Identity of Lysosomal NAADP-Sensitive Ca2+ Release Channels is TRP-ML1 in Coronary Arterial Smooth Muscle Cells.

Recently, we characterized lysosomal nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive Ca²⁺ release channels in coronary arterial smooth muscle cells (CASMCs). The present study was designed to further confirm the identity of these lysosomal Ca²⁺ release channels that possibly relate to transient receptor potential-mucolipin-1 (TRP-ML1) protein, a non-selective Ca²⁺ permeable cation channel. Western blot analysis demonstrated the existence of TRP-ML1 protein in purified lysosomes from CASMCs. By lipid bilayer reconstitution of these purified CASMCs lysosomes, this NAADP-activated channel activity in TRP-ML1-deprived lysosomal preparations was significantly reduced. In intact CASMCs, endothelin-1 (a lysosomal Ca²⁺ release agonist) induced a large lysosome-associated Ca²⁺ release, which was significantly attenuated by transfection of TRP-ML1 siRNA in the cells. However, oxotremorine-induced Ca²⁺ release in CASMCs was not altered by TRP-ML1 siRNA. In addition, ultrasound microbubble introduction of NAADP into TRP-ML4-human fibroblastcells did not produce any Ca²⁺ release response. However, when these TRP-ML^-/- cells were transfected with a full-length TRP-ML1 gene, NAADP-induced Ca²⁺ release response was restored. We conclude that lysosomal TRP-ML1 is functioning as NAADP-sensitive Ca²⁺release channels. [ABSTRACT FROM AUTHOR]