Mechanism of glutamate receptor-channel function in rat hippocampal neurons investigated using the laser-pulse photolysis (LaPP) technique.

Ionotropic glutamate receptors are members of a large family of plasma membrane proteins expressed by cells of the nervous system. Upon binding glutamate, the receptors transiently open transmembrane channels that allow the entry of sodium ions. The resulting changes in the transmembrane potential of the cell initiates a process that is involved in signal transmission to another cell. The binding of glutamic acid triggers the channel opening in the microsecond time domain and the reversible inactivation (desensitization) of the receptors in the millisecond time region. The channel-opening mechanism of glutamate receptors was investigated in rat hippocampal neurons voltage-clamped to -60 mV at room temperature and pH 7.4. Two rapid chemical reaction techniques were used: (1) a cell-flow method with a 4-10 ms time resolution to apply L-glutamate and (2) a laser-pulse photolysis technique to release glutamate from gamma-O-(alpha-carboxy-2-nitrobenzyl)glutamate (alphaCNB-caged L-glutamate) with a time constant of 30 micros. The rate and equilibrium constants for channel opening were determined. The results are consistent with the receptor binding two molecules of glutamic acid before the channel opens, with an apparent dissociation constant of 600 microM. Channel opening and closing rate constants, k(op) and k(cl), were determined to be (9.5 +/- 1) x 10(3) s(-1) and (1.1 +/- 0.1) x 10(3) s(-1), respectively. The value of the channel-opening equilibrium constant, Phi (=k(op)/k(cl)), was 8.6 when determined by laser-pulse photolysis and 6.6 in cell-flow experiments. The results suggest that there are at least two forms of glutamate receptors in rat hippocampal neurons that desensitize with different rates. At a concentration of 500 microM glutamate, 80% of the receptors desensitized with a rate of approximately 200 s(-1) and 20% with a rate of approximately 50 s(-1).