TRP‐ML1 functions as a lysosomal NAADP‐sensitive Ca2+ release channel in coronary arterial myocytes

AbstractNicotinic acid adenine dinucleotide phosphate (NAADP) is a potent intracellular Ca2+ signalling second messenger, but the mechanism of NAADP‐induced Ca2+ release is still poorly understood. The present study tested the hypothesis that NAADP induces Ca2+ release from the lysosomal store via a TRP‐ML1 (transient receptor potential‐mucolipin 1)‐mediated Ca2+ release channel in coronary arterial myocytes (CAMs). RT‐PCR and Western blot analyses demonstrated that TRP‐ML1 was present in CAMs, and fluorescence resonance energy transfer (FRET) detection revealed that the TRP‐ML1 was closely associated with some lysosomal proteins in these CAMs. ET‐1, a well‐known NAADP stimulator, was found to induce a local Ca2+ burst from lysosomes followed by a global Ca2+ release. This lysosome‐associated Ca2+ release was significantly inhibited in the TRP‐ML1 siRNA pre‐treated CAMs by 46.8 ± 12.6% in the local Ca2+ burst and 73.3 ± 14.9% in the global Ca2+ wave. In the reconstituted lysosomal channels from CAMs, NAADP activated Ca2+ release channels at concentrations of 1–1000 nM, but neither activators (1 μM IP3, 5 μM Rya) nor blockers (100 μM 2‐APB, 50 μM Rya) of sarcoplasmic reticulum (SR) Ca2+ release channels had effect on the channel activity. Moreover, TRP‐ML1 gene silencing reduced this NAADP‐sensitive Ca2+ release channel activity in lysosomes by 71.5 ± 18.5%. Immunoprecipitation or blockade of TRP‐ML1 by anti‐TRP‐ML1 antibodies almost abolished NAADP‐induced activation of lysosomal Ca2+ channels (to 14.0 ± 4.4% of control). These results for the first time provide direct evidence that an NAADP‐sensitive Ca2+ release channel is characteristic of TRP‐ML1 channels.