Cholesterol depletion alters amplitude and pharmacology of vascular calcium-activated chloride channels.
OBJECTIVE
Calcium-activated chloride channels (CACCs) share common pharmacological properties with Kcnma1-encoded large conductance K(+) channels (BK(Ca) or K(Ca)1.1) and it has been suggested that they may co-exist in a macromolecular complex. As K(Ca)1.1 channels are known to localize to cholesterol and caveolin-rich lipid rafts (caveolae), the present study investigated whether Ca(2+)-sensitive Cl(-) currents in vascular myocytes were affected by the cholesterol depleting agent methyl-beta-cyclodextrin (M-betaCD).


RESULTS
Calcium-activated chloride and potassium currents were recorded from single murine portal vein myocytes in whole cell voltage clamp. Western blot was undertaken following sucrose gradient ultracentrifugation using protein lysates from whole portal veins. Ca(2+)-activated Cl(-) currents were augmented by 3 mg mL(-1) M-betaCD with a rapid time course (t(0.5) = 1.8 min). M-betaCD had no effect on the bi-modal response to niflumic acid or anthracene-9-carboxylate but completely removed the inhibitory effects of the K(Ca)1.1 blockers, paxilline and tamoxifen, as well as the stimulatory effect of the K(Ca)1.1 activator NS1619. Discontinuous sucrose density gradients followed by western blot analysis revealed that the position of lipid raft markers caveolin and flotillin-2 was altered by 15 min application of 3 mg mL(-1) M-betaCD. The position of K(Ca)1.1 and the newly identified candidate for CACCs, TMEM16A, was also affected by M-betaCD.


CONCLUSIONS
These data reveal that CACC properties are influenced by lipid raft integrity.
Sones
William R
WR
Division of Basic Medical Sciences, St George's, University of London, London SW17 0RE, UK.
Davis
Alison J
AJ
Leblanc
Normand
N
Greenwood
Iain A
IA
D000818:Animals; D000075369:Anoctamin-1; D015153:Blotting, Western; D022461:Caveolins / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D018118:Chloride Channels / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000172:deficiency*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials; D008565:Membrane Proteins / Q000378:metabolism; D049990:Membrane Transport Modulators / Q000494:pharmacology; D051379:Mice; D008807:Mice, Inbred BALB C; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011169:Portal Vein / Q000378:metabolism; D013997:Time Factors; D047392:beta-Cyclodextrins / Q000494:pharmacology
C476213:ANO1 protein, human; D000075369:Anoctamin-1; D022461:Caveolins; D018118:Chloride Channels; C465449:Kcnma1 protein, mouse; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D008565:Membrane Proteins; D049990:Membrane Transport Modulators; D009363:Neoplasm Proteins; D047392:beta-Cyclodextrins; C106354:flotillins; C108732:methyl-beta-cyclodextrin; D002784:Cholesterol