Figure 4. E2 increases the mRNA expression of volatge-activated Ca²⁺ channels and Hcn1 channels in Kiss1^ARH neurons.

(A) E2 increases the expression of low and high voltage-activated calcium channels in Kiss1^ARH neurons. Kiss1^ARH neurons (three 10-cell pools) were harvested from each of five vehicle- and five E2-treated, OVX females to quantify ion channel mRNA expression of low and high voltage-activated calcium channels as described in the ‘Materials and methods’. The analysis included T-type (Cacna1g) low voltage-activated, as well as the following high voltage-activated channels: R-type (Cacna1e), L-type (Cacna1c), N-type (Cacna1b), and P/Q-type (Cacna1a) calcium channels. Interestingly, all of these channels were upregulated with E2 treatment, which significantly increased the whole-cell calcium current (see Figure 5). Bar graphs represent the mean ± SEM, with data points representing individual animals (oil versus E2, unpaired t-test for Cacna1g, t₍₈₎ = 7.105, ***p=0.0001; for Cacna1e, t₍₈₎ = 3.007, *p=0.0169; for Cacna1c, t₍₈₎ = 2.721, *p=0.0262; for Cacna1b, t₍₈₎ = 4.001, **p=0.0039; for Cacna1a, t₍₈₎ = 4.225, **p=0.0028). (B) The same Kiss1^ARH neuronal pools were also analyzed for mRNA expression of Hcn1 ion channels, and E2 also increased the expression of hyperpolarization-activated, cyclic-nucleotide gated Hcn1 channels in Kiss1^ARH neurons. Hcn1 channel mRNA expression was the most highly upregulated by E2 treatment in Kiss1^ARH neurons. The expression values were calculated via the ΔΔCT method, normalized to Gapdh and relative to the oil control values (oil versus E2, unpaired t-test, t₍₈₎ = 11.450, ****p<0.0001).