Reconstitution of lysosomal NAADP-TRP-ML1 signaling pathway and its function in TRP-ML1−/−cells

Abstract

It is well known that the mutation of TRP-ML1 (transient receptor potential-mucolipin-1) causes mucolipidosis IV, a lysosomal storage disease. Given that lysosomal nicotinic acid adenine dinucleotide phosphate (NAADP)-Ca(2+) release channel activity is associated with TRP-ML1, the present study was designed to test the hypothesis that NAADP regulates lysosome function via activation of TRP-ML1 channel activity. Using lysosomal preparations from wild-type (TRP-ML1(+/+)) human fibroblasts, channel reconstitution experiments demonstrated that NAADP (0.01-1.0 μM) produced a concentration-dependent increase in TRP-ML1 channel activity. This NAADP-induced activation of TRP-ML1 channels could not be observed in lysosomes from TRP-ML1(-/-) cells, but was restored by introducing a TRP-ML1 transgene into these cells. Microscopic Ca(2+) fluorescence imaging showed that NAADP significantly increased intracellular Ca(2+) concentration to 302.4 ± 74.28 nM (vs. 180 ± 44.13 nM of the basal) in TRP-ML1(+/+) cells, but it had no effect in TRP-ML1(-/-) cells. If a TRP-ML1 gene was transfected into TRP-ML1(-/-) cells, the Ca(2+) response to NAADP was restored to the level comparable to TRP-ML1(+/+) cells. Functionally, confocal microscopy revealed that NAADP significantly enhanced the dynamic interaction of endosomes and lysosomes and the lipid delivery to lysosomes in TRP-ML1(+/+) cells. This functional action of NAADP was abolished in TRP-ML1(-/-) cells, but restored after TRP-ML1 gene was rescued in these cells. Our results suggest that NAADP increases lysosomal TRP-ML1 channel activity to release Ca(2+), which promotes the interaction of endosomes and lysosomes and thereby regulates lipid transport to lysosomes. Failure of NAADP-TRP-ML1 signaling may be one of the important mechanisms resulting in intracellular lipid trafficking disorder and consequent mucolipidosis.