Role of trace metal sensitive voltage-gated Ca2+ channels for neuroprotective and neurotoxic actions of endogenous Zn2+ and Cu2+ in the limbic system.

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Losely bound (histochemically reactive) Zn2+ and Cu2+ ions are putative regulators of neuronal transmission and limit pathologic hyper-excitability in limbic pathways, but they can also contribute to seizure-related excitotoxic cell death. The renewal proposal continues to focus on the two voltage-gated Ca2+ channels (Cav2.3 and Cav3.2), which are critically involved in synaptic transmission and among the few molecular targets sensitive to resting Cu2+ and Zn2+ levels in the brain. We have previously shown and are confirming in our report that their genetic or pharmacologic ablation provides significant protection from kainic acid (KA)-induced limbic seizures. Up to now our experiments have focused on the effects of Zn2+ on Cav2.3 channels in-vitro and in Cav2.3-deficient and Cav2.3-competent mice. Micromolar levels of Zn2+ alter gating and permeation of the cloned human Cav2.3-channel in a concentration-dependent manner. Pathophysiological changes in pH, as they occur during epileptic seizures, differently alter the potency of these Zn2+ effects on Cav2.3. In Cav2.3-competent mice, micromolar Zn2+ concentrations provoke behavioral and electrophysiological changes, which are not found in Cav2.3-deficient mice.During the extension phase, we will mainly assess the role of these channels for the anti-convulsive and neuroprotective effects of Cu2+ at rest, and for the toxic effects of cellular Cu2+ (and Zn2+) entry during seizures. In vivo telemetric electrocorticography will be complemented by immunhistochemical and autometallographic post-mortem analysis to assess effects of Cu2+ (and Zn2+) on normal neurotransmission and KA-induced seizures in wild-type mice and mice lacking the respective Ca2+ channel subunits. We will continue with in vitro patch-clamp recordings in stably transfected HEK-293 cells, and will begin with native neurons and hippocampal slices to characterize cellular and molecular mechanisms of action under conditions of minimal interference from other parameters. Our project still provides functional insights into the relevance of Zn2+ for neuronal excitability under physiological conditions and during excessive activation of the limbic system, which will be extended to the relevance of Cu2+ for these processes. The applied extension may not only provide a better understanding of the apparent dualism of protective vs. toxic Zn2+ (and Cu2+) actions in the brain but could also help in guiding the development of preventive or interventional approaches based on a manipulation of brain Cu2+ or Zn2+ levels.