Intracellular two-phase Ca²⁺ release and apoptosis controlled by TRP-ML1 channel activity in coronary arterial myocytes

Abstract

Activation of the death receptor Fas has been reported to produce a two-phase intracellular Ca2+ release response in coronary arterial myocytes (CAMs), which consists of local Ca2+ bursts via lysosomal transient potential receptor-mucolipin 1 (TRP-ML1) channels and consequent Ca2+ release from the sarcoplasmic reticulum (SR). The present study was designed to explore the molecular mechanism by which lysosomal Ca2+ bursts are coupled with SR Ca2+ release in mouse CAMs and to determine the functional relevance of this lysosome-associated two-phase Ca2+ release to apoptosis, a common action of Fas activation with Fas ligand (FasL). By confocal microscopy, we found that transfection of CAMs with TRP-ML1 small interfering (si)RNA substantially inhibited FasL (10 ng/ml)-induced lysosome Ca2+ bursts and consequent SR Ca2+ release. In contrast, transfection of CAMs with plasmids containing a full-length TRP-ML1 gene enhanced FasL-induced two-phase Ca2+ release. We further demonstrated that FasL significantly increased the colocalization of the lysosomal marker Lamp1 with ryanodine receptor 3 and enhanced a dynamic trafficking of lysosomes to the SR. When CAMs were treated with TRP-ML1 siRNA, FasL-induced interactions between the lysosomes and SR were substantially blocked. Functionally, FasL-induced apoptosis and activation of calpain and calcineurin, the Ca2+ sensitive proteins that mediate apoptosis, were significantly attenuated by silencing TRP-ML1 gene but enhanced by overexpression of TRP-ML1 gene. These results suggest that TRP-ML1 channel-mediated lysosomal Ca2+ bursts upon FasL stimulation promote lysosome trafficking and interactions with the SR, leading to apoptosis of CAMs via a Ca2+-dependent mechanism.

The lysosomal Ca2+ release channel TRPML1 regulates lysosome size by activating calmodulin

“…Compared with the fusion process, very limited information is available for lysosomal membrane fission. Recent studies have shown that Ca 2ϩ release via lysosomal P2X4 activates CaM to trigger lysosome fusion (12 (17,20) or GECO-TRPML1 (21)), and lipid bilayers (25)(26)(27)(28), TRPML1 has been demonstrated to be a Ca 2ϩ -permeable channel in the lysosome. In this study, we investigated the role of TRPML1 in the regulation of lysosome size.…”

“…Transient receptor potential mucolipin 1 (TRPML1) (10,(13)(14)(15) belongs to the large family of transient receptor potential ion channels that permeates Ca 2ϩ , Na ϩ , and other cations (10,(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28). Mutations in TRPML1 gene lead to mucolipidosis type IV (ML4) disease, which is characterized with defects in membrane trafficking in the late endocytic pathway (10,29), enlarged lysosomes (10,29), and impaired lysosome biogenesis (30 -32).…”

Regulation of dynein-mediated autophagosomes trafficking by ASM in CASMCs

“…RNA interference of TRPML1 gene was achieved by transfection of double-stranded siRNA of targeting TRPML1 (Accession number: BC118374) consisted of 5′-CAGCUUCCGGCUCCUG-3′ as we previously described (22). The scrambled small RNA (5′-AATTCTCCGAACGTGTCACGT-3′) was used as a negative control.…”

“…The scrambled small RNA (5′-AATTCTCCGAACGTGTCACGT-3′) was used as a negative control. Overexpression of TRPML1 was achieved by transfections of CASMCs with TRPML1 cDNA plasmids as described in our previous study (22, 23). Transfection of siRNA or cDNA was performed using the siLentFect Lipid Reagent or TransFectin Lipid Reagent (Bio-Rad, CA, USA) according to the manufacturer's instructions, as we described previously (24).…”

Isolation of Murine Coronary Vascular Smooth Muscle Cells

“…Teng et al was the first to report a method for isolating VSMCs from the mouse coronary circulation; however, we have amended the protocol significantly as they also isolated endothelial cells. Other labs have also used the protocol from Teng et al to isolate coronary arterial myocytes and airway smooth muscle cells 4,5 . The alterations we have incorporated will yield a population of cells highly enriched for VSMCs from the coronary circulation.…”