Effects of methyl beta-cyclodextrin on EDHF responses in pig and rat arteries; association between SK(Ca) channels and caveolin-rich domains.

The small and intermediate conductance, Ca2+-sensitive K+ channels (SK(Ca) and IK(Ca), respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IK(Ca) and SK(Ca) channels was investigated. The effect of the caveolae-disrupting agent methyl-beta-cyclodextrin (MbetaCD) on IK(Ca) and SK(Ca) localization and function was determined. EDHF-mediated, SK(Ca)-dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries (following blockade of IK(Ca) with TRAM-34) were inhibited by MbetaCD. Hyperpolarizations evoked by direct SK(Ca) channel activation (using NS309 in the presence of TRAM-34) were also inhibited by MbetaCD, an effect reversed by cholesterol. In contrast, IK(Ca)-dependent hyperpolarizations (in the presence of apamin) were unaffected by MbetaCD. Similarly, in porcine coronary arteries, EDHF-mediated, SK(Ca)-dependent (but not IK(Ca)-dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MbetaCD. In mesenteric artery homogenates subjected to sucrose-density centrifugation, caveolin-1 and SK3 (SK(Ca)) proteins but not IK1 (IK(Ca)) protein migrated to the buoyant, caveolin-rich fraction. MbetaCD pretreatment redistributed caveolin-1 and SK3 proteins into more dense fractions. In immunofluorescence images of porcine coronary artery endothelium, SK3 (but not IK1) and caveolin-1 were co-localized. Furthermore, caveolin-1 immunoprecipitates prepared from native porcine coronary artery endothelium contained SK3 but not IK1 protein. These data provide strong evidence that endothelial cell SK(Ca) channels are located in caveolae while the IK(Ca) channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade.