Specific proteolysis of the NR2 subunit at multiple sites by calpain

The NMDA subtype of glutamate receptor plays an important role in the molec ular mechanisms of learning, memory and excitotoxicity. NMDA receptors are highly permeable to calcium, which can lead to the activation of the calciu m-dependent protease, calpain. In the present study, the ability of calpain to modulate NMDA receptor function through direct proteolytic digestion of the individual NMDA receptor subunits was examined. HEK293t cells were cot ransfected with the NR1a/2A, NR1a/2B or NR1a/2C receptor combinations. Cell ular homogenates of these receptor combinations were prepared and digested by purified calpain I in vitro. All three NR2 subunits could be proteolyzed by calpain I while no actin or NR1a cleavage was observed. Based on immuno blot analysis, calpain cleavage of NR2A, NR2B and NR2C subunits was limited to their C-terminal region. In vitro calpain digestion of fusion protein c onstructs containing the C-terminal region of NR2A yielded two cleavage sit es at amino acids 1279 and 1330. Although it has been suggested that calpai n cleavage of the NMDA receptor may act as a negative feedback mechanism, t he current findings demonstrated that calpain cleavage did not alter [I-125 ]MK801 binding and that receptors truncated to the identified cleavage site s had peak intracellular calcium levels, Ca-45 uptake rates and basal elect rophysiological properties similar to wild type.