Domain-Specific Agonist Binding Affinities Explain Structural and Functional Regulation of TRPM2.

Area of Science:

• Molecular Biology
• Biophysics
• Cell Physiology

Background:

• TRPM2 is a calcium-permeable channel activated by ADP-ribose (ADPR) and oxidative stress.
• The roles of its two nucleotide-binding domains, MHR1/2 and NUDT9H, in TRPM2 activation are not fully understood.

Purpose of the Study:

• To quantitatively determine the ligand-binding affinities of isolated human TRPM2 MHR1/2 and NUDT9H domains.
• To elucidate the relative contributions of these domains to TRPM2 channel activation by ADPR and its analogs.

Main Methods:

• Biophysical techniques (e.g., surface plasmon resonance) were used to measure binding affinities.
• Mutational analysis was performed to assess the functional impact of domain alterations.
• Intracellular ADPR concentrations were quantified in resting and stimulated cells.

Main Results:

• The MHR1/2 domain exhibits high-affinity binding to ADPR (K_d ≈ 0.5 µM), while NUDT9H shows significantly lower affinity (K_d ≈ 192 µM).
• Mutations in MHR1/2 strongly impact ligand binding and channel gating, whereas NUDT9H mutations have modest effects.
• Intracellular ADPR levels are generally below the affinity threshold for significant NUDT9H binding.

Conclusions:

• High-affinity ADPR binding to the MHR1/2 domain is sufficient for TRPM2 channel activation under physiological conditions.
• The NUDT9H domain likely plays a structural role rather than directly mediating ligand-dependent activation.
• These findings provide a quantitative basis for understanding TRPM2 regulation by ADPR.