Induction of T-type calcium channel Cav3.2 expression by zinc via MTF-1 promoter activation

Rationale: Brain neurons contain substantial amounts of Zn²⁺, which plays multiple roles in cellular functions. Whereas the release of Zn²⁺ into the interstitium reportedly modulate 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 Cav2.3 and Cav3.1–3.3. In NG108–15 neuroblastoma cells, transient exposure to 200 uM Zn²+ under depolarizing conditions (50 mM KCl) led to Zn²+ influx via L-type (BayK-sensitive) Ca²+ channels, and to increased mRNA transcription of Cav3.2, but not of other R/T-type Ca²+ channels.

Results: Promoter analysis of Cav3.2 revealed a 1.164bp fragment within the 5' UTR-flanking genomic sequence harboring several metal regulatory elements (MRE) for the metal-responsive transcription factor-1 (MTF-1). In a Luciferase assay, exposure to Zn²+ strongly induced transcription of this 1.164bp Cav3.2 promoter fragment. Similar effects were induced by the NO-donor Na⁺ nitroprusside, which causes liberation of intracellular Zn²+ 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 Cav3.2 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 Cav3.2 promoter activation.

Conclusions: Our findings implicate Cav3.2 as a novel target for MTF-1-mediated transcriptional upregulation via transient increases in intracellular free Zn²⁺. Since brain injuries are accompanied by marked elevations in intracellular free Zn²⁺, and since Cav3.2 upregulation profoundly enhances neuronal discharge, we propose that the novel upregulation of Cav3.2 transcription by zinc may be a key process in injury-induced epileptogenesis.