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 17 beta- 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 17 beta-estradiol-induced Cl- current. Extracellular application of membrane-impermeable 17 beta-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 ER alpha agonist, but not ER beta agonist, activated a Cl- current similar to that induced by 17 beta-estradiol. Silencing ER alpha expression prevented activation of estrogeninduced currents. Immunofluorescence and coimmunoprecipitation experiments demonstrated that ClC-3 Cl- channels and ER alpha were colocalized and closely related in cells. Estrogen promoted translocation of ClC-3 and ER alpha to the cell membrane from the nucleus. In conclusion, our findings show that Cl- channels can be activated by estrogen via ER alpha 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.