Analysing steroid modulation of BK(Ca) channels reconstituted into planar lipid bilayers.
A number of recent studies have described the activation of BK(Ca) channels by steroid hormones such as estrogen. The proposed mechanisms are diverse and include both the direct interaction with the ion channel subunits and the stimulation via receptor activation and cell signalling pathways. To investigate the activation of BK(Ca) channels by estrogen we devised a cell-free system by incorporating recombinant channels of known subunit composition into artificial bilayers and recorded single channel currents. This chapter describes the methods used to prepare purified membrane fractions from cultured cells and the construction of artificial phospholipids bilayers for the incorporation and recording of ion channels.
de Wet
Heidi
H
Institute of Membrane and Systems Biology, University of Leeds, UK.
Lippiat
Jonathan D
JD
Allen
Marcus
M
D002460:Cell Line; D004594:Electrophysiology / Q000379:methods; D004967:Estrogens / Q000494:pharmacology*; D006801:Humans; D007668:Kidney / Q000196:embryology; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000187:drug effects / Q000502:physiology*; D008051:Lipid Bilayers / Q000138:chemical synthesis; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology
D004967:Estrogens; C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008051:Lipid Bilayers; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine