Abstract
The pore-forming Ca(2+) channel subunit Ca(V)3.2 mediates a low voltage-activated (T-type) Ca(2+) current (I(CaT)) that contributes pivotally to neuronal and cardiac pacemaker activity. Despite the importance of tightly regulated Ca(V)3.2 levels, the mechanisms regulating its transcriptional dynamics are not well understood. Here, we have identified two key factors that up- and down-regulate the expression of the gene encoding Ca(V)3.2 (Cacna1h). First, we determined the promoter region and observed several stimulatory and inhibitory clusters. Furthermore, we found binding sites for the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) to be highly overrepresented within the Ca(V)3.2 promoter region. mRNA expression analyses and dual-luciferase promoter assays revealed that the Ca(V)3.2 promoter was strongly activated by Egr1 overexpression in vitro and in vivo. Subsequent chromatin immunoprecipitation assays in NG108-15 cells and mouse hippocampi confirmed specific Egr1 binding to the Ca(V)3.2 promoter. Congruently, whole-cell I(CaT) values were significantly larger after Egr1 overexpression. Intriguingly, Egr1-induced activation of the Ca(V)3.2 promoter was effectively counteracted by the repressor element 1-silencing transcription factor (REST). Thus, Egr1 and REST can bi-directionally regulate Ca(V)3.2 promoter activity and mRNA expression and, hence, the size of I(CaT). This mechanism has critical implications for the regulation of neuronal and cardiac Ca(2+) homeostasis under physiological conditions and in episodic disorders such as arrhythmias and epilepsy.
Highlights
Expression of the T-type Ca2ϩ-channel CaV3.2 has to be tightly regulated for proper calcium homeostasis
We found binding sites for the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) to be highly overrepresented within the CaV3.2 promoter region. mRNA expression analyses and dual-luciferase promoter assays revealed that the CaV3.2 promoter was strongly activated by Egr1 overexpression in vitro and in vivo
We have defined a regulatory element in the upstream CaV3.2 promoter that mediates activation of CaV3.2 transcription by Egr1
Summary
Expression of the T-type Ca2ϩ-channel CaV3.2 has to be tightly regulated for proper calcium homeostasis. Results: Overexpression of the transcription factor Egr strongly activates the CaV3.2 promoter and can be counteracted by the repressor REST. Egr and REST can bi-directionally regulate CaV3.2 promoter activity and mRNA expression and, the size of ICaT. This mechanism has critical implications for the regulation of neuronal and cardiac Ca2؉ homeostasis under physiological conditions and in episodic disorders such as arrhythmias and epilepsy. Our analyses show for the first time that the transcription factor early growth response 1 (Egr1/Zif268/Krox-24) mediates CaV3.2 promoter activation. This effect of Egr is potently antagonized by the transcriptional repressor REST. The functional interactions described here may have important implications for CaV3.2 regulation under physio- and pathological conditions
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