Changjoon Justin Lee, PhD - US grants

DESCRIPTION (provided by applicant): Serine proteases are blood components that function in the clotting cascade. These proteases are known to circulate in blood and are not allowed to enter brain tissue by the blood-brain barrier. However, accumulating evidence indicate that certain proteases and receptors for these proteases, termed as protease activated receptors (PAR), are expressed by neurons and clia and participate in a large signaling cascade. It has been recently shown that information activation of PAR potentiates NMDA receptor responses by voltage-independent and voltage-dependent ways. NMDA receptor overactivation has been implicated in cell death during a stroke or ischemia, in which serine proteases enter the blood-brain barrier and activate PARs expressed by neurons and glia. The mechanism for voltage-dependent NMDA receptor potentiation by PARI activation has not been elucidated, although preliminary results suggest that the voltage dependent block by Mg2+ is relieved after PAR1 activation. The aims of this proposal investigate the detailed mechanism of how individual NMDA receptor channel is modulated by activation of PARI. The experiments are designed to first establish the single channel kinetic model of NMDA receptor channel by performing cell attached single channel patch, in which only one channel is present. Then the channel properties, such as open time distribution, open probability, burst structure, and shut time distribution, are compared before and after PAR1 activation by thromicin. The relief of Mg2+ block will be tested by performing dual recordings of perforated-patch/voltage-clamp to manipulate the holding potential on one patch and cell attached patch of single channel on the other. The molecular determinants of the effects by PARI will be determined by using C-terminal chimera of NR2C and NR2A and C-terminal deletion mutants of NR2A subunit.