CALCIUM RELEASE-ACTIVATED CALCIUM CURRENT (I-CRAC) IS A DIRECT TARGETFOR 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, including N ,N-dimethylsphingosine, DL-threo-dihydrosphingosine, and N-acetylsphin gosine (C-2-ceramide) also block I-CRAC. This effect is always accompa nied by an elevation of whole cell membrane capacitance. These sphingo lipids appear, therefore, to accumulate in the plasma membrane and dir ectly block I,, channels, Sphingosylphosphorylcholine also increases c apacitance but does not inhibit I-CRAC, demonstrating structural speci ficity and that the elevation of capacitance is necessary but not suff icient for block. Nerve growth factor, which is known to break down sp hingomyelin, inhibits I-CRAC and this inhibition can be antagonized by reducing sphingosine production with L-cycloserine, suggesting that I -CRAC is a physiologically relevant and direct target of sphingosine. We propose that sphingosine directly blocks I-CRAC suggesting that the sphingomyelin pathway is involved in I-CRAC regulation.