Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC68
Estrogen induces internalization of KCNQ1 K+ channels in the colonic tumour cell line HT29cl19A.
R. Rapetti-Mauss1, V. Urbach1, B. J. Harvey1
1. Molecular medicine, RCSI, Dublin, Ireland.
17β-estradiol (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 intestine (2). Estrogen rapidly inhibits KCNQ1 channel current in the distal colon by a female sex-specific mechanism involving PKCδ and PKA-dependent phosphorylation of KCNQ1 (3, 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. Data are given as Mean ± S.E.M. One-way ANOVA was used to establish the significance of the results. Confocal immunofluorescence microscopy revealed that after 30 min E2 treatment (10 nM), the abundance of KCNQ1 decreased at the basolateral membranes and increased in the cytosol. Pre-treatment with the PKCδ inhibitor rottlerin (5 μM, 30 min) partially reversed this sub-cellular re-distribution of KCNQ1 (n=3). As shown by biotinylation, E2 treatment halved the amount of KCNQ1 protein remaining in the cell membrane (KCNQ1 remaining = 50.4% ± 3.6% of control, n= 5, p< 0.001). The E2-induced removal of KCNQ1 from the cell membrane was partially reversed by rottlerin (KCNQ1 remaining = 75.6% ± 8.9 %, n=5, p<0.05). Fluorescence co-localization studies indicated that E2 induced internalization of a subset of KCNQ1 channels into early endosomes, (early endosome marker, EEA-1; overlap coefficient with KCNQ1 = 0.30 ± 0.07% control, 0.60 ± 0.04 E2, n=4, p< 0.05). Pre-treatment with rottlerin (5 μM, 30 min) completely reversed this effect (EEA-1; overlap coefficient with KCNQ1 was not different from untreated cells at 0.29 ± 0.06, n=4). E2 treatment increased the phosphorylation of PKCδ by 56.2 % ± 8.8 %, n=6, p< 0.001, within 5 min. This response was transient since no significant difference in PKCδ phosphorylation from control was observed after 30min E2 exposure (14% ± 8.5%, n=6). PKA activity was also increased after E2 treatment within 5 min (34.4% ± 7.5%, n=3 p<0.001) and remained activated after 30 min (33.9% ± 3.5 %, n=3, p<0.001). Following E2 treatment, PKA associated with KCNQ1 within 5 min (2.1 ± 0.1 fold higher than control, n=3 p<0.01) and this association was prevented by pre-treatment with rottlerin (1.2 ± 0.3, n=3). E2 stimulated KCNQ1 phosphorylation (2.6 ± 6.9 fold higher compared to control, n=2) which was inhibited by rottlerin (1.08 ± 0.08, n=2). These results suggest that PKA:KCNQ1 association depends on prior PKCδ phosphorylation, and leads to KCNQ1 phosphorylation. E2 rapidly induces retrieval of KCNQ1 channel from the plasma membrane and promotes its internalization into an early endosome compartment. The internalisation of KCNQ1 is dependent on PKCδ phosphorylation and PKA:KCNQ1 association.
Where applicable, experiments conform with Society ethical requirements