Figure 3. Increases in [Zn²⁺]_i activate the Ca_V3.2 promoter.

(a) NG108-15 cells transfected with the 1,188-bp Ca_V3.2 promoter–luciferase reporter construct²⁰ and stimulated with K⁺+Zn²⁺ in the presence of increasing Zn²⁺ concentrations (0, 50, 100, 150, 200, 250 and 300 μM). The effect on the promoter activity was determined using a luciferase assay 4 h after stimulation (one-way analysis of variance (ANOVA): P<0.001; F_(7,16)=25.64; Tukey's multiple comparisons test, *P≤0.05, ***P≤0.001; n=3). (b) Activity of the Ca_V3.2 promoter–luciferase reporter gene determined after stimulation of NG108-15 cells first with K⁺ or K⁺+Zn²⁺ (50 mM/500 μM) solutions for 1 h and subsequently incubated in the presence or absence of TPEN (10 μM) for 1 h. Luciferase activity was measured 4 h after stimulation (one-way ANOVA: P<0.001; F_(5,10)=24.63; Tukey's multiple comparisons test,*P≤0.05, ***P≤0.001; n≥3). (c) Luciferase activity of three Ca_V3.2 promoter deletion fragments²⁰ after stimulation with K⁺+Zn²⁺ (50 mM/200 μM). Only the Ca_V3.2-1020 deletion fragment showed significant activation of the Ca_V3.2 promoter after stimulation with K⁺+Zn²⁺. The short Ca_V3.2-105 showed a significantly reduced activity after stimulation with K⁺+Zn²⁺, likely due to the presence of Zn²⁺-inhibitory regions within this fragment (one-way ANOVA: P<0.001; F_(5,12)=39.82; Tukey's multiple comparisons test, **P≤0.01,***P≤0.001; n=3).