Estradiol Binding to Maxi-K Channels Induces Their Down-regulation via Proteasomal Degradation

Abstract

Estrogens exert their biological action via both genomic and non-genomic mechanisms. Proteins different from classical estradiol receptors are believed to mediate the latter effects. Here we demonstrate that the maxi-K channel functions as an estrogen-binding protein in transfected HEK293 cells. Whole-cell maxi-K channel currents and protein expression were attenuated by exposure to either 17␣-or 17␤-estradiol. This effect was dose-dependent for 17␤-estradiol at concentrations ranging from 10 nM to 1 M, while 17␣-estradiol inhibited channel expression only at 1 M. These effects were mediated by direct low affinity binding of estradiol to the maxi-K channel but not to its accessory ␤1-subunit, as revealed by cell membrane estradiol binding assays. However, specific binding of estradiol to the channel was facilitated by the presence of the ␤1 subunit. Addition of MG-132, a blocker of proteasomal degradation, stabilized channel expression. These data suggest that channel down-regulation is mediated by estrogen-induced proteasomal degradation, similar to the pathway used for estrogen receptor degradation. Membrane expression of endogenous maxi-K channels in cultured vascular smooth muscle cells was also attenuated by prolonged exposure to 17␣-and 17␤-estradiol. Thus our studies demonstrate that estrogen binds to maxi-K channels and may directly regulate channel expression and function. These results will have important implications in understanding estradiol-induced effects in multiple tissues including vascular smooth muscle.Existing epidemiological data suggest a beneficial role for estrogens in maintaining cardiovascular protection in pre-menopausal women. However, other data show that estrogen-containing preparations can negatively affect the health of premenopausal as well as post-menopausal women (1-6). These results indicate the existence of complex estradiol-signaling mechanisms, possibly mediated by proteins other than classical intracellular estradiol receptors (7).One protein that has been hypothesized to be a potential effector of the action of estrogen is the large conductance Ca 2ϩ -and voltage-activated K ϩ channel (maxi-K channel). This channel provides a buffering repolarizing current in response to various physiological stimuli (8 -11). The mechanism by which estrogen interacts with this channel is unknown. However, both 17␤-estradiol and tamoxifen, a partial agonist of nuclear estradiol receptors, have been shown to activate maxi-K channels in both heterologous expression systems and smooth muscle cells, resulting in greater repolarizing currents (12, 13). Because acute incubation with either 17␤-estradiol or membrane-impermeable estrogen derivatives activate maxi-K channels, signal transduction pathways other than those mediated by classical nuclear estradiol receptors ␣ (ER␣) 1 or ␤ (ER␤) are likely to be involved. Valverde et al. (14) showed that activation of maxi-K channels by 17␤-estradiol occurs when the channel is associated with its accessory ␤1-subunit. This effect was not observed in t...

Acid-sensing ion channels interact with and inhibit BK K + channels

“…1B); the results were similar to previous reports (25,26). However, when we coexpressed the two channels, BK current amplitude fell ( Fig.…”

“…cDNA constructs were either previously reported or were generated by using standard procedures. HEK293 cells stably expressing BK were previously reported (25).…”

Ouabain Binds with High Affinity to the Na,K-ATPase in Human Polycystic Kidney Cells and Induces Extracellular Signal–Regulated Kinase Activation and Cell Proliferation

“…Strong evidence now supports the idea that steroids can bind to cell surface proteins to trigger downstream signaling cascades in the cell (44). Rapid effects of estrogen and aldosterone via plasma membrane transport mechanisms, such as Ca 2ϩ -and voltage-activated K ϩ channels and the Na ϩ /H ϩ exchanger, have been reported (45,46). The effect of ouabain in ADPKD cells is consistent with that observed in several other cell types, in which ouabain binding to the Na,K-ATPase triggers ERK phosphorylation (15).…”

17‐β‐Estradiol activates maxi‐K channels through a non‐genomic pathway in human breast cancer cells

“…Indeed, non-genomic effects of E 2 on maxi-K current have been demonstrated by its ability to induce channel openings when the channel a subunit is associated with either the accessory b1 or the b4 subunit, but not in their absence [14,18,19]. More recently, using HEK-293 cells transfected with maxi-K channels, Korovkina et al [20] demonstrated that estrogens bind to maxi-K channels and may directly regulate channel function. Estrogens are also known to regulate maxi-K channel activity by several cellular signalling mechanisms: (i) the activation of nitric oxide synthase (NOS) in uterine cells [21]; (ii) the cGMP-dependent phosphorylation in coronary arteries [4,15].…”