Calcium-induced inactivation of NMDA receptor-channels evolves independently of run-down in cultured rat brain neurones

1. Calcium-induced transient inactivation of NMDA receptor (NMDAR) channels was studied in cultured rat hippocampal and cerebellar granule neurones using patch-clamp techniques and confocal scanning microscopy. 2. During whole-cell recordings, in the presence of 2 mM external Ca2+, conditioning (2-20 s) pulses of NMDA (20-100 mu M) caused a transient decrease in NMDA responses. Recovery developed in two phases with time constants of 0 . 6 and 40 s. The slow phase of the recovery could be prevented either by strong intracellular Ca2+ ([Ca2+](i)) buffering with 30 mM BAPTA or by using Ca2+-free extracellular solution. 3. Simultaneous measurement of currents and Ca2+-dependent fluorescence revealed a close correlation between the time constants of [Ca2+](i) decay and the slow component-of NMDA-activated test current recovery. 4. During prolonged recordings, the transient inactivation was not related to irreversible NMDA-activated current run-down. After 25 min of recording with ATP-free intracellular solution, NMDA-activated currents in hippocampal neurones irreversibly decreased by 49 +/- 5% while inactivation decreased by 8% (n = 9). Calyculin A and FK-506 (phosphatase inhibitors) significantly delayed run-down but-did not modulate the transient inactivation. 5. In cerebellar granule cells that did not show run-down (4 mM MgATP in the pipette) the percentage of transient inactivation strongly decreased during 25 min of recording (from 28 +/- 6 to 7 +/- 5%, n = 15). 6. In cell-attached recordings (5 mu M NMDA in the pipette), elevation of [Ca2+](i) (application of 100 mu M NMDA to the soma) caused a reversible reduction of single NMDAR channel open probability (NPo) due to a decrease in the frequency of channel opening. 7. In inside-out patches, application of Ca2+ to the cytoplasmic side of the membrane caused a rapid and reversible decrease in NPo (13 out of 29 patches). In the absence of run-down, the ability of Ca2+ to transiently inhibit NMDAR channel activity disappeared after 3-5 min of recording. 8. These results indicate that Ca2+-induced transient inactivation of NMDAR currents develops independently from the run-down and suggest that a diffusible Ca2+-dependent factor mediates NMDAR channel inactivation.