TRP-ML1 Functions as a Lysosomal NAADP-Sensitive Ca Release Channel in Coronary Arterial Myocytes

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent intracellular Ca signalling second messenger, but the mechanism of NAADP-induced Ca release is still poorly understood. The present study tested the hypothesis that NAADP induces Ca release from the lysosomal store via a TRP-ML1 (transient receptor potential-mucolipin 1)-mediated Ca 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 Ca burst from lysosomes followed by a global Ca release. This lysosome-associated Ca release was significantly inhibited in the TRP-ML1 siRNA pre-treated CAMs by 46.8 12.6% in the local Ca burst and 73.3 14.9% in the global Ca wave. In the reconstituted lysosomal channels from CAMs, NAADP activated Ca release channels at concentrations of 1–1000 nM, but neither activators (1 M IP3, 5 M Rya) nor blockers (100 M 2-APB, 50 M Rya) of sarcoplasmic reticulum (SR) Ca release channels had effect on the channel activity. Moreover, TRP-ML1 gene silencing reduced this NAADP-sensitive Ca release channel activity in lysosomes by 71.5 18.5%. Immunoprecipitation or blockade of TRP-ML1 by antiTRP-ML1 antibodies almost abolished NAADP-induced activation of lysosomal Ca channels (to 14.0 4.4% of control). These results for the first time provide direct evidence that an NAADP-sensitive Ca release channel is characteristic of TRP-ML1 channels.