Biochemical regulation at neuronal synapses

Higher order brain functions such as learning and memory arise from fundamental biochemical events occurring in the nervous system. Signal transduction mediated by Ca²⁺ is involved in a variety of physiological events at neuronal synapses. The spatio-temporal variations in the magnitude of Ca²⁺ signals give rise to varied physiological responses ranging from Long Term Potentiation (LTP) that underlie learning and memory to excitotoxicity that leads to neuronal death.

Calcium/calmodulin dependent protein kinase II (CaMKII) plays an important role in decoding Ca²⁺signals. Our group is interested in understanding how the biochemical properties of calcium /calmodulin dependent protein kinase II (CaMKII) support cellular events such as synaptic plasticity. Towards this goal, we are investigating the structure-activity relationships and protein-protein interactions of CaMKII. Although the interaction of CaMKII with the N-methyl-D-aspartate (NMDA)-type glutamate receptor is known to occur in vivo,the structural details as well as the functional significance of this interaction are still not clearly understood. We have found that the binding of the 2B subunit of NMDA receptor modulates the function of CaMKII in a way that might support its role in synaptic plasticity. Currently we are focusing our efforts on identification of the amino acid residues of both the proteins that make mutual contacts using a combination of site-directed mutagenesis and binding assays under in vitro as well as under intracellular conditions. For expression of the proteins we use E.coli, Baculovirus/insect cell system and HEK-293 cells. The functional consequences of this interaction are also being investigated. We are also searching for other proteins, which may be involved in binding interactions with CaMKII.

The NMDA receptor is regulated by phosphorylation. A site on NR2B, Ser¹³⁰³, is phosphorylated by CaMKII. We have found that this site is dephosphorylated by protein phosphatise 1 (PP1) present in the postsynaptic density (PSD). Currently we are investigating the physiological role of this phosphorylation.

Bioprospecting for Neuroactive compounds

Excitotoxicity is a cause of neuronal damage in many neuropathological conditions such as epilepsy, stroke, etc. Excessive influx of through channel proteins such as NMDA receptor into neurons leads to cell death. We are using an in vitro excitotoxicity model using primary neurons in culture to screen extracts from plants for their antagonistic effects on excitotoxicity. Since Ca²⁺-channel blockers could prevent excitotoxicity, we are also screening plant extracts for the presence of blockers of Ca²⁺-channels such as NMDA receptor. For this purpose, we have developed a cell biological assay for Ca²⁺-channels. Extracts which show neuroprotective effect in vitro,are being tested for their efficacy in vivo using animal models of excitotoxicity.