Gb3 accumulation reduces K(Ca)3.1 channel expression by down-regulating ERK and AP-1 and up-regulating REST and the channel activity by decreasing intracellular levels of PI(3)P. Gb3 thereby evokes K(Ca)3.1 channel dysfunction, and the channel dysfunction in vascular endothelial cells may contribute to vasculopathy in Fabry disease.

Objective— Globotriaosylceramide (Gb3) induces K Ca 3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces K Ca 3.1 endocytosis and degradation. Approach and Results— K Ca 3.1, especially plasma membrane–localized K Ca 3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced K Ca 3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress–inducing agents did not induce K Ca 3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated K Ca 3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered K Ca 3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored K Ca 3.1 expression, the current, and endothelium-dependent relaxation. Conclusions —Gb3 accelerates the endocytosis and lysosomal degradation of endothelial K Ca 3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.

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The effect of sphingosine-1-phosphate (S1P) on large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels was examined in primary cultured human umbilical vein endothelial cells by measuring intracellular Ca(2+) concentration ([Ca(2+)](i)), whole cell membrane currents, and single-channel activity. In nystatin-perforated current-clamped cells, S1P hyperpolarized the membrane and simultaneously increased [Ca(2+)](i). [Ca(2+)](i) and membrane potentials were strongly correlated. In whole cell clamped cells, BK(Ca) currents were activated by increasing [Ca(2+)](i) via cell dialysis with pipette solution, and the activated BK(Ca) currents were further enhanced by S1P. When [Ca(2+)](i) was buffered at 1 microM, 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 BK(Ca) channel activity in a reversible manner and shifted the relationship between Ca(2+) 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) (GDPbetaS; 1 mM) using a patch pipette, GDPbetaS application or pretreatment of cells with pertussis toxin (100 ng/ml) for 15 h did not affect S1P-induced BK(Ca) current and channel activation. These results suggest that S1P enhances BK(Ca) channel activity by increasing Ca(2+) sensitivity. This channel activation hyperpolarizes the membrane and thereby increases Ca(2+) influx through Ca(2+) entry channels. Inasmuch as S1P activates BK(Ca) channels via a mechanism independent of G protein-coupled receptors, S1P may be a component of the intracellular second messenger that is involved in Ca(2+) mobilization in human endothelial cells.