Estrogen regulation of KCNQ1:KCNE3 channels in intestinal secretion.

Estrogen, 17-βestradiol (E2), rapidly reduces cAMP-dependent intestinal Cl- secretion by inhibiting K+ recycling (1). KCNQ1:KCNE3 is the rate-limiting K+ channel involved in Cl- secretion in the colon (2) and E2 rapidly inhibits KCNQ1 current in the female rat distal colon by a gender-specific mechanism (3). Regulation of KCNQ1 surface density has been shown to play a role in the control of KCNQ1 activity. Recently we demonstrated that estrogen rapidly uncouples KCNQ1 from its regulatory beta subunit KCNE3 causing a decrease in channel conductance (4). The aim of this study was to determine if membrane trafficking plays a role in the E2 inhibition of KCNQ1 function in the human colonic cell line, HT29cl19A. Ussing chamber ion transport measurements, biotinylation assays and immuno-staining have been used to study the effect of 10nM E2 treatment on KCNQ1 current and protein cellular localization. Protein expression was determined by Western blotting assay. All results are given as the mean ± S.E.M. Statistical significance was established using one-way ANOVA followed by a Tukey's post hoc test or a Student t test where relevant. E2 (10 nM) reduced both forskolin-induced Cl- secretion (30 ± 6 % n=6 p<0.05) and KCNQ1 activity (45 ± 8 % n=4 p<0.05) in HT29 cells monolayer. KCNQ1 was removed from the plasma membrane and internalized in cytosolic pools after 15 min E2 treatment (n=5). A biotin internalization assay confirmed this observation (internalized KCNQ1 after E2 treatment = 200 ± 9.9 % of control, n=5, p<0.001). Our results suggested that KCNQ1 internalization is clathrin and dynamin dependent since chlorpromazine and dynasore reversed E2 mediated KCNQ1 internalization as shown by immunofluorescence (n=4). Fluorescence co-localization studies indicated that KCNQ1 rapidly co-localized with the clathrin adaptor AP2 µ2 (Adaptor Protein 2 sub-unit µ2) 10 min after E2 treatment (overlap coefficient (O.C.) = 0.28 ± 0.04, control - 0.77 ± 0.01 E2, n=4). Following internalization, a subset of KCNQ1 appeared to accumulate in early endosomes (EE), (EE marker, EEA-1; O.C. with KCNQ1 = 0.30 ± 0.07% control, 0.60 ± 0.04 E2, n=4, p< 0.05). Further experiments revealed that KCNQ1 is recycled to the membrane rather than degraded. Following E2 exposure KCNQ1 rapidly co-localized with Rab4 (O.C. = 0.31 ± 0.02, control, 0.64 ± 0.02 E2 15 min), but Rab11 co-localization was only observable after 120 min (O.C. = 0.3 ± 0.03 control, 0.67 ± 0.01, E2) suggesting that KCNQ1 is sorted from the EE to the recycling endosomes. After 240 min exposure to E2, 70 ± 5 % (n=6, p<0.001) of internalized KCNQ1 was recycled back to the membrane as shown by the biotin recycling assay. Following E2 treatment, PKCδ (287 ± 51 % n=4, p<0.05) and AMPK (232 ± 24%, n=5, p<0.05) phosphorylation were increased within 2 min exposure to E2. Immuno-staining and biotinylation experiments revealed that E2 failed to induced KCNQ1 endocytosis in HT29cl19A cells when pre-treated with BIS1 a general PKC inhibitor, rottlerin a potent PKCδ inhibitor or Compound-C a AMPK inhibitor (n=4). Interestingly, we also demonstrated that KCNQ1 rapidly associates with Nedd4.2, an ubiquitin ligase, in response to estrogen treatment (346 ± 56% n=3, p<0.001). The last results suggest that E2 induces KCNQ1 ubiquitination to stimulate channel internalization. This study establishes a role for E2 in the regulation of KCNQ1 cell surface abundance. In conclusion, we propose that the internalisation of KCNQ1 is a rapid estrogen response in modulating intestinal secretion. [ABSTRACT FROM AUTHOR]