ABSTRACT

Estradiol regulates thyroid function, and chloride channels are involved in the regulation of thyroid function. However, little is known about the role of chloride channels in the regulation of thyroid functions by estrogen. In this study, the effects of estrogen on chloride channel activities in human thyroid Nthy-ori3-1 cells were therefore investigated using the whole cell patch-clamp technique. The results showed that the extracellular application of 17β-estradiol (E2) activated Cl⁻ currents, which reversed at a potential close to Cl⁻ equilibrium potential and showed remarkable outward rectification and an anion permeability of I⁻ > Br⁻ > Cl⁻ > gluconate. The Cl⁻ currents were inhibited by the chloride channel blockers, NPPB and tamoxifen. Quantitative Real-time PCR results demonstrated that ClC-3 expression was highest in ClC family member in Nthy-ori3-1 cells. The down-regulation of ClC-3 expression by ClC-3 siRNA inhibited E2-induced Cl⁻ current. The Cl⁻ current was blocked by the estrogen receptor antagonist, ICI 182780 (fulvestrant). Estrogen receptor alpha (ERα) and not estrogen receptor beta was the protein expressed in Nthy-ori3-1 cells, and the knockdown of ERα expression with ERα siRNA abolished E2-induced Cl⁻ currents. Estradiol can promote the accumulation of ClC-3 in cell membrane. ERα and ClC-3 proteins were partially co-localized in the cell membrane of Nthy-ori3-1 cells after estrogen exposure. The results suggest that estrogen activates chloride channels via ERα in normal human thyroid cells, and ClC-3 proteins play a pivotal role in the activation of E2-induced Cl⁻ current.

摘要

雌二醇调节甲状腺功能，氯离子通道参与甲状腺功能的调节。然而，关于氯离子通道在雌激素调节甲状腺功能中的作用知之甚少。因此，在本研究中，使用全细胞膜片钳技术研究了雌激素对人甲状腺 Nthy-ori3-1 细胞中氯离子通道活性的影响。结果表明，17β-雌二醇（E2）的细胞外应用激活了Cl ^-电流，该电流在接近Cl ^-平衡电位的电位处反转，并显示出显着的向外整流和I ^- > Br ^- > Cl ^- > 葡萄糖酸盐的阴离子渗透性。 . 氯^-电流受到氯离子通道阻滞剂、NPPB 和他莫昔芬的抑制。定量实时PCR结果表明ClC-3在Nthy-ori3-1细胞中ClC家族成员中的表达最高。ClC-3 siRNA对ClC-3表达的下调抑制了E2诱导的Cl ^-电流。Cl ^-电流被雌激素受体拮抗剂ICI 182780（氟维司群）阻断。Nthy-ori3-1 细胞中表达的蛋白质是雌激素受体 α (ERα) 而不是雌激素受体 β，用 ERα siRNA 敲低 ERα 表达消除了 E2 诱导的 Cl ^-电流。雌二醇可以促进ClC-3在细胞膜中的积累。雌激素暴露后，ERα 和 ClC-3 蛋白部分共定位于 Nthy-ori3-1 细胞的细胞膜中。结果表明，雌激素通过ERα激活正常人甲状腺细胞中的氯离子通道，而ClC-3蛋白在E2诱导的Cl ^-电流激活中起关键作用。