Control of TRPML1 Channel Activity and Lysosome Trafficking by Acid Ceramidase in Mouse Coronary Arterial Endothelial Cells

Abstract

Previous studies have demonstrated that lysosomal acid ceramidase (AC) regulates lysosome-multivesicular body (MVB) interaction and exosome release in mouse coronary arterial endothelial cells (CAECs). However, it remains unknown how exosome secretion is controlled by AC. In this study, we tested a hypothesis that enhancement of lysosome trafficking by AC activity increases lysosome-MVB interaction determining MVB fate to promote exosome release, which may be attributed to its action on lysosomal TRPML1 channel-mediated Ca2+ release in CAECs. By GCaMP3 Ca2+ imaging, we found that ML-SA1 as a TRPML1 channel agonist remarkably increased lysosomal Ca2+ release through TRPML1 channel in WT/WT CAECs. In Asah1fl/fl/ECcre CAECs, however, ML-SA1 failed to influence TRPML1 channel-mediated Ca2+ release. As an AC product, sphingosine remarkably increased lysosomal Ca2+ release through TRPML1 channel in both WT/WT and Asah1fl/fl/ECcre CAECs. With whole-lysosome patch clamp recording, we detected ML-SA1-induced Ca2+ currents in a dose-dependent manner through the membrane of lysosomes isolated from WT/WT CAECs, while intact lysosomes isolated from Asah1fl/fl/ECcre CAECs had evidently blunted opening of TRPML1 channels in response to ML-SA1. Different effects of AC associated sphingolipids on TRPML1 channel activity were also examined in lysosomes isolated from WT/WT CAECs. It was found that sphingomyelin inhibited, but sphingosine enhanced lysosomal TRPML1 channel activity. Ceramide did not alter this lysosomal channel activity. In lysosomes isolated from Asah1fl/fl/ECcre CAECs, only sphingosine increased ML-SA1-induced TRPML1 channel-mediated Ca2+ release. Using confocal microscopy for measurement of lysosomes movement, we demonstrated that lysosomal TRPML1 channel activation by ML-SA1 in CAECs markedly enhanced lysosome trafficking toward nucleus, which was blocked by genetic deletion of AC. These results suggest that AC-mediated sphingolipid metabolism controls lysosomal TRPML1 channel-mediated Ca2+ release and lysosome trafficking in CAECs, which may contribute to the regulation of lysosome-MVB interaction and exosome release by AC (supported by NIH grant DK120491).