Journal
JOURNAL OF CELLULAR AND MOLECULAR MEDICINE
Volume 13, Issue 9B, Pages 3174-3185Publisher
WILEY
DOI: 10.1111/j.1582-4934.2008.00486.x
Keywords
transient receptor potential channel; lysosomal Ca2+store; Ca2+mobilization; organelles; coronary circulation
Categories
Funding
- National Institute of Health [HL-57244, HL-75316, DK54927]
- NATIONAL HEART, LUNG, AND BLOOD INSTITUTE [R01HL057244, R01HL091464, R01HL075316, R29HL057244] Funding Source: NIH RePORTER
- NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES [R01DK054927] Funding Source: NIH RePORTER
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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 mu M IP3, 5 mu M Rya) nor blockers (100 mu M 2-APB, 50 mu M 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.
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