Synaptic NMDA receptor channels have a low open probability

Realistic estimates of channel-gating parameters of synaptic receptors are essential to an understanding of synaptic transmission and modulation. However, the gating of N-methyl-D-aspartate (NMDA) channels appears to differ, depending on recording conditions; thus, it remains unclear what measurements are most relevant to synaptic receptors. To further explore this discrepancy, we examined the open probability (P(o)) of NMDA channels in whole-cell and outside-out patch recording from cultured hippocampal neurons. Currents were evoked by rapid application of saturating concentrations of NMDA in the presence or absence of the 'irreversible' open channel blocker, MK-801 ((+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine). The reduction of the peak amplitude and the acceleration of the decay of the current in MK-801 were used to derive P(o) by fitting the current traces to a multistate kinetic model. The P(o) in whole cell was low (0.04), similar to that previously measured for synaptically activated NMDA channels. In contrast, ensemble average currents from outside-out patches were much more rapidly blocked in the presence of MK-801, indicative of a significantly higher P(o). The P(o) also gradually increased with the duration of recording in both whole-cell and outside-out configurations, suggesting that channel gating is sensitive to mechanical alterations of the patch or that washout of cytoplasmic factors leads to an increase in channel open probability. To test whether the disparity in P(o) could be attributed to different gating of synaptic and extrasynaptic channels, the P(o) of whole-cell currents was measured before and after all synaptic NMDA channels (> 90%) were blocked by evoking EPSCs in the presence of MK-801. In microisland cultures containing a single excitatory neuron, 81 ± 4% of NMDA channels were synaptic. However, the P(o) of synaptic and extrasynaptic channels was equivalent, suggesting that P(o) recorded in the whole-cell configuration is the same as at the synapse. The low open probability for synaptic channels implies that with each presynaptic stimulus only 50% of channels that bind glutamate actually open; thus, the NMDA receptor-mediated EPSC has a significant functional 'reserve.'