Using the human mammary epithelial cell line MCF-7, we have investigated volume-activated changes in response to hyposmotic stress. Switching MCF-7 cells from an isosmotic to a hyposmotic solution resulted in an initial cell swelling response, followed by a regulatory volume decrease (RVD). This RVD response was inhibited by the nonselective K(+) channel inhibitors Ba(2+), quinine, and tetraethylammonium chloride, implicating K(+) channel activity in this volume-regulatory mechanism. Additional studies using chromonol 293B and XE991 as inhibitors of the KCNQ1 K(+) channel, and also a dominant-negative NH(2)-terminal truncated KCNQ1 isoform, showed complete abolition of the RVD response, suggesting that KCNQ1 plays an important role in regulation of cell volume in MCF-7 cells. We additionally confirmed that KCNQ1 mRNA and protein is expressed in MCF-7 cells, and that, when these cells are cultured as a polarized monolayer, KCNQ1 is located exclusively at the apical membrane. Whole cell patch-clamp recordings from MCF-7 cells revealed a small 293B-sensitive current under hyposmotic, but not isosmotic conditions, while recordings from mammalian cells heterologously expressing KCNQ1 alone or KCNQ1 with the accessory subunit KCNE3 reveal a volume-sensitive K(+) current, inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, regulating cell volume and potentially mediating transepithelial K(+) secretion.

Pharmacological inhibitors of the human ether-ago-go (hEAG) potassium channel, astemizole and imipramine, have been used to demonstrate that hEAG plays a role in cancer cell proliferation. Astemizole and imipramine are, however, relatively non-specific ion channel blockers, as astemizole can also block the related potassium channel, human ether-ago go related (hERG). Therefore, we aimed to determine the molecular target of astemizole, in the human mammary carcinoma cell line MCF-7. We initially confirmed the expression of KCNH1 and KCNH2 mRNA and hEAG and hERG channel protein in MCF-7 cells. Using a [ 3 H]thymidine incorporation assay we determined that astemizole inhibited MCF-7 cell proliferation, whereas the hERGspecific channel blocker E-4031 had no effect. We then determined that E-4031 inhibited the regulatory volume decrease (RVD) observed in these cells following exposure to hypotonic solutions, confirming that functional hERG channels are present and may be important for cell volume regulation in MCF-7 cells. Our results suggest, for the first time, that hERG is involved in cell volume regulation. In addition, the function of hEAG and hERG in MCF-7 cell proliferation can be separated pharmacologically by utilizing the channel inhibitors astemizole and E-4031. The hEAG channel function in MCF-7 cells appears to be involved in the regulation of cell proliferation, whereas hERG is involved in cell volume regulation.

Transepithelial ion transport across mammary epithelial cells underlies milk secretion and breast cyst formation. However, little is known about the molecular nature of the K+ channels involved. We have investigated the location and functionality of the KCNQ1 K+ channel in the mammary epithelial cell line MCF‐7. Using RT‐PCR, we determined that KCNQ1, as well as its accessory subunits KCNE1‐3, were present in MCF‐7 cells. KCNQ1 protein expression was confirmed by Western blotting and immunolocalization with confocal microscopy. When cells were cultured as a polarized monolayer, KCNQ1 was located in the apical membrane. We next investigated the potential role this channel may play in the regulatory volume decrease (RVD) of these cells. Switching from an isotonic to a hypotonic solution resulted in an initial cell swelling, followed by an RVD response. This could be inhibited by the KCNQ1 inhibitors chromonol 293B and XE991, and also when a dominant‐negative N‐terminus truncated KCNQ1 isoform was transfected into MCF‐7 cells. Since MCF‐7 cells are also known to express hIK (KCNN4) and BK (KCNMA1) channels, implicated in the RVD response in other cell types, we additionally investigated the ability of MCF‐7 cells to RVD in the presence of clotrimazole and charybdotoxin. Neither agent inhibited the RVD in these cells. Finally, whole cell recordings from baby hamster kidney cells expressing KCNQ1 and the accessory subunit KCNE3 revealed a volume‐sensitive K+ current, which was inhibited by 293B. These data suggest that KCNQ1 may play important physiological roles in the mammary epithelium, potentially mediating apical K+ secretion and well as being critically involved in the regulation of cell volume. Supported by the Canadian Breast Cancer Foundation, Atlantic Chapter.