Abstracts

Rationale: Brain neurons contain substantial amounts of Zn²⁺, which plays multiple roles in cellular functions. Whereas the release of Zn²⁺ into the interstitium reportedly modulates various neurotransmitter receptors and ion channels, only little is known about the effects of intracellular Zn²⁺ (Zn²⁺_in) on transcription of genes critical for neuronal excitability. Methods: Here, we address whether Zn²⁺_in controls the transcription of Ni²⁺-sensitive Ca²⁺ channels, i.e. R/T-type Ca²⁺ currents mediated by the α₁ subunits Ca_V2.3 and Ca_V3.1-3.3. In NG108-15 neuroblastoma cells, transient exposure to 200 µM Zn²⁺ under depolarizing conditions (50 mM KCl) led to Zn²⁺ influx via L-type (BayK-sensitive) Ca²⁺ channels, and to increased mRNA transcription of Ca_V3.2, but not of other R/T-type Ca²⁺ channels. Results: Promoter analysis of Ca_V3.2 revealed a 1.164bp fragment within the 5' UTR-flanking genomic sequence harboring several MREs for MTF-1. In a Luciferase assay, exposure to Zn²⁺ strongly induced transcription of this 1.164bp Ca_V3.2 promoter fragment. Similar effects were induced by the NO-donor Na⁺ nitroprusside, causes liberation of Zn²⁺_in bound to endogenous chelators. In an electrophoretic mobility shift assay in NG108-15 cells, Zn²⁺ exposure resulted in increased binding of MTF-1 to MREs. Overexpression of MTF-1 was sufficient to augment Ca_V3.2 promoter activation levels, as after exposure to Zn²⁺. Functional inhibition of MTF-1 by overexpression of a dominant negative MTF-1 construct reversed the effects of Zn²⁺ on Ca_V3.2 promoter activation. Conclusions: Our findings implicate Ca_V3.2 as a novel target for MTF-1-mediated transcriptional upregulation via transient increases in free Zn²⁺_in. Since brain injuries are accompanied by marked elevations in free Zn²⁺_in, and since Ca_V3.2 upregulation profoundly enhances neuronal discharge, we propose that the novel upregulation of Ca_V3.2 transcription by zinc may be a key process in injury-induced epileptogenesis.