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. 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. Data are given as Mean ± S.E.M. Ussing chamber experiments revealed that 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). Confocal microscopy showed that KCNQ1 is 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 = 226 ± 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=3). 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 a 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). This study establishes a role for E2 in the regulation of KCNQ1surface density. In conclusion, we propose that the internalisation of KCNQ1 is a rapid estrogen response in modulating intestinal secretion.