TY - JOUR
T1 - Caveolae targeting and regulation of large conductance Ca 2+-activated K+ channels in vascular endothelial cells
AU - Wang, Xiao Li
AU - Ye, Dan
AU - Peterson, Timothy E.
AU - Cao, Sheng
AU - Shah, Vijay H.
AU - Katusic, Zvonimir S.
AU - Sieck, Gary C.
AU - Lee, Hon Chi
PY - 2005/3/25
Y1 - 2005/3/25
N2 - 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 Ca2+-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 μM, 6.9 ± 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 μM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 μM) 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.
AB - 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 Ca2+-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 μM, 6.9 ± 2.4 pA/pF, n = 6). Inclusion of a caveolin-1 scaffolding domain peptide (10 μM) in the pipette solution completely abrogated the effects of ISO on BK channel activation, whereas inclusion of the scrambled control peptide (10 μM) 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.
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U2 - 10.1074/jbc.M410987200
DO - 10.1074/jbc.M410987200
M3 - Article
C2 - 15665381
AN - SCOPUS:15744367070
SN - 0021-9258
VL - 280
SP - 11656
EP - 11664
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 12
ER -