Cholesterol depletion alters amplitude and pharmacology of vascular calcium-activated chloride channels

Calcium-activated chloride channels (CACCs) share common pharmacological properties with Kcnma1-encoded large conductance K+ channels (BKCa or KCa1.1) and it has been suggested that they may co-exist in a macromolecular complex. As KCa1.1 channels are known to localize to cholesterol and caveolin-rich lipid rafts (caveolae), the present study investigated whether Ca2+-sensitive Cl− currents in vascular myocytes were affected by the cholesterol depleting agent methyl-β-cyclodextrin (M-βCD). 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. Ca2+-activated Cl− currents were augmented by 3 mg mL−1 M-βCD with a rapid time course (t0.5 = 1.8 min). M-βCD had no effect on the bi-modal response to niflumic acid or anthracene-9-carboxylate but completely removed the inhibitory effects of the KCa1.1 blockers, paxilline and tamoxifen, as well as the stimulatory effect of the KCa1.1 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-βCD. The position of KCa1.1 and the newly identified candidate for CACCs, TMEM16A, was also affected by M-βCD. These data reveal that CACC properties are influenced by lipid raft integrity.