Effects of methyl β-cyclodextrin on EDHF responses in pig and rat arteries; association between SKCa channels and caveolin-rich domains

Background and purpose: The small and intermediate conductance, Ca2+- sensitive K+ channels ( SKCa and IKCa, respectively) which are pivotal in the EDHF pathway may be differentially activated. The importance of caveolae in the functioning of IKCa and SKCa channels was investigated. Experimental approach: The effect of the caveolae-disrupting agent methyl-beta-cyclodextrin ( MbCD) on IKCa and SKCa localization and function was determined. Key results: EDHF- mediated, SKCa-dependent myocyte hyperpolarizations evoked by acetylcholine in rat mesenteric arteries ( following blockade of IKCa with TRAM-34) were inhibited by M beta CD. Hyperpolarizations evoked by direct SKCa channel activation ( using NS309 in the presence of TRAM- 34) were also inhibited by M beta CD, an effect reversed by cholesterol. In contrast, IKCa-dependent hyperpolarizations ( in the presence of apamin) were unaffected by M beta CD. Similarly, in porcine coronary arteries, EDHF- mediated, SKCa-dependent ( but not IKCa-dependent) endothelial cell hyperpolarizations evoked by substance P were inhibited by MbCD. In mesenteric artery homogenates subjected to sucrose- density centrifugation, caveolin- 1 and SK3 ( SKCa) proteins but not IK1 ( IKCa) protein migrated to the buoyant, caveolin- rich fraction. M beta CD 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. Conclusions and Implications: These data provide strong evidence that endothelial cell SKCa channels are located in caveolae while the IKCa channels reside in a different membrane compartment. These studies reveal cellular organisation as a further complexity in the EDHF pathway signalling cascade. British Journal of Pharmacology ( 2007).