Defect And Rescue Of Naadp-Activated Ca2+Channel Activity In Mucolipin-1 Deficient Human Fibroblasts

Recent studies have indicated that mucolipin‐1 (TRP‐ML1) may be a lysosomal Ca 2+ channel activated by a novel Ca 2+ signaling nucleotide, NAADP. The present study attempted to further test the hypothesis that NAADP functions as an endogenous activator of TRP‐ML1 using human fibroblasts. By reconstituting lysosomal channels, we found that lysosomes from TRP‐ML −/− cells had much lower Ca 2+ channel activity (NP O =0.00406 ±0.0009) compared to TRP‐ML +/+ cells (NP O =0.00562 ±0.00176). NAADP (100 nM) significantly increased the activity of lysosomal Ca 2+ channel from TRP‐ML1 +/+ cells (NP O =0.0547±0.0105), but it had no effect on these channels from TRP‐ML1 −/− cells. When TRP‐ML −/− cells was transfected with TRP‐ML1 gene, the channel activity was restored. Fluorescence imaging showed, ET‐1, a potent stimulator of NAADP production activated lysosome‐related local Ca 2+ burst followed by global Ca 2+ release in TRP‐ML1 +/+ cells, but not in TRP‐ML1 −/ − cells. In TRP‐ML1 transfected TRP‐ML1 −/− cells, ET‐1‐induced Ca 2+ response was rescued. Similarly, confocal microscopy also demonstrated that ET‐1 induced local Ca 2+ release from lysosomes in TRP‐ML1 +/+ or gene rescued TRP‐ML1 −/− fibroblasts, rather than TRP‐ML1 −/− cells. All these results indicate that NAADP may target TRP‐ML1 to release Ca 2+ from lysosomes and participates in NAADP‐mediated signaling regulation (Supported by NIH grants HL057244 and HL075316).