Date of Award

2013

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Brian J. Harvey

Second Supervisor

Dr. Valerie Urbach

Keywords

Molecular Medicine

Abstract

Background: The KCNQ1:KCNE3 K+ channel is an essential component of the Cl" secretion machinery in the distal colon. This channel provides the driving force for apical Cl’ secretion by basolateral recycling of K+. The steroid hormone estrogen (17|3-estradiol, E2) has previously been reported to exert a female specific antisecretory response in colonic crypts through the inhibition of the KCNQ1:KCNE3 channel. The purpose of this study was to uncover and describe molecular mechanisms of estrogen regulation of KCNQ1 channel function and its consequences for intestinal Cl' secretion, colonocyte proliferation and migration. The thesis reveals a novel estrogen regulation of KCNQ1:KCNE3 activity by channel endocytosis and complex dissociation.

Methods: Isolated rat colonic crypts as well as the colonic cell line HT29cll9A (HT29) were used to investigate estrogen effects on Cl' secretion and KCNQ1 channel function using a combination of electrophysiological, cellular and molecular biology and imaging techniques.

Results: The forskolin-stimulated Cl' secretion and KCNQ1 current in rat colon and HT29 epithelia were rapidly reduced following estrogen treatment (lOnM) and remained inhibited over 2 hours after estrogen exposure. Our findings revealed a rapid estrogen-promoted retrieval of KCNQ1 from the plasma membrane via a PKC6-AMPK-Nedd4 .2 signaling pathway, followed by the recycling of the channel. The mechanism underlying recycling was biphasic; a rapid recycling phase mediated by Rab4 and a slow recycling phase mediated by R abil. Estrogen also causes dissociation of the KCNQ1:KCNE3 channel complex resulting in collapse of the K+ channel conductance and Cl' secretion. The thesis also demonstrated that KCNQ1 plays a role in E2-modulated colonocyte migration.

Conclusion: This study establishes a role for estrogen in the regulation of colonic electrolyte secretion via modulation of KCNQ1 cell membrane surface abundance and KCNQ1:KCNE3 complex formation. Here, we highlighted a new role of KCNQ1 in colonocyte migration which is also modulated by estrogen through KCNQ1. Thus, estrogen plays an important role in colonic crypt homeostasis by regulating colonocyte secretion and migration rate.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 License.

File Size

13.6 MB

Comments

A thesis submitted for the degree of Doctor of Philosophy from the Royal College of Surgeons in Ireland in 2013.

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