Estradiol activates chloride channels via estrogen receptor-α in the cell membranes of osteoblasts

Estrogen plays important roles in regulation of bone formation. Cl − channels in the ClC family are expressed in osteoblasts and are associated with bone physiology and pathology, but the relationship between Cl − channels and estrogen is not clear. In this study the action of estrogen on Cl − channels was investigated in the MC3T3-E1 osteoblast cell line. Our results show that 17β-estradiol could activate a current that reversed at a potential close to the Cl − equilibrium potential, with a sequence of anion selectivity of I − > Br − > Cl − > gluconate, and was inhibited by the Cl − channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate and 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid. Knockdown of ClC-3 Cl − channel expression by a specific small interfering RNA to ClC-3 attenuated activation of the 17β-estradiol-induced Cl − current. Extracellular application of membrane-impermeable 17β-estradiol-albumin conjugates activated a similar current. The estrogen-activated Cl − current could be inhibited by the estrogen receptor (ER) antagonist fulvestrant (ICI 182780). The selective ERα agonist, but not ERβ agonist, activated a Cl − current similar to that induced by 17β-estradiol. Silencing ERα expression prevented activation of estrogen-induced currents. Immunofluorescence and coimmunoprecipitation experiments demonstrated that ClC-3 Cl − channels and ERα were colocalized and closely related in cells. Estrogen promoted translocation of ClC-3 and ERα to the cell membrane from the nucleus. In conclusion, our findings show that Cl − channels can be activated by estrogen via ERα on the cell membrane and suggest that the ClC-3 Cl − channel may be one of the targets of estrogen in the regulation of osteoblast activity.