Control of lysosomal TRPML1 channel activity and exosome release by acid ceramidase in mouse podocytes

The transient receptor potential mucolipin 1 (TRPML1) channel has been reported to mediate lysosomal Ca²⁺ release that is involved in Ca²⁺-dependent lysosome trafficking and autophagic flux. However, this regulatory mechanism of lysosomal TRPML1 channel activity in podocytes remains poorly understood. In the present study, we tested whether the TRPML1 channel in podocytes mediates lysosome trafficking, which is essential for multivesicular body (MVB) degradation by lysosomes. We first demonstrated the abundant expression of TRPML1 channel in podocytes. By GCaMP3 Ca²⁺ imaging, we characterized the lysosomal specificity of TRPML1 channel-mediated Ca²⁺ release in podocytes. Given the important role of acid ceramidase (AC) in lysosome function and podocyte injury, we tested whether AC regulates this TRPML1 channel-mediated Ca²⁺ release and consequent lysosome-dependent MVB degradation in podocytes. Pharmacologically, it was found that TRPML1 channel activity was remarkably attenuated by the AC inhibitor carmofur. Sphingosine, as an AC product, was demonstrated to induce TRPML1-mediated Ca²⁺ release, which was inhibited by a TRPML1 blocker, verapamil. Using a Port-a-Patch planar patch-clamp system, we found that AC-associated sphingolipids, sphingomyelin, ceramide, and sphingosine had different effects on TRPML1 channel activity in podocytes. Functionally, the inhibition of AC or blockade of TRPML1 channels was found to suppress the interaction of lysosomes and MVBs, leading to increased exosome release from podocytes. These results suggest that AC is critical for TRPML1 channel-mediated Ca²⁺ release, which controls lysosome-MVB interaction and exosome release in podocytes.