Abstract 423: Molecular Mechanism of Large Conductance Ca2+-activated K+ Channel Inhibition by Caveolin-1
Recently, we reported that the activities of large conductance Ca2+-activated K+(BK) channels are inhibited by caveolae microdomain targeting in vascular endothelial cells. The molecular mechanism of this negative regulation of BK channels by caveolae is unknown. In this study, we tested the hypotheses that BK channels are inhibited by physical interaction with the scaffolding domain of caveolin-1, which alters the properties of the channel. Using HEK293 cells stably expressing hSlo (which encodes human BK channels) with and without transient transfection with caveolin-1, We found that hSlo channels were enriched in the low buoyant density fraction when co-expressed with caveolin-1 and the channels were co-immunoprecipitated by anti-caveolin-1 antibodies. Co-expression of caveolin-1 resulted in inhibition of whole-cell hSlo current densities from 77.0±18.0 pA/pF in control (HP=−60 mV, TP=+80 mV, 1 μM free Ca2+, n=10) to 12.0±3.9 pA/pF (n=8, p<0.01 vs. control). In contrast, co-expression of caveolin-1 scaffolding domain deletion mutant Cav-1Δ80 −100 did not affect hSlo current density (58.4±14.6 pA/pF, n=8, p=NS vs. control). In addition, co-expression with Cav-1F92A mutant did not affect hSlo current density. Single channel recordings of hSlo channels in inside-out patches showed that exposure to cavtratin, a peptide containing the caveolin-1 scaffolding domain, dose-dependently inhibited hSlo opening probability (Po) with an IC50 of 91.1±6.9 nM (n=5). In contrast, the scrambled control peptide had no effect on channel Po. Co-expression with caveolin-1 reduced the hSlo sensitivity to Ca2+from an EC50 of 0.75±0.12μM (control, n=8) to 4.4±1.7 μM (n=6, p<0.05 vs. control), and also reduced hSlo sensitivity to voltage activation from a V1/2 of 34.1± 6.9 mV (control n=5) to 102.7±7.5 mV (n=5, p<0.001 vs. control). These results suggest that the caveolin-1 scaffolding domain, and particularly F92, is a critical molecular site for inhibition of hSlo activities. Interaction with caveolin-1 reduces the sensitivity of hSlo to Ca2+ and voltage activation.