Sphingosine-1-phosphate activates BKCa channels independently of G protein-coupled receptor in human endothelial cells

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Abstract
The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca2+-activated K+ (BKCa) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca2+ concentration ([Ca2+]i), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca2+]i. [Ca2+]i and membrane potentials were strongly correlated. In whole cell clamped cells, BKCa currents were activated by increasing [Ca2+]i via cell dialysis with pipette solution, and the activated BKCa currents were further enhanced by S1P. When [Ca2+]i was buffered at 1 μM, the S1P concentration required to evoke half-maximal activation was 403 ± 13 nM. In inside-out patches, when S1P was included in the bath solution, S1P enhanced BKCa channel activity in a reversible manner and shifted the relationship between Ca2+ concentration in the bath solution and the mean open probability to the left. In whole cell clamped cells or inside-out patches loaded with guanosine 5′- O -(2-thiodiphosphate) (GDPβS; 1 mM) using a patch pipette, GDPβS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BKCa current and channel activation. These results suggest that S1P enhances BKCa channel activity by increasing Ca2+ sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca2+ influx through Ca2+ entry channels. Inasmuch as S1P activates BKCa channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca2+ mobilization in human endothelial cells.