title: Caveolae targeting and regulation of large conductance Ca(2+)-activated K+ channels in vascular endothelial cells.
abstract: The vascular endothelium is richly endowed with caveolae, which are specialized membrane microdomains that facilitate the integration of specific cellular signal transduction processes. We found that the large conductance Ca(2+)-activated K+ (BK) channels are associated with caveolin-1 in bovine aortic endothelial cells (BAECs). OptiPrep gradient cell fractionation demonstrated that BK channels were concentrated in the caveolae-rich fraction in BAECs. Immunofluorescence imaging showed co-localization of caveolin-1 and BK channels in the BAEC membrane. Immunoprecipitation and glutathione S-transferase pull-down assay results indicated that caveolin-1 and BK channels are physically associated. However, whole cell patch clamp recordings could not detect BK (iberiotoxin-sensitive) currents in cultured BAECs under baseline conditions, even though the presence of BK mRNA and protein expression was confirmed by reverse transcription-PCR and Western blots. Cholesterol depletion redistributed the BK channels to non-caveolar fractions of BAECs, resulting in BK channel activation (7.3 +/- 1.6 pA/picofarad (pF), n = 5). BK currents were also activated by isoproterenol (ISO, 1 microM, 6.9 +/- 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 microM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 microM) did not inhibit the ISO effects. We have also found that caveolin-1 knockdown by small interference RNA activated BK currents (5.3 +/- 1.4 pA/pF, n = 6). We conclude that: 1) BK channels are targeted to caveolae microdomains in vascular endothelial cells; 2) caveolin-1 interacts with BK channels and exerts a negative regulatory effect on channel functions; and 3) BK channels are inactive under control conditions but can be activated by cholesterol depletion, knockdown of caveolin-1 expression, or ISO stimulation. These novel findings may have important implications for the role of BK channels in the regulation of endothelial function.
authors.affiliation: Department of Medicine, Mayo Clinic, Rochester, Minnesota 55905, USA.
authors.forename: Xiao-Li
authors.initials: XL
authors.lastname: Wang
authors.forename: Dan
authors.initials: D
authors.lastname: Ye
authors.forename: Timothy E
authors.initials: TE
authors.lastname: Peterson
authors.forename: Sheng
authors.initials: S
authors.lastname: Cao
authors.forename: Vijay H
authors.initials: VH
authors.lastname: Shah
authors.forename: Zvonimir S
authors.initials: ZS
authors.lastname: Katusic
authors.forename: Gary C
authors.initials: GC
authors.lastname: Sieck
authors.forename: Hon-Chi
authors.initials: HC
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D021941:Caveolae / Q000502:physiology*; D051242:Caveolin 1; D022461:Caveolins / Q000032:analysis / Q000502:physiology; D042783:Endothelial Cells / Q000502:physiology*; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels; D008969:Molecular Sequence Data; D024681:Potassium Channels, Calcium-Activated / Q000032:analysis / Q000737:chemistry / Q000502:physiology*; D015222:Sodium Channels / Q000502:physiology
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D051036:Large-Conductance Calcium-Activated Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D015222:Sodium Channels