Acute Activation of Maxi-K Channels ( <i>hSlo</i> ) by Estradiol Binding to the β Subunit
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Jiang C., Surrel P. M., Lindsay D. C., Poole-Wilson P. A., Collins P., Br. J. Pharmacol. 104, 1033 (1994).
Watters J. J., Campbell J. S., Cunningham M. J., Krebs E. G., Dorsa D. M., Endocrinology 138, 4030 (1997);
Russell K. S., Caulin-Glaser T., Sessa W., Bender W. C., Circulation 96, 256 (1997).
Toro L., Wallner M., Meera P., Tanaka Y., News Physiol. Sci. 13, 112 (1998).
Human myometrial mRNAs of the α and β subunits at a 1:2 or 1:4 ratio (GenBank accession numbers: and ) were injected into collagenase-treated X. laevis oocytes following a protocol described by M. Wallner et al. [ Receptors Channels 3 185 (1995)].
Macroscopic current recording was made with the patch-clamp technique [
] or the cut-open oocyte voltage clamp (18) 3 to 5 days after injection of the mRNA. The pipettes had 1 to 2 Mohm resistance and were filled with 110 mM potassium methanesulfonate (KMES) 10 mM Hepes 2 mM KCl pH 7 and the indicated 17β-estradiol concentrations. Bath solutions had 110 mM KMES 10 mM Hepes and 5 mM EDTA pH 7.0. The free calcium concentration (Ca 2+ ) ranging from 56 nM to 265 μM was set by adding aliquots of concentrated CaCl 2 to this base solution. Free [Ca 2+ ] was measured with a Ca 2+ electrode. The current signal was digitized to a frequency equal to five times the filter cut-off frequency. The external solution used when the cut-open oocyte voltage clamp was used consisted of 110 mM KMES 10 mM Hepes 2 mM CaCl 2 pH 7. The acquisition and basic analysis of the data were performed with pClamp 6.0 software.
Oocyte membranes prepared as described in (21) were incubated for 60 min at 4°C with 0.3 mM [2 4 6 7 16 17- 3 H( N )]-estradiol in a medium containing 50 mM tris-HCl 120 mM NaCl 5 mM KCl 1 mM MgCl 2 plus 0.08% BSA [
]. Separation of free and bound [ 3 H]estradiol was done by filtration with GF75 filters previously incubated in the binding medium containing 3 mM estradiol.
Human embryonic kidney cells (HEK-293) were cultured in phenol red–free Dulbecco's modified Eagle's medium containing 10% fetal calf serum in a humidified atmosphere of 95% air and 5% CO 2 at 37°C. HEK-293 cells stably expressing α or α and β Maxi-K channel subunits [
] were cultured for 24 hours in 13-mm cover slips then the cells were incubated in 10 μM 17β-estradiol 6-( O -carboxymethil)oxime-BSA-FITC conjugate for 1 hour at 37°C. Cells were washed twice with phosphate-buffered saline (PBS) and viewed with a Leica DMIL inverted microscope. Fluorescence studies are representative of three independent experiments.
Membrane vesicles from rat skeletal muscle [
] bovine aortic smooth muscle [
Slaughter R., Shevell J. L., Felix J. P., Garcia M. L., Kaczorowski G. J., Biochemistry 28, 3995 (1989);
] or from Xenopus oocyte membranes [
] expressing the α and β subunit were incorporated into lipid bilayers made of phophatidylcholine:phosphatidylethanolamine in a ratio of 2:8 in decane (20 mg/mP). Solutions contained 100 mM KCl and 10 mM MOPS pH 7. The internal Ca 2+ was in the nanomolar range for aortic smooth muscle and oocyte Maxi-K channels and 100 nM to 20 μM for skeletal muscle Maxi-K channels. 17β-Estradiol was added to the external side of the channel to the indicated concentrations.
Supported by Human Frontiers Science Program (M.A.V. and R.L.) The Royal Society and The Physiological Society (M.A.V.) Wellcome Trust and Tommy's Campaign (G.E.M.) and Chilean grants 198-1053 (C.V.) and 197-0739 Catedra Presidencial and a group of Chilean companies (CODELCO CMPC CGE Minera Escondida NOVAGAS Bussiness Design Ass. and XEROX Chile) (R.L.). We thank L. Toro for providing us with the clones for the α and β subunit of hSlo channel and for discussions S. Hall and M. J. Shipston for providing HEK-293 cells stably expressing α or α and β subunits and E. Rosemann for technical assistance.