Abstract

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 the hypothesis that acid ceramidase (AC) plays a pivotal role in regulation of TRPML1 channel activity in podocytes. To detect TRPML1 channel‐mediated Ca2+ release, cultured murine podocytes were transfected via nucleofection with GCaMP3‐ML1, a TRPML1‐targeted genetically encoded Ca2+ indicator. To characterize GCaMP3 expression in lysosomes, confocal microscopy confirmed the colocalization of GCaMP3‐ML1 and Lamp‐1 suggesting its successful transfection and localization in lysosomes. ML‐SA1 as a specific cell‐permeable TRPML1 inducer remarkably increased GCaMP3 fluorescence intensity in podocytes. For dynamic recording of Ca2+ release, a high speed wavelength switching fluorescent microscopic imaging system was used to continuously monitor GCaMP3 fluorescence signal in living podocytes. It was found that in the presence of a lysosome‐disrupting cathepsin C substrate, GPN that depleted lysosomal Ca2+, GCaMP3‐detected Ca2+ release response to ML‐SA1 was substantially attenuated. Pre‐treatment of podocytes with carmofur, a specific AC inhibitor almost totally blocked ML‐SA1‐induced Ca2+ release in podocytes. Sphingosine as the AC product of ceramide hydrolysis was found to increase GCaMP3 fluorescence by 3.95 folds in podocytes. Whole‐lysosome patch clamp recording using a Port‐A‐Patch system was performed to study the effects of various AC‐associated sphingolipids on TRPML1 channel activity. After characterization of integrity and purity of isolated lysosomes, we detected ML‐SA1‐induced Ca2+ currents through the isolated lysosome membrane in a dose‐dependent manner. Sphingomyelin, the substrate of ceramide, blocked Ca2+ release induced by ML‐SA1, ceramide had no effects on ML‐SA1‐induced Ca2+ currents, but sphingosine as a product of ceramide hydrolysis via AC remarkably increased ML‐SA1‐induced Ca2+ release by 2.35 folds. Furthermore, interactions of lysosome and autophagosome in podocytes indicated by colocalization of LC3B‐GFP and Lamp‐1‐RFP were inhibited by carmofur. These results suggest that normal function of lysosomal AC and associated production of sphingosine are important for TRPML1 channel activity and Ca2+‐dependent lysosome trafficking.Support or Funding InformationThis study was supported by NIH grants DK54927 to Pin‐Lan Li and HL89563 to Ningjun Li.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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