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 ([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.

Regulation of Endoplasmic Reticulum–Mitochondria Ca2+ Transfer and Its Importance for Anti-Cancer Therapies

“…Such complexity is controlled primarily by the presence of ion channels located in the PM and by structured inter-organelle interactions, such as between the ER and mitochondria ( 16 ). The importance of caveolae in Ca 2+ signaling was confirmed by the strategic localization of Ca 2+ effectors, such as PM Ca 2+ ATPase pumps and IP3Rs, providing a platform for the assembly of diverse Ca 2+ signaling complexes ( 17 – 20 ). In particular, the tumor-suppressor caveolin-1, a fundamental member of caveolae, plays a key role in the control of the Ca 2+ -dependent apoptotic pathway and regulates fundamental mitochondrial functions during tumor growth ( 21 ).…”

Functional Microdomains in Heart’s Pacemaker: A Step Beyond Classical Electrophysiology and Remodeling

“…Significant decrease in the occurrence of spontaneous Ca 2+ sparks was reported in rat and human atrial myocytes treated with MβCD, which depletes cholesterol and destroys caveolae structures ( Glukhov et al, 2015a ). Stimulation of IP 3 Rs through sphingosine kinase 1 is shown to control the Ca 2+ release in caveolar microdomains ( Pulli et al, 2015 ). IP 3 Rs were also associated with structural protein AnkB: their expression and membrane targeting were found to be significantly reduced in AnkB +/- mice ( Le Scouarnec et al, 2008 ).…”

Oxysterol-binding protein related-proteins (ORPs) 5 and 8 regulate calcium signaling at specific cell compartments

“…To assess the importance of ORP5/8 in regulating Ca 2+ in intracellular compartments, we employed the luminescent calcium indicator protein, aequorin. Upon transient expression, wild-type aequorin localizes to the cytosol, but aequorin has been genetically engineered to target different cell compartments such as the mitochondria and the caveolae [24,25]. First, we set out to measure cytoplasmic and mitochondrial Ca 2+ upon overexpression of GFP-ORP5 or −ORP8 in the presence of extracellular Ca 2+ .…”

“…The luminescent Ca 2+ binding recombinant protein, aequorin, was employed to measure Ca 2+ concentrations in the cytoplasm, in the mitochondria, and at the caveolae as previously described in [24,25]. Briefly, 5000-10,000 HeLa cells were seeded on to a 96-well cell culture plate for measurements when a plate reader setup was employed (HIDEX Sense plate reader, HIDEX corp., Turku, Finland).…”