Figure 8. E2 upregulates Kcnma1 mRNA and BK current in Kiss1^ARH neurons.

(A) Representative traces of the inhibition of outward currents before (left, control) and after the specific BK blocker iberiotoxin (IbTx; 200 nM, middle). IbTx-sensitive currents were calculated from the subtraction of control and IbTx at depolarized potentials (right). Cells were clamped at –70 mV and given 500 ms voltage pulses from –60 mV to +40 mV in 10 mV steps at 0.2 Hz, as shown in (A) at the bottom. (B) Mean current density–voltage relationships measured at the end of the 500 ms voltage step ranging from –60 mV to +40 mV were obtained in the absence and presence of IbTx (two-way ANOVA: main effect of treatment [F_{(1, 8)} = 0.8841, p=0.3746], main effect of voltage [F_{(10, 80)} = 71.56, p<0.0001] and interaction [F_{(10, 80)} = 1.127, p=0.3528]; mean ± SEM, n = 5; post hoc Bonferroni test, p>0.05). (C) IbTX-sensitive current densities were obtained from (B) (mean ± SEM, n = 5). (D) Representative traces of the inhibition of outward currents before (left, control) and after the specific BK blocker iberiotoxin (IbTx; 200 nM, middle). IbTx-sensitive currents resulted from the subtraction of control and IbTx at depolarized potentials (right). (E) Mean current density–voltage relationships measured at the end of the 500 ms voltage step ranging from –60 mV to +40 mV were obtained in the absence and presence of IbTX (two-way ANOVA: main effect of treatment [F_{(1, 10)} = 4.660, p=0.0562], main effect of voltage [F_{(10, 100)} = 63.98, p<0.0001] and interaction [F_{(10, 100)} = 4.907, p<0.0001]; mean ± SEM, n = 6; post hoc Bonferroni test, *p<0.05, **p<0.01, ****p<0.001). (F) IbTx-sensitive current densities were obtained from (C) and (E) (two-way ANOVA: main effect of treatment [F_{(1, 9)} = 22.04, p=0.0011], main effect of voltage [F_{(10, 90)} = 78.26, p<0.0001] and interaction [F_{(10, 90)} = 17.84, p<0.0001]; mean ± SEM, OVX, n = 5; OVX + E2, n = 6; Bonferroni post hoc test, *p<0.05, ***p<0.005, ****p<0.001). (G) Kiss1^ARH neurons (three 10-cell pools) were harvested from each of five vehicle- and five E2-treated, OVX females to quantify the mRNA expression of Kcnma1 (BKα1) channel. E2-treatment increased the mRNA expression of Kcnma1. The expression values were calculated via the ΔΔCT method, normalized to Gapdh and relative to the oil control values. Bar graphs represent the mean ± SEM, with data points representing individual animals (unpaired two-tailed t-test for BK, t₍₈₎ = 3.479, **p=0.0083). (H) The mathematical model was calibrated to reproduce the current–voltage relationship observed in Kiss1^ARH neurons before and after treatment with IbTx. For the calibration, it was assumed that the applied concentration of IbTx (200 nM) completely blocked the BK current. The modeled IbTx -sensitive current with g_BK = 20.0 nS (right panel) matches the electrophysiological data from OVX + E2 animals.