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Critical intracellular Ca2+ dependence of transient receptor potential melastatin 2 (TRPM2) cation channel activation
Damian McHugh1, Richard Flemming, Shang-Zhong Xu
1School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.
Abstract:
TRPM2 is a member of the melastatin-related TRP (transient receptor potential) subfamily. It is expressed in brain and lymphocytes and forms a cation channel that is activated by intracellular ADP-ribose and associated with cell death. In this study we investigated the calcium dependence of human TRPM2 expressed under a tetracycline-dependent promoter in HEK-293 cells. TRPM2 expression was associated with enhanced hydrogen peroxide-evoked intracellular calcium signals. In whole-cell patch clamp recordings, switching from barium- to calcium-containing extracellular solution markedly activated TRPM2 as long as ADP-ribose was in the patch pipette and exogenous intracellular calcium buffering was minimal. We suggest this effect reveals a critical dependence of TRPM2 channel activity on intracellular calcium. In the absence of extracellular calcium we observed concentration-dependent activation of TRPM2 channels by calcium delivered from the patch pipette (EC(50) 340 nM, slope 4.9); the maximum effect was at least as large as that evoked by extracellular calcium. Intracellular dialysis of cells with high concentrations of EGTA or 1,2-bis(o-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) strongly reduced the amplitude of the extracellular calcium response, and the residual response was abolished by a mixture of high and low affinity calcium buffers. TRPM2 channel currents in inside-out patches showed a strong requirement for Ca(2+) at the intracellular face of the membrane. We suggest that calcium entering via TRPM2 proteins acts at an intracellular calcium sensor closely associated with the channel, providing essential positive feedback for channel activation.
Insights
Transient Receptor Potential Melastatin 2 (TRPM2) channels critically depend on intracellular calcium for activation. This calcium acts as a positive feedback mechanism, enhancing channel activity and influencing cell death pathways.
Area of Science:
- Molecular Biology
- Cell Physiology
Background:
- Transient Receptor Potential Melastatin 2 (TRPM2) is a cation channel expressed in the brain and lymphocytes.
- TRPM2 is activated by intracellular ADP-ribose and implicated in cell death.
- Its precise regulatory mechanisms, particularly calcium dependence, require further elucidation.
Purpose of the Study:
- To investigate the calcium dependence of human TRPM2 channels expressed in HEK-293 cells.
- To determine the role of intracellular calcium in TRPM2 channel activation.
- To characterize the interaction between intracellular calcium and TRPM2 activity.
Main Methods:
- HEK-293 cells expressing human TRPM2 under a tetracycline-dependent promoter.
- Whole-cell patch clamp recordings to assess channel activity.
- Manipulation of extracellular and intracellular calcium concentrations, including the use of calcium buffers (EGTA, BAPTA).
Main Results:
- TRPM2 expression enhanced hydrogen peroxide-evoked intracellular calcium signals.
- Switching to calcium-containing extracellular solution markedly activated TRPM2 in the presence of ADP-ribose.
- Intracellular calcium activated TRPM2 in a concentration-dependent manner (EC50 340 nM) in the absence of extracellular calcium.
- High concentrations of intracellular calcium buffers significantly reduced TRPM2 responses.
- Inside-out patch recordings confirmed a strong requirement for intracellular Ca(2+) for TRPM2 channel currents.
Conclusions:
- Human TRPM2 channel activity is critically dependent on intracellular calcium.
- Intracellular calcium acts as a positive feedback regulator for TRPM2 channel activation.
- Calcium entering through TRPM2 may activate an associated intracellular sensor, enhancing channel function and potentially influencing cell death.