Calcium Release-activated Calcium Current (I CRAC) Is a Direct Target for Sphingosine*

Whole cell patch-clamp recordings were made to study the regulation of the store-operated calcium release-activated calcium current(I CRAC) by metabolites involved in the sphingomyelin pathway in RBL-2H3 cells. Sphingosine, a regulator of cell growth, inhibits I CRAC completely within 200 s and independently from conversion to either sphingosine 1-phosphate or ceramide. Structural analogs of sphingosine, includingN,N-dimethylsphingosine,dl-threo-dihydrosphingosine, andN-acetylsphingosine (C2-ceramide) also blockI CRAC. This effect is always accompanied by an elevation of whole cell membrane capacitance. These sphingolipids appear, therefore, to accumulate in the plasma membrane and directly block I CRAC channels. Sphingosylphosphorylcholine also increases capacitance but does not inhibit I CRAC, demonstrating structural specificity and that the elevation of capacitance is necessary but not sufficient for block. Nerve growth factor, which is known to break down sphingomyelin, inhibits I CRAC, and this inhibition can be antagonized by reducing sphingosine production withl-cycloserine, suggesting thatI CRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocksI CRAC, suggesting that the sphingomyelin pathway is involved in I CRAC regulation.