ causes a positive shift in V_rev in Ca-A/K(+) neurones: a Ca-A/K(+) neurone was patched in whole-cell mode and kainate I-V response elicited in the presence of Zn²⁺ (0.3 or 3 mM as indicated) as the only permeant cation. The inset shows a shift in V_rev of KA currents in six Ca-A/K(+) neurones upon switching from 0.3Zn-EB to 3Zn-EB.

Several further observations support the contention that these currents reflected Zn²⁺ passage through Ca-A/K channels. First, inward currents were not observed in neurones with RI < 0.8 (n = 3, in which kainate stimulated an outward Cs⁺ current at -70 mV; Fig. 5A). Second, the currents at -70 mV were fully (120 ± 14 %) blocked by 300 µM of the Ca-A/K channel antagonist NAS (n = 6; Fig. 5B). In addition, providing further confirmation that these Zn²⁺ currents are mediated by AMPA channels, both inward Zn²⁺ currents and outward currents were fully blocked (98 ± 26 % block at -70 mV, n = 5) by 20 µM of the AMPA-specific noncompetitive antagonist GYKI 53655 (Wilding & Huettner, 1995). Finally, the current was substantially reduced (to -1.7 ± 7.6 pA, n = 6) when [Zn²⁺]_o was lowered to 0.3 mM (0.3Zn-EB; Fig. 5C).

As with Ca²⁺, V_rev measurements were used to assess the relative Zn²⁺ permeability of Ca-A/K channels. In six Ca-A/K(+) neurones (RI = 0.52 ± 0.04), V_rev shifted from -71.8 ± 1.7 in 0.3Zn-EB to -58.5 ± 1.5 in 3Zn-EB (Fig. 5B). In contrast, if recording was carried out in 3Ca-EB, V_rev in Ca-A/K(+) neurones was -43.3 ± 2.6 mV (RI = 0.46 ± 0.07; n = 6: corresponding current amplitudes at -70 mV were -112.50 ± 19.48 and -444.83 ± 112.85 pA in 3Zn-EB and 3Ca-EB, respectively; see Fig. 6A). As above, values of V_rev in 3 mM Ca²⁺ or Zn²⁺ were related to relative permeability (P_Ca/P_Cs or P_Zn/P_Cs) by the extended constant field equation, which determined P_Ca/P_Cs = 2.3 and P_Zn/P_Cs = 1.3, yielding P_Ca/P_Zn = 1.8.

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Figure 6. Zn²⁺ does not block Ca²⁺ entry through Ca-A/K channels
A, comparative amplitude of currents carried by Zn²⁺ and Ca²⁺ through Ca-A/K channels. A Ca-A/K(+) neurone was patched in whole-cell mode and kainate response at -70 mV recorded sequentially in 3Zn-EB and 3Ca-EB. B, lack of effect of Zn²⁺ on Ca²⁺ currents. Whole-cell KA responses were elicited in Ca-A/K(+) neurones at -70 mV, in 3Ca-EB (left) or in 3Zn-EB (right) first alone, and then in the additional presence of the other cation, as indicated. Bars depict the mean amplitude of the resultant currents, normalized to those obtained in 3Ca-EB or 3Zn-EB alone (n = 7 and 8 neurones, respectively). Note the paucity of change in the Ca²⁺ current upon addition of 0.3 mM Zn²⁺, and the increase in the Zn²⁺ current upon addition of Ca²⁺ (*indicates difference from control condition, P < 0.01 by Student's paired t test).

