Figure 5: MTF1 binds the Ca_V3.2 promoter.

(a) Luciferase activity of the Ca_V3.2 promoter deletion fragments after overexpression with MTF1. Note the almost similar activation pattern of the Ca_V3.2 promoter deletion fragments as seen after stimulation with K⁺+Zn²⁺ (50 mM/200 μM; Fig. 3c). No activation was observed for the pGL3-basic control plasmid (one-way analysis of variance (ANOVA): P=0.012; F_(7,16)=3.856; Tukey’s multiple comparisons test, *P≤0.05; n=3). (b) Luciferase activity of the Ca_V3.2-1020 fragment and the Ca_V3.2-1020 fragment with the mutated Zn²⁺-sensitive MRE-binding site (Ca_V3.2-1020-MRE-mut) after overexpression with MTF1. Mutation of the Zn²⁺-sensitive MRE-binding site resulted in a reduced Ca_V3.2 promoter activity (two-way ANOVA: P=0.0002; F_(1,8)=38.9. (c) ChIP analysis of MTF1 binding to the Zn²⁺-sensitive MRE within the Ca_V3.2 promoter. PCR amplicons were generated of anti-MTF1 ChIP immunoprecipitates from NG108-15 cells and mouse hippocampi, using primer pairs spanning the Zn²⁺-sensitive MRE and a control region in the Ca_V3.2 promoter lacking a MRE. A rabbit-IgG immunoprecipitate was used as negative control. (d) Luciferase activity of unstimulated and K⁺+Zn²⁺-challenged (50 mM/200 μM) NG108-15 cells transfected with the full-length Ca_V3.2 promoter–luciferase reporter construct and MTF1 or MTF1ΔC (one-way ANOVA: P<0.001; F_(4,10)=117.9; Tukey’s multiple comparisons test, **P≤0.01, ***P≤0.001; n≥3).