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

Nicotinic 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.