In the current study, we replaced tryptophan at 163 with an isoleucine residue, resulting in the loss of binding between E2 and the 1 subunit and abolishing the estrogen-activating effect on the BK channel. its active configuration and increasing the coupling between voltage sensor activation and pore opening. Notably, we found that E2 binds to a hydrophobic cluster of residues, E2-binding pocket, in the second transmembrane segment of 1 1. Furthermore, we showed that residues tryptophan 163 and phenylalanine 166 in 1 subunit, are necessary to stabilize E2 in the E2-binding pocket, since substitution of these residues eliminated the E2 effect on BK/1 channel. Results E2 binds to the 1 subunit We studied the E2.binding in BK channels by transfecting HEK 293 cells with BK, 1 and BK/1 subunit, respectively. Membrane expression was evaluated by immunocytochemistry and flow cytometry (Fig.?1). The and 1 subunit reach the plasma membrane when expressed alone or as a /1 complex (Fig.?1aCc). Membrane expression was quantified using extracellular labeling and flow cytometry and was detected in 30C40% of the studied cells (Fig.?1d). Open in a separate window Physique 1 BK channel expression in HEK 293 cells. (a) Immunofluorescence of channels formed by the subunit; green: anti-KCa1.1 and Alexa Fluor 488 antibodies. (b) Immunofluorescence of the 1 subunit; red: anti-MaxiK- 1 and Alexa Fluor 568 antibodies. (c) Immunofluorescence of channels formed by co-expression of /1 subunits; yellow: merged image of green and red antibodies in cells expressing and 1 subunits. Blue: DAPI nuclear Rabbit Polyclonal to TCEAL4 stain. (d) Quantification of membrane expression by flow cytometry (% cells). : 49.2??2.7; 1: 41.51??0.8, in /1 co-transfected cells: 41.4??2.8; 1 in /1 co-transfected cells: 46.1??2.6. Error bars: standard error of mean (SEM). We explored the binding characteristics of E2 to the BK channel by E2-binding assays using a membrane-impermeant conjugate of fluorescein isothiocyanate-labelled E2 covalently linked to albumin (E2-BSA-FITC) as described Refametinib Refametinib in Methods. The binding was analyzed by confocal microscopy and quantified by flow cytometry (Fig.?2). In support of our previous results19, E2-BSA-FITC binds to /1 expressing cells (Fig.?2c,e) with an normalized Median Fluorescence Intensity (nMFI) of 2.89??0.29 but not to control untransfected cells or to BK expressing cells (Fig.?2a,b,e). Also, E2 binds to cells expressing the 1 subunit alone, with an nMFI of 3.21??0.43, suggesting that this E2-binding site is located in the 1 subunit (Fig.?2d,e). Open in a separate window Physique 2 E2 binding to BK channel. (a) Representative images of HEK 293 cells without transfection, (b) transfected with subunit, (c) cells transfected with /1 subunits, and (d) cells transfected with 1 subunit, showing bright field and FITC channels. All cells were treated with 10 M E2-BSA-FITC. (e) Membrane binding quantification of E2-BSA-FITC by flow cytometry using a normalized Median Fluorescence Intensity (nMFI). : 1.14??0.07 (black bar), 1: 3.21??0.43 (red bar), /1: 2.89??0.29 (squared pattern red Refametinib bar). Error bars: SEM, ***P? ?0.001. E2 stabilizes the voltage sensor in its active configuration E2 can act as an activator of the BK channel even in the absence of internal Ca2+?19. However, its mechanism of action is usually unknown. 1 modulates BK channel activity by displacing VSD equilibrium towards active state14,16. In order to examine the possible effects of E2 on resting-active VSD equilibrium, we measure gating currents. Gating currents reflect the displacement of the charges contained in the voltage sensor. We measured gating currents to examine the effect of E2 on BK modulation by 1 in oocytes expressing BK and BK/1 channel (Fig.?3). We were able to detect strong gating currents in the absence or.