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

• Published in: Biochimica et biophysica acta 2015-09, Vol.1853 (9), p.2173-2182
• Main Authors: Pulli, Ilari, Blom, Tomas, Löf, Christoffer, Magnusson, Melissa, Rimessi, Alessandro, Pinton, Paolo, Törnquist, Kid
• Format: Article
• Language: English
• Subjects: Aequorin; Aequorin - biosynthesis; Aequorin - genetics; Calcium; Calcium - metabolism; Calcium Signaling - drug effects; Calcium Signaling - physiology; Caveolae - metabolism; Caveolin 1 - biosynthesis; Caveolin 1 - genetics; Caveolin-1; HeLa Cells; Humans; Lysophospholipids - pharmacology; Phosphotransferases (Alcohol Group Acceptor) - genetics; Phosphotransferases (Alcohol Group Acceptor) - metabolism; Plasma membrane; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Sphingosine - analogs & derivatives; Sphingosine - pharmacology; Sphingosine kinase 1; Sphingosine-1-phosphate
• ISSN: 0167-4889; 0006-3002; 1879-2596
• DOI: 10.1016/j.bbamcr.2015.04.005

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 ([Ca2+]cav). In HeLa cells, Cav1–Aeq reported different [Ca2+] as compared to the plasma membrane [Ca2+] in general (reported by SNAP25–Aeq) or as compared to the cytosolic [Ca2+] (reported by cyt-Aeq). The Ca2+ 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 Ca2+ signaling. HeLa cells overexpressing SK1 showed increased [Ca2+]cav during histamine-induced Ca2+ mobilization in the absence of extracellular Ca2+ as well as during receptor-operated Ca2+ entry (ROCE). The SK1-induced increase in [Ca2+]cav during ROCE was reverted by S1P receptor antagonists. In accordance, pharmacologic inhibition of SK1 reduced the [Ca2+]cav during ROCE. S1P treatment stimulated the [Ca2+]cav upon ROCE. The Ca2+ responses at the plasma membrane in general were not affected by SK1 expression. In summary, our results show that SK1/S1P-signaling regulates Ca2+ signals at the caveolae. This article is part of a Special Issue entitled: 13th European Symposium on Calcium. •Caveolin-1–aequorin (Cav1–Aeq) is a novel Ca2+ probe.•Cav1–Aeq reports caveolae-specific Ca2+ signals.•Sphingosine kinase 1 regulates Ca2+ signaling at the caveolae.