Objectives: 

17β-estradiol (E2) rapidly reduces intestinal Cl- secretion by inhibiting KCNQ1 K+ channels (1-2). 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.

Materials: 

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.

Results: 

E2 reduced both forskolin-induced Cl- secretion (30±6% n=6 p<0.05) and KCNQ1 current (45±8% n=5 p<0.05). KCNQ1 protein was removed from the plasma membrane and internalized in cytosolic pools following 15 min exposure to E2 (n=5). KCNQ1 internalization is clathrin and dynamin dependent since Chlorpromazine and Dynasore reversed the E2 mediated KCNQ1 internalization (n=4). Following internalization, a subset of KCNQ1 appeared to accumulate in early endosomes (EE). Further experiments revealed that KCNQ1 is recycled within 4 hours to the membrane rather than degraded. Following E2 exposure, KCNQ1 rapidly co-localized with Rab4, but Rab11 co-localization was only observable after 120 min 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 determined by a biotin recycling assay.

Conclusions: 

This study establishes a role for E2 in the regulation of KCNQ1 plasma membrane surface density. We propose that the internalisation of KCNQ1 is a rapid estrogen response in modulating intestinal Cl- secretion.

1. Condliffe SB. et al J Physiol. 2001; 530 (Pt1; 47-54

2. Alzamora R. et al J Physiol. 2011 ; 589.21 5091-5107