Interaction between Zn²⁺ and Ca²⁺

To ensure that Zn²⁺ can act as the charge carrier, the above studies were carried out in buffer containing Zn²⁺ as the sole permeant cation. However, because Ca²⁺ is always present in vivo, it is pertinent to consider whether the presence of Zn²⁺ or Ca²⁺ interferes with permeation of the other. Indeed, in the recent report demonstrating Zn²⁺ passage through VSCC on murine cortical neurones (Kerchner et al. 2000), relatively low [Zn²⁺]_o levels markedly attenuated Ca²⁺ currents through the channels. As noted above, kainate-activated Ca²⁺ currents through Ca-A/K channels are of greater amplitude than those carried by Zn²⁺ (Fig. 6A). In Ca-A/K(+) neurones in which kainate-triggered Ca²⁺ currents were recorded in 3Ca-EB, addition of 0.3 mM Zn²⁺ had little effect on the magnitude of the inward current recorded at -70 mV. Because the Ca²⁺ current is far greater than that expected with this concentration of Zn²⁺ alone, it appears that the Ca²⁺ current is little blocked by the presence of Zn²⁺. In contrast, when kainate-induced Zn²⁺ currents (3 mM) were recorded in the presence of 1 mM extracellular Ca²⁺, the magnitude of the current increased (Fig. 6B). However, since Ca-A/K channels have greater permeability for Ca²⁺ than for Zn²⁺, it is possible that a substantial portion of the resultant current is carried by Ca²⁺, and we therefore cannot infer from these data the degree to which the Ca²⁺ interferes with Zn²⁺ permeation through the channels.

Thus, a final set of experiments used fluorescence imaging techniques to address the ability of Ca²⁺ to decrease Zn²⁺ entry through Ca-A/K channels. Hippocampal cultures loaded with the zinc-selective (calcium-insensitive) probe Newport Green were exposed to kainate (100 µM, with Gd³⁺ and MK-801) and Zn²⁺ (0.3 mM), in the further absence or presence of 1.8 mM Ca²⁺. As in Ca²⁺ imaging experiments to pre-identify Ca-A/K(+) neurones, these exposures resulted in a distinct segregation of responses, with 20-30 % of neurones showing particularly abrupt increases (Fig. 7A). Our prior studies have indicated that these rapidly responding neurones are nearly all Ca-A/K(+) as determined by subsequent Co²⁺ staining (Sensi et al. 1999). The presence of this six-fold excess of Ca²⁺ induced only a modest decrement in the mean increase in [Zn²⁺]_i (Fig. 7B), suggesting that physiologically relevant levels of Ca²⁺ interfere little with Zn²⁺ permeation through Ca-A/K channels.

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Figure 7. The presence of extracellular Ca²⁺ has little apparent effect on Zn²⁺ entry through Ca-A/K channels
A, kainate-triggered [Zn²⁺]_i rises in individual neurones. Cultures were loaded with the zinc-sensitive probe Newport Green, and imaged before, during and after a 5 min exposure to kainate (100 µM) with Zn²⁺ (0.3 mM); the extracellular medium was either calcium-free (i, iii) or contained 1.8 mM Ca²⁺ (ii, iv). Pseudocolour fluorescence images (i, ii) depict the Newport Green fluorescence increases ( $Delta$ F) at the end of the 5 min exposure. Zinc-dependent signal increases are shown as pseudocolour ratios of peak to basal fluorescence. Traces (iii, iv) show the time course of $Delta$ F in individual neurones, expressed as the ratio of fluorescence at each time point (F_x) to its own baseline fluorescence (F₀). Note that the marked segregation in [Zn²⁺]_i rises among neurones; those with abrupt $Delta$ F (continuous traces) are considered to be Ca-A/K(+) (see Methods). B, summary data. Bars depict mean increase in Newport Green fluorescence (F_x/F₀) at the termination of 5 min kainate exposures, in the additional presence of the indicated cations. In each experiment, fluorescence changes were averaged within each culture (4-13 Ca-A/K(+), 6-24 Ca-A/K(-) neurones per dish) before compiling results across experiments (n = 4 matched sets of cultures). * $Delta$ F different from the same exposure to Ca-A/K(+) neurones (P < 0.001); # $Delta$ F not different from Ca-A/K(+) neurones upon exposure in Zn²⁺ alone (P > 0.05); P values determined by one-way ANOVA with Tukey's multiple comparison test.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

Recent studies have demonstrated that Zn²⁺ affects the function of many ion channels. However, since vesicular Zn²⁺ appears to be localized in and released from certain excitatory presynaptic terminals, its actions on postsynaptic glutamate receptor function have been of particular interest. The recently demonstrated tonic inhibition of NMDA channels (Vogt et al. 2000) may well be the first clearly documented physiological effect of extracellular synaptically released Zn²⁺.

