TRPM2-mediated intracellular Zn2+ release triggers pancreatic β-cell death

Abstract

Reactive oxygen species (ROS) can cause pancreatic β-cell death by activating transient receptor potential (melastatin) 2 (TRPM2) channels. Cell death has been attributed to the ability of these channels to raise cytosolic Ca 2 + . Recent studies however revealed that TRPM2 channels can also conduct Zn 2 + , but the physiological relevance of this property is enigmatic. Given that Zn 2 + is cytotoxic, we asked whether TRPM2 channels can permeate sufficient Zn 2 + to affect cell viability. To address this, we used the insulin secreting (INS1) β-cell line, human embryonic kidney (HEK)-293 cells transfected with TRPM2 and pancreatic islets. H 2 O 2 activation of TRPM2 channels increases the cytosolic levels of both Ca 2 + and Zn 2 + and causes apoptotic cell death. Interestingly, chelation of Zn 2 + alone was sufficient to prevent β-cell death. The source of the cytotoxic Zn 2 + is intracellular, found largely sequestered in lysosomes. Lysosomes express TRPM2 channels, providing a potential route for Zn 2 + release. Zn 2 + release is potentiated by extracellular Ca 2 + entry, indicating that Ca 2 + -induced Zn 2 + release leads to apoptosis. Knockout of TRPM2 channels protects mice from β-cell death and hyperglycaemia induced by multiple low-dose streptozotocin (STZ; MLDS) administration. These results argue that TRPM2-mediated, Ca 2 + -potentiated Zn 2 + release underlies ROS-induced β-cell death and Zn 2 + , rather than Ca 2 + , plays a primary role in apoptosis.

Reciprocal regulation of actin cytoskeleton remodelling and cell migration by calcium and zinc: role of TRPM2 channels

“…Given that H 2 O 2 is cytotoxic and its toxicity can vary depending on the cell type (Manna et al, 2015), we first screened PC-3 and HeLa cells for their sensitivity to H 2 O 2 (data not shown) and chose a concentration (<200 µM) that is not cytotoxic under the experimental conditions used. We used Alexa-Fluor-488-conjugated phalloidin, a fluorescent actin stain, to monitor H 2 O 2 -induced changes in the actin cytoskeleton.…”

“…Western blotting of lysosomes isolated from transfected HeLa cells by OptiPrep density gradient centrifugation showed a band corresponding to the estimated size (∼170 kDa) of HA-TRPM2 (Fig. 2L) It is known that Fluo-4 is not specific for Ca 2+ ; for example, it binds Zn 2+ ∼100-fold more avidly than Ca 2+ (Manna et al, 2015;Sensi et al, 2009). We therefore asked whether the Fluo-4 signal seen in Fig.…”

“…Prior to their use, we validated the specificity of the probes (Fig. S3A-D ; and TPEN, as reported previously (Manna et al, 2015), failed to chelate Ca 2+ . Increasing the cytosolic Ca 2+ levels through treatments with A23187 or thapsigargin (data not shown) had no major effect on actin cytoskeleton, but, interestingly, co-treatment with BAPTA-AM attenuated stress fibres and induced filopodia formation (Fig.…”

TRPM7 senses oxidative stress to release Zn 2+ from unique intracellular vesicles

“…However, we have previously shown that TRPM7 is also on intracellular membranes (33)(34)(35). Indeed, ROS activation of divalent ion influx through TRPM7 (5, 9) is reminiscent of ROS activation of TRPM2, which releases Ca 2+ and Zn 2+ from lysosomes (36,37 uncharacterized vesicles (M7Vs) that are distinct from familiar organelles. These vesicles sequester Zn 2+ during cytosolic overload.…”

“…However, we have previously shown that TRPM7 is also on intracellular membranes (33-35). Indeed, ROS activation of divalent ion influx through TRPM7 (5, 9) is reminiscent of ROS activation of TRPM2, which releases Ca 2+ and Zn 2+ from lysosomes (36,37 ] and ROS during development and injury. …”