title: Depletion of membrane cholesterol eliminates the Ca2+-activated component of outward potassium current and decreases membrane capacitance in rat uterine myocytes.
abstract: Changes in membrane cholesterol content have potent effects on cell signalling and contractility in rat myometrium and other smooth muscles. We have previously shown that depletion of cholesterol with methyl-beta-cyclodextrin (MCD) disrupts caveolar microdomains. The aim of this work was to determine the mechanism underlying the increase in Ca(2+) signalling and contractility occurring in the myometrium with MCD. Patch clamp data obtained on freshly isolated myocytes from the uterus of day 19-21 rats showed that outward K(+) current was significantly reduced by MCD. Membrane capacitance was also reduced. Cholesterol-saturated MCD had no effect on the amplitude of outward current suggesting that the reduction in the outward current was due to cholesterol depletion induced by MCD rather than a direct inhibitory action of MCD on the K(+) channels. Confocal visualization of the membrane bound indicator Calcium Green C18, revealed internalization of the surface membrane with MCD treatment. Large conductance, Ca(2+)-sensitive K(+) channel proteins have been shown to localize to caveolae. When these channels were blocked by iberiotoxin outward current was significantly reduced in the uterine myocytes; MCD treatment reduced the density of outward current. Following reduction of outward current by MCD pretreatment, iberiotoxin was unable to produce any additional decrease in the current, suggesting a common target. MCD treatment also increased the amplitude and frequency of spontaneous rises in cytosolic Ca(2+) level ([Ca(2+)](i) transients) in isolated myocytes. In intact rat myometrium, MCD treatment increased Ca(2+) signalling and contractility, consistent with previous findings, and this effect was also found to be reduced by BK channel inhibition. These data suggest that (1) disruption of cholesterol-rich microdomains and caveolae by MCD leads to a decrease in the BK channel current thus increasing cell excitability, and (2) the changes in membrane excitability produced by MCD underlie the changes found in Ca(2+) signalling and uterine contractility.
authors.affiliation: Clinical Sciences Research Institute, Warwick Medical School, University of Warwick, Coventry CV2 2DX, UK.
authors.forename: A
authors.initials: A
authors.lastname: Shmygol
authors.forename: K
authors.initials: K
authors.lastname: Noble
authors.forename: Susan
authors.initials: S
authors.lastname: Wray
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000172:deficiency / Q000378:metabolism*; D025802:Electric Capacitance; D005260:Female; D066298:In Vitro Techniques; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D009119:Muscle Contraction / Q000187:drug effects; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009215:Myometrium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism*; D026902:Potassium Channel Blockers / Q000494:pharmacology; D024681:Potassium Channels, Calcium-Activated / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D010455:Peptides; D026902:Potassium Channel Blockers; D024681:Potassium Channels, Calcium-Activated; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C064719:iberiotoxin; D002784:Cholesterol; D011188:Potassium