The present study pertains to another way in which synaptically released Zn²⁺ might affect neuronal function, by crossing the synapse and entering postsynaptic neurones through receptor- or voltage-gated channels. Although imaging and neurotoxicity studies have provided compelling evidence that Zn²⁺ can enter neurones through VSCC and Ca-A/K channels while having minimal ability to permeate NMDA channels, to date direct electrophysiological measurements of Zn²⁺ currents have only been made for high-voltage-activated VSCC (Kerchner et al. 2000). The present study provides novel evidence that extracellular Zn²⁺ can carry substantial currents through Ca-A/K channels. Supporting this conclusion, the putative inward Zn²⁺ currents were observed only in neurones pre-identified as possessing large numbers of Ca-A/K channels (on the basis of fluorescence imaging and/or rectification properties). Furthermore, the kainate-triggered inward currents that were elicited in the presence of VSCC and NMDA antagonists, were dependent upon the presence of Zn²⁺ in the extracellular fluid, and were reversibly blocked by the Ca-A/K channel antagonist, NAS.

Zn²⁺ permeation: comparison with NMDA channels and VSCC

Although the Zn²⁺ currents are smaller than those carried by equimolar Ca²⁺ through these channels (P_Ca/P_Zn ~ 1.8), inward currents elicited at -70 mV in 3 mM [Zn²⁺]_o averaged near 100 pA. Since the net current comprises an outward Cs⁺ component, the actual size of the Zn²⁺ flux is undoubtedly greater. These currents are substantially larger than the maximal whole-cell Zn²⁺ currents through VSCC (~20 pA) reported in cultured cortical neurones (Kerchner et al. 2000).

Interestingly, Ca-A/K channels appear to have very distinct permeation properties from either NMDA channels or VSCC. NMDA channels are characterized by a high degree of selectivity among divalent cations, being very permeable to Ca²⁺ and Ba²⁺ (with peak P_Ca/P_Cs near 6; McDermott et al. 1986; Iino et al. 1990; Wollmuth & Sakmann, 1998), but with little permeability for other divalent cations, including the physiologically relevant ions Mg²⁺ and Zn²⁺, both of which are effective channel blockers (Nowak et al. 1984; Peters et al. 1987; Westbrook & Mayer, 1987). This is distinct from Ca-A/K channels, which have been found to be permeable to Mg²⁺ as well as a number of other divalent cations (Iino et al. 1990; Hollmann et al. 1991), although Zn²⁺ permeation through these channels has not been studied previously.

In contrast to NMDA channels, the VSCC channel pore appears to be less selective, being permeable to Na⁺ in the absence of Ca²⁺, and showing permeability for Zn²⁺ as well Ca²⁺ (Fukuda & Kawa, 1997; Kerchner et al. 2000). However, these ions do not permeate independently of each other; Ca²⁺ blocks Na⁺ flux (Almers & McCleskey, 1984; Tsien et al. 1987), and Zn²⁺ is able to block Ca²⁺ flux (Winegar & Lansman, 1990; Büsselberg et al. 1992; Kerchner et al. 2000). This is in contrast to our present observations on Ca-A/K channels, which appear able to simultaneously flux multiple divalent cations with relatively little mutual interference. Peak synaptic Zn²⁺ levels occurring with strong presynaptic activation are estimated to be in the order of 300 µM (Assaf & Chung, 1984; Howell et al. 1984). This level of Zn²⁺ (reported in Kerchner et al. (2000) to decrease VSCC Ca²⁺ currents by ~60 %) appears to have little effect on Ca²⁺ flux. Furthermore, using imaging techniques to examine the converse, a six-fold excess of Ca²⁺ (1.8 mM) had relatively little apparent effect on the ability of this level of Zn²⁺ to permeate the channels. Similar behaviour has been observed in nicotinic acetylcholine channels, in which the permeation of either Ca²⁺ or Zn²⁺ is little affected by the presence of the other (Ragozzino et al. 2000).

