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

Abstract

Background and purpose: 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. Experimental approach: The effect of the caveolae‐disrupting agent methyl‐β‐cyclodextrin (MβCD) on IK_Ca and SK_Ca localization and function was determined. Key results: 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 MβCD. Hyperpolarizations evoked by direct SK_Ca channel activation (using NS309 in the presence of TRAM‐34) were also inhibited by MβCD, an effect reversed by cholesterol. In contrast, IK_Ca‐dependent hyperpolarizations (in the presence of apamin) were unaffected by MβCD. 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 MβCD. 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. MβCD 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 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. British Journal of Pharmacology (2007) 151, 332–340; doi:10.1038/sj.bjp.0707222