Intracellular calcium activates TRPM2 and its alternative spliced isoforms.

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.