1. The whole‐cell patch clamp recording technique was employed to investigate the intracellular regulation of N‐methyl‐D‐aspartate (NMDA) receptors in cultured murine hippocampal neurones. Excitatory amino acids were repeatedly applied at regular intervals during intracellular dialysis with solutions of various composition. 2. Currents evoked by L‐aspartate, an agonist of NMDA receptors, gradually ‘washed out’ to approximately 50% of their initial amplitude during dialysis with an intracellular solution containing CsCl and EGTA as a calcium buffer. In contrast, responses to kainate did not wash out. The wash‐out of L‐aspartate currents followed an exponential time course with a time constant of about 150 s. Wash‐out did not appear to be related to desensitization of NMDA receptors. 3. Following wash‐out, L‐aspartate responses were blocked by Mg2+, ketamine or D‐2‐amino‐5‐phosphonovalerate indicating that these responses were still mediated by NMDA receptors. Furthermore, responses to NMDA itself showed wash‐out to the same extent and with a time course similar to that for L‐aspartate responses. 4. Neither the time course nor the extent of the wash‐out of responses to L‐aspartate was affected when the Ca2+ concentration of the dialysate was varied from zero to 1.5 x 10(‐5) M. In addition, wash‐out was unaffected by substitution of BAPTA for EGTA, indicating that wash‐out was not a consequence of changes in intracellular pH related to the binding of Ca2+ to the buffer or to the kinetics of this binding. Therefore, the wash‐out of NMDA currents could not be attributed to a gradual elevation of the concentration of intracellular Ca2+. 5. The extent of the wash‐out of L‐aspartate currents was similar for cells held at +40 versus ‐60 mV although the rate of wash‐out was slower at the depolarized potential. In addition, the reversal potential of these currents was not altered, demonstrating that a change in driving force did not account for a component of the wash‐out. 6. Inclusion of an ATP regeneration solution (Forscher & Oxford, 1985) in the dialysate prevented the wash‐out of L‐aspartate currents. ATP alone was less effective in preventing wash‐out whereas phosphocreatine and creatine phosphokinase were ineffective by themselves. Wash‐out also occurred when ATP was replaced with the non‐hydrolysable analogue, beta, gamma‐methyleneATP, or with GTP. In cells where wash‐out of L‐aspartate currents had been established, subsequent dialysis with the ATP regenerating solution partially reversed this wash‐out.(ABSTRACT TRUNCATED AT 400 WORDS)
The Journal of Physiology – Wiley
Published: Jul 1, 1989
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