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Kinetics of the block by intracellular Mg2+ of the NMDA‐activated channel in cultured rat neurons.

1. Single‐channel currents activated by the glutamate agonist N‐methyl‐D‐aspartate (NMDA) were recorded from outside‐out patches of cultured rat cortical neurons in the presence of intracellular Mg2+ (Mgi2+). The rate constants of the block by Mgi2+ were measured using amplitude distribution analysis. 2. At a membrane potential of 0 mV, the blocking rate constant (k+) of Mgi2+ was estimated to be 2.1 x 10(7) M‐1 S‐1 and the unblocking rate constant (k‐) 1.7 x 15(5)s‐1. The very fast rate constants of the block by Mgi2+ explain why channel flicker was not fully resolvable during block of the NMDA‐activated single‐channel current by Mgi2+. 3. The blocking rate constant of Mgi2+ increased with increasing concentrations of Mgi2+. The unblocking rate constant was Mgi2+ concentration independent. 4. The blocking rate constant increased e‐fold per 64 mV depolarization, whereas the unblocking rate constant decreased e‐fold per 133 mV depolarization. The dissociation constant (KD) calculated from the blocking rates (k‐/k+) decreased e‐fold per 43 mV depolarization, and had a value at 0 mV of 7.8 mM. These values are consistent with previous estimates obtained from the voltage‐dependent inhibition of the single‐channel current amplitude. Both results predict, based on the Woodhull model, that Mgi2+ traverses about one‐third of the membrane field to reach its blocking site. 5. The unblocking rate constant of Mgi2+ is one to two orders of magnitude faster than the previously reported unblocking rate constant of extracellular Mg2+ (Mgo2+) in the physiological voltage range, and their voltage dependencies are of opposite signs. These findings are consistent with the hypothesis that there are separate binding sites in the channel for Mgi2+ and Mgo2+. 6. Based on the blocking kinetics of Mgi2+ and Mgo2+, an energy profile of three barriers and two binding sites for Mg2+ is proposed.