Intracellular calcium activates TRPM2 and its alternative spliced isoforms
Jianyang Du1, Jia Xie, Lixia Yue
1Department of Cell Biology, Calhoun Cardiology Center, University of Connecticut Health Center, Farmington, CT 06030, USA.
Abstract:
Melastatin-related transient receptor potential channel 2 (TRPM2) is a Ca(2+)-permeable, nonselective cation channel that is involved in oxidative stress-induced cell death and inflammation processes. Although TRPM2 can be activated by ADP-ribose (ADPR) in vitro, it was unknown how TRPM2 is gated in vivo. Moreover, several alternative spliced isoforms of TRPM2 identified recently are insensitive to ADPR, and their gating mechanisms remain unclear. Here, we report that intracellular Ca(2+) ([Ca(2+)](i)) can activate TRPM2 as well as its spliced isoforms. We demonstrate that TRPM2 mutants with disrupted ADPR-binding sites can be activated readily by [Ca(2+)](i), indicating that [Ca(2+)](i) gating of TRPM2 is independent of ADPR. The mechanism by which [Ca(2+)](i) activates TRPM2 is via a calmodulin (CaM)-binding domain in the N terminus of TRPM2. Whereas Ca(2+)-mediated TRPM2 activation is independent of ADPR and ADPR-binding sites, both [Ca(2+)](i) and the CaM-binding motif are required for ADPR-mediated TRPM2 gating. Importantly, we demonstrate that intracellular Ca(2+) release activates both recombinant and endogenous TRPM2 in intact cells. Moreover, receptor activation-induced Ca(2+) release is capable of activating TRPM2. These results indicate that [Ca(2+)](i) is a key activator of TRPM2 and the only known activator of the spliced isoforms of TRPM2. Our findings suggest that [Ca(2+)](i)-mediated activation of TRPM2 and its alternative spliced isoforms may represent a major gating mechanism in vivo, therefore conferring important physiological and pathological functions of TRPM2 and its spliced isoforms in response to elevation of [Ca(2+)](i).
Insights
Intracellular calcium ions ([Ca2+]i) activate the TRPM2 channel and its variants, independent of ADP-ribose. This calcium-dependent gating, mediated by a calmodulin-binding domain, is crucial for TRPM2 function in vivo.
Area of Science:
- Molecular Biology
- Ion Channel Physiology
- Cell Signaling
Background:
- Melastatin-related transient receptor potential channel 2 (TRPM2) is a calcium-permeable cation channel implicated in oxidative stress, cell death, and inflammation.
- While TRPM2 is known to be activated by ADP-ribose (ADPR) in vitro, its in vivo gating mechanisms and the function of its ADPR-insensitive splice variants remain unclear.
- Understanding TRPM2 gating is critical for elucidating its role in various physiological and pathological processes.
Purpose of the Study:
- To investigate the in vivo gating mechanisms of TRPM2 and its alternative splice isoforms.
- To determine the role of intracellular calcium ([Ca2+]i) and ADP-ribose (ADPR) in TRPM2 activation.
- To elucidate the molecular basis of [Ca2+]i-mediated TRPM2 gating.
Main Methods:
- Utilized site-directed mutagenesis to disrupt ADPR-binding sites in TRPM2.
- Assessed TRPM2 channel activity in response to [Ca2+]i and ADPR using electrophysiological recordings in intact cells.
- Investigated the role of the N-terminal calmodulin (CaM)-binding domain in TRPM2 activation.
- Examined the activation of both recombinant and endogenous TRPM2 channels following intracellular calcium release.
Main Results:
- Intracellular calcium ([Ca2+]i) directly activates both TRPM2 and its splice isoforms, independent of ADPR.
- TRPM2 mutants lacking functional ADPR-binding sites are readily activated by [Ca2+]i, confirming ADPR-independent gating.
- Calcium-mediated activation of TRPM2 involves an N-terminal calmodulin (CaM)-binding domain.
- Both [Ca2+]i and the CaM-binding motif are essential for ADPR-mediated TRPM2 gating.
- Intracellular calcium release, including receptor activation-induced release, activates both recombinant and endogenous TRPM2 channels.
Conclusions:
- Intracellular calcium ([Ca2+]i) serves as a key activator of TRPM2 and is the sole known activator for its splice isoforms.
- The CaM-binding domain is critical for [Ca2+]i-dependent TRPM2 activation.
- [Ca2+]i-mediated gating of TRPM2 and its splice variants likely represents a significant in vivo activation mechanism, contributing to their physiological and pathological roles.
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