Activation of Recombinant Rat GluN1/gluN2D NMDA Receptors

Abstract

N-methyl-D-aspartate (NMDA) receptors are members of a class of ionotropic glutamate receptors and mediate slow, Ca2+-permeable synaptic transmission. Four separate GluN2 subunit genes (N2A-D) have been identified, which give rise to many of the observed differences in functional properties of the NMDA receptors, including conductance levels, open probability, and deactivation time course upon removal of agonists. A number of macroscopic and single channel properties of NMDA receptors can be grouped according to functional similarities. To study the distinctions between N2A and N2D-containing NMDA receptors, we have conducted single channel voltage-clamp recordings of N1/N2A and N1/N2D receptors to compare channel properties such as open probability and conductance. N1/N2A receptors have a higher channel conductance level (69 + 0.5 pS) and open probability (0.48 + 0.05) than N1/N2D receptors, which have a lower main conductance level and prominent subconductance level (55 + 2.3 pS and 33 + 1.4 pS) and a low open probability (0.015 + 0.004). One explanation of this difference in open probability is that rates constants describing activation steps are slower for NR1/N2D than for N1/N2A receptors, thereby reducing the probability of activating the receptor. However, our whole cell voltage clamp recordings indicate N1/N2D show a surprisingly rapid rise time (6.7 + 0.49 ms), similar to N1/N2A (8.5 + 0.50 ms). To understand how these two receptors with such strikingly different open probabilities can activate at a similar rate, we have fitted models of NMDA receptor activation to our single channel recordings. Preliminary analysis of our data has identified several rate constants describing pre-gating activation steps for N1/N2A that are more than 10-fold faster than in N1/N2D, suggesting that it may be possible to identify pre-gating steps responsible for the distinct characteristics of the N1/N2A and N1/N2D NMDA receptors.