Molecular mechanism of glucocorticoid action
Attarzadeh Yazdi, Ghassem
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Adrenal corticosteroids are involved in multiple aspects of homeostatic control. The early delayed ( <2h) action of glucocorticoids is mediated by rapid induction of mRNA and protein synthesis. Within this time -frame, glucocorticoids potently modify the electrical excitability of target cells through regulation of ion channels. Increasing evidence suggests that in neurones and endocrine cells, large conductance calcium - and voltage- activated potassium channels (BK channels) are important targets for glucocorticoid action. The aim of this thesis project was to investigate the mechanisms by which glucocorticoid hormones regulate the activity of BK channels in human embryonic kidney 293 (HEK 293) cells as the model system for glucocorticoid- action. It was shown that glucocorticoids act via endogenously expressed type II receptors in a concentration- and time -dependent manner in these cells. Dexamethasone (100 nM) had no significant effect on Dexrasl mRNA but significantly increased serum- and glucocorticoid- induced protein kinase 1 (SGK-1) mRNA. Biochemical analysis showed that SGK-1 protein is increased by dexamethasone in a Triton X-100 insoluble fraction. Further work was directed toward analysing the possible association of SGK -1 and protein phosphatases with two BK channel cc-subunit variants: ZERO-BK and STREX-BK, the latter contains the 59 amino -acid splice insert encoded by the stress hormone induced exon (STREX). HEK 293 cells stably expressing the respective channel subunits were analysed. Immunoprecipitations with antisera directed against the BK channel cc-subunits showed that protein phosphatase 2A (PP2A) but not SGK-1 is constitutively associated with the STREX as well as the ZERO variant BK channel. Furthermore, the cytoplasmic C- terminal segment of the STREX-BK channel was necessary for cell -surface expression of the channel and the association of the channel with PP2A. Dexamethasone failed to change the apparent amount of immunoreactive PP2A co-immunoprecipitating with the channel. In conclusion: SGK-1 but not Dexrasl is a protein rapidly induced by dexamethasone in HEK293 cells. PP2A but not SGK -1 is in complex with both ZERO and STREX-BK channels, and dexamethasone does not alter this association. The cytoplasmic tail of the BK channels is essential for PP2A interaction.