A novel chimeric aequorin fused with caveolin-1 reveals a sphingosine kinase 1-regulated Ca2 + microdomain in the caveolar compartment☆

Abstract Caveolae are plasma membrane invaginations enriched in sterols and sphingolipids. Sphingosine kinase 1 (SK1) is an oncogenic protein that converts sphingosine to sphingosine 1-phosphate (S1P), which is a messenger molecule involved in calcium signaling. Caveolae contain calcium responsive proteins, but the effects of SK1 or S1P on caveolar calcium signaling have not been investigated. We generated a Caveolin-1–Aequorin fusion protein (Cav1–Aeq) that can be employed for monitoring the local calcium concentration at the caveolae ([Ca 2+ ]cav). In HeLa cells, Cav1–Aeq reported different [Ca 2+ ] as compared to the plasma membrane [Ca 2+ ] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca 2+ ] (reported by cyt-Aeq). The Ca 2+ signals detected by Cav1–Aeq were significantly attenuated when the caveolar structures were disrupted by methyl-β-cyclodextrin, suggesting that the caveolae are specific targets for Ca 2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca 2+ ]cav during histamine-induced Ca 2+ mobilization in the absence of extracellular Ca 2+ as well as during receptor-operated Ca 2+ entry (ROCE). The SK1-induced increase in [Ca 2+ ]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca 2+ ]cav during ROCE. S1P treatment stimulated the [Ca 2+ ]cav upon ROCE. The Ca 2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca 2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.