Physiological implications

While the physiological significance of Zn²⁺ translocation is presently poorly understood, this phenomenon is likely to be of importance to both physiological signalling as well as the pathophysiology of selective neuronal degeneration in certain disease states. Vesicular Zn²⁺ appears to be localized to forebrain and limbic regions, where it is present in some, but not all pathways. For instance, in the hippocampus, where its potential roles have been studied most, it is present at particularly high levels in the dentate granule cell mossy fibres, and is also found in CA3 and CA1 neurones and their projections, but is not present in subicular neurones (Frederickson, 1989; Frederickson et al. 2000). Specific roles for Zn²⁺ entry through Ca-A/K channels, however, might be expected to depend upon pairing of presynaptic vesicular Zn²⁺ and postsynaptic Ca-A/K channels. When such a congruence of presynaptic source and postsynaptic entry route occurs, given the relatively high permeability of Ca-A/K channels to Zn²⁺, it seems likely that these channels would constitute the dominant route of Zn²⁺ entry.

One place where such as apposition is likely to occur is on GABAergic interneurones, which constitute a substantial portion of the Ca-A/K(+) hippocampal neurones from which we have recorded. Indeed, in some cases, postsynaptic Ca-A/K channels have been specifically identified on GABAergic neurones at sites of innervation by zinc-containing mossy fibres (Ascady et al. 1998; Toth & McBain, 1998). Thus, it is likely that with strong presynaptic activity, Zn²⁺ directly enters these neurones through Ca-A/K channels. Although specific physiological effects of such Zn²⁺ entry have not been elucidated, perhaps it is a factor in the high susceptibility of many hilar GABAergic interneurones to injury after epilepsy (Sloviter, 1991; for review see Houser, 1999).

One consequence of Zn²⁺ translocation that has received considerable attention is its apparent role in the degeneration of hippocampal pyramidal neurones associated with epilepsy or ischaemia. Specifically, it appears that in these conditions, synaptically released Zn²⁺ enters and contributes to injury of CA3 and CA1 pyramidal neurones (Frederickson, 1989; Tonder et al. 1990; Koh et al. 1996). Although in general, electrophysiological studies have not found evidence for the presence of Ca-A/K channels on adult pyramidal neurones, several lines of evidence suggest the possibility that they may be present in the distal dendrites of at least some of these neurones (Lerma et al. 1994; Yin et al. 1999). Indeed, we have recently found that the Ca-A/K channel antagonist NAS decreases histochemically detected Zn²⁺ accumulation in CA3 and CA1 pyramidal neurones in a slice model of ischaemia, suggesting the possibility that synaptically released Zn²⁺ enters these neurones through Ca-A/K channels (Yin et al. 2002). In addition, other studies have raised the intriguing possibility that irrespective of their levels under basal conditions, numbers of Ca-A/K channels in pyramidal neurones might increase after epilepsy or ischaemia (Bennett et al. 1996; Pellegrini-Giampietro et al. 1997), perhaps permitting consequent increases in Zn²⁺ entry through these channels.

It seems likely that Zn²⁺ entry through Ca-A/K channels will prove to have physiological significance independent of injury induction. Recent studies have suggested that endogenous Zn²⁺ translocation might play a role in hippocampal CA3 long-term potentiation (Lu et al. 2000; Li et al. 2001), and Zn²⁺ might be well suited to be a mediator of recently described forms of plasticity in GABAergic interneurones (Ross & Soltesz, 2001). Trans-synaptic signalling by this highly biologically active ion has been little explored. In comparison to Ca²⁺, which is present in the extracellular fluid at high levels and enters neurones through widely expressed NMDA channels, Zn²⁺ could be an ionic signal providing much greater specificity, mediating effects predominantly where presynaptic sources come together with postsynaptic Ca-A/K channels.

REFERENCES

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WEISS, J. H., HARTLEY, D. M., KOH, J. Y. & CHOI, D. W. (1993). AMPA receptor activation potentiates zinc neurotoxicity. Neuron 10, 43-49 [Medline]

WEISS, J. H. & SENSI, S. L. (2000). Ca²⁺-Zn²⁺ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration. Trends in Neurosciences 23, 365-371 [Medline]

WESTBROOK, G. L. & MAYER, M. L. (1987). Micromolar concentrations of Zn²⁺ antagonize NMDA and GABA responses of hippocampal neurones. Nature 328, 640-643 [Medline]

WILDING, T. J. & HUETTNER, J. E. (1995). Differential antagonism of $alpha$ -amino-3-hydroxy-5-methyl-4-isoxazole propionic acid preferring and kainate preferring receptors by 2, 3-benzodiazepines. Molecular Pharmacology 47, 582-587 [Abstract]

