Oestrogen promotes KCNQ1 potassium channel endocytosis and postendocytic trafficking in colonic epithelium

Key points center dot Oestrogen (E2) exposure leads to a decrease in both Cl- secretion and KCNQ1 current. This inhibition is maintained by a rapid and sustained retrieval of the channel from the plasma membrane. center dot The E2-stimulated internalization of KCNQ1 occurs via a dynamin- and clathrin-dependent mechanism. center dot KCNQ1 is recycled back to the cell membrane via Rab4 and Rab11 rather than being degraded. center dot The signalling pathway activated by E2 and leading to KCNQ1 internalization involves a signalling cascade, in which the activation of protein kinase C induces the phosphorylation of AMP-dependent kinase. Oestrogen stimulated an increase in the association of KCNQ1 with the ubiquitin ligase Nedd4.2. center dot The findings provide evidence for a hormone-stimulated regulation of KCNQ1 surface density in colonic epithelium. Moreover, this study complements the understanding of the mechanisms for E2-induced inhibition of KCNQ1 previously described, and provides new insights on hormonal regulation of ion channel retrieval from the plasma membrane. Abstract The cAMP-regulated potassium channel KCNQ1:KCNE3 plays an essential role in transepithelial Cl- secretion. Recycling of K+ across the basolateral membrane provides the driving force necessary to maintain apical Cl- secretion. The steroid hormone oestrogen (17-oestradiol; E2), produces a female-specific antisecretory response in rat distal colon through the inhibition of the KCNQ1:KCNE3 channel. It has previously been shown that rapid inhibition of the channel conductance results from E2-induced uncoupling of the KCNE3 regulatory subunit from the KCNQ1 channel pore complex. The purpose of this study was to determine the mechanism required for sustained inhibition of the channel function. We found that E2 plays a role in regulation of KCNQ1 cell membrane abundance by endocytosis. Ussing chamber experiments have shown that E2 inhibits both Cl- secretion and KCNQ1 current in a colonic cell line, HT29cl.19A, when cultured as a confluent epithelium. Following E2 treatment, KCNQ1 was retrieved from the plasma membrane by a clathrin-mediated endocytosis, which involved the association between KCNQ1 and the clathrin adaptor, AP-2. Following endocytosis, KCNQ1 was accumulated in early endosomes. Following E2-induced endocytosis, rather than being degraded, KCNQ1 was recycled by a biphasic mechanism involving Rab4 and Rab11. Protein kinase C and AMP-dependent kinase were rapidly phosphorylated in response to E2 on their activating phosphorylation sites, Ser643 and Thr172, respectively (as previously shown). Both kinases are necessary for the E2-induced endocytosis, because E2 failed to induce KCNQ1 internalization following pretreatment with specific inhibitors of both protein kinase C and AMP-dependent kinase. The ubiquitin ligase Nedd4.2 binds KCNQ1 in response to E2 to induce channel internalization. This study has provided the first demonstration of hormonal regulation of KCNQ1 trafficking. In conclusion, we propose that internalization of KCNQ1 is a key event in the sustained antisecretory response to oestrogen.