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

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.