WINEGAR, B. D. & LANSMAN, J. B. (1990). Voltage-dependent block by zinc of single calcium channels in mouse myotubes. Journal of Physiology 425, 563-578 [Abstract]

WOLLMUTH, L. P. & SAKMANN, B. (1998). Different mechanisms of Ca²⁺ transport in NMDA and Ca²⁺-permeable AMPA glutamate receptor channels. Journal of General Physiology 112, 623-636 [Abstract/Full Text]

YIN, H. Z., HA, D. H., CARRIEDO, S. G. & WEISS, J. H. (1998). Kainate-stimulated Zn²⁺ uptake labels cortical neurons with Ca²⁺-permeable AMPA/kainate channels. Brain Research 781, 45-55 [Medline]

YIN, H. Z., SENSI, S. L., CARRIEDO, S. G. & WEISS, J. H. (1999). Dendritic localization of Ca²⁺-permeable AMPA/kainate channels in hippocampal pyramidal neurons. Journal of Comparative Neurology 409, 250-260 [Medline]

YIN, H. Z., SENSI, S. L., OGOSHI, F. & WEISS, J. H. (2002). Blockade of Ca²⁺-permeable AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn²⁺ accumulation and neuronal loss in hippocampal pyramidal neurons. Journal of Neuroscience 22, 1273-1279 [Abstract/Full Text]

YIN, H. Z., TURETSKY, D., CHOI, D. W. & WEISS, J. H. (1994). Cortical neurons with Ca²⁺-permeable AMPA/kainate channels display distinct receptor immunoreactivity and are GABAergic. Neurobiology of Disease 1, 43-49 [Medline]

YIN, H. Z. & WEISS, J. W. (1995). Zn²⁺ permeates Ca²⁺-permeable AMPA/kainate channels and triggers selective neural injury. Neuroreport 6, 2553-2556 [Medline]

Acknowledgements

We thank Dr. Simin Amindari for expert assistance with cell cultures. This work was supported by NIH grants NS30884 and AG00836 (J. H.W.), AG00919 (S.L.S.), a grant from the Alzheimer's Association (J.H.W.), and an AHA Predoctoral Fellowship 0110032Y (J.-M.J.).

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WOLLMUTH, L. P. & SAKMANN, B. (1998). Different mechanisms of Ca²⁺ transport in NMDA and Ca²⁺-permeable AMPA glutamate receptor channels. Journal of General Physiology 112, 623-636	[Abstract/Full Text]
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YIN, H. Z., SENSI, S. L., CARRIEDO, S. G. & WEISS, J. H. (1999). Dendritic localization of Ca²⁺-permeable AMPA/kainate channels in hippocampal pyramidal neurons. Journal of Comparative Neurology 409, 250-260	[Medline]
YIN, H. Z., SENSI, S. L., OGOSHI, F. & WEISS, J. H. (2002). Blockade of Ca²⁺-permeable AMPA/kainate channels decreases oxygen-glucose deprivation-induced Zn²⁺ accumulation and neuronal loss in hippocampal pyramidal neurons. Journal of Neuroscience 22, 1273-1279	[Abstract/Full Text]
YIN, H. Z., TURETSKY, D., CHOI, D. W. & WEISS, J. H. (1994). Cortical neurons with Ca²⁺-permeable AMPA/kainate channels display distinct receptor immunoreactivity and are GABAergic. Neurobiology of Disease 1, 43-49	[Medline]
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				L. J. Blakemore, M. Resasco, M. A. Mercado, and P. Q. Trombley Evidence for Ca2+-permeable AMPA receptors in the olfactory bulb Am J Physiol Cell Physiol, March 1, 2006; 290(3): C925 - C935. [Abstract] [Full Text] [PDF]

				T. G. Smart, A. M. Hosie, and P. S. Miller Zn2+ Ions: Modulators of Excitatory and Inhibitory Synaptic Activity Neuroscientist, October 1, 2004; 10(5): 432 - 442. [Abstract] [PDF]

				A.-L. Prost, A. Bloc, N. Hussy, R. Derand, and M. Vivaudou Zinc is both an intracellular and extracellular regulator of KATP channel function J. Physiol., August 15, 2004; 559(1): 157 - 167. [Abstract] [Full Text] [PDF]