Permeant ion regulation of N-methyl-D-aspartate receptor channel block by Mg(2+)

Abstract

Block of the channel of N-methyl-D-aspartate (NMDA) receptors by external Mg(2+) (Mg(o)(2+)) has broad implications for the many physiological and pathological processes that depend on NMDA receptor activation. An essential property of channel block by Mg(o)(2+) is its powerful voltage dependence. A widely cited explanation for the strength of the voltage dependence of block is that the Mg(o)(2+)-binding site is located deep in the channel of NMDA receptors; Mg(o)(2+) then would sense most of the membrane potential field during block. However, recent electrophysiological and mutagenesis studies suggest that the blocking site cannot be deep enough to account for the voltage dependence of Mg(o)(2+) block. Here we describe the basis for this discrepancy: the magnitude and voltage dependence of channel block by Mg(o)(2+) are strongly regulated by external and internal permeant monovalent cations. Our data support a model in which access to the channel by Mg(o)(2+) is prevented when permeant ion-binding sites at the external entrance to the channel are occupied. Mg(o)(2+) can block the channel only when the permeant ion-binding sites are unoccupied and then can either unblock back to the external solution or permeate the channel. Unblock to the external solution is prevented if external permeant ions bind while Mg(2+) blocks the channel, although permeation is still permitted. The model provides an explanation for the strength of the voltage dependence of Mg(o)(2+) block and quantifies the interdependence of permanent and blocking ion binding to NMDA receptors.

Figure 1

Decreasing permeant ion concentrations enhances Mg_o²⁺-blocking rate. (a) Single-channel currents activated by 10 μM NMDA + 10 μM glycine during block by 10 μM Mg_o²⁺ at −50 mV with [Na⁺]_o = 140 mM and the indicated [Cs⁺]_i. (b) Open-time histograms corresponding to the currents shown in a to the left of each histogram. Lines are double-exponential (upper two histograms) or single-exponential (lower histogram) fits to the histograms. Number of events in each histogram is more than 2,000. The principal mean open time (τ_o) is shown above each histogram. (c and d) Dependence of 1/τ_o on [Mg²⁺]_o under the indicated conditions. ○, 140 mM Na_o⁺/130 mM Cs_i⁺; ▵, 140 mM Na_o⁺/25 mM Cs_i⁺; □, 140 mM Na_o⁺/8 mM Cs_i⁺; ▴, 105 mM Na_o⁺/25 mM Cs_i⁺; ▾, 70 mM Na_o⁺/25 mM Cs_i⁺. Lines are regression fits to the plotted points.

Figure 2

k_+,app depends on the concentration of both [Cs⁺]_i and [Na⁺]_o. (a and b) Plots of the voltage dependence of k_+,app for block by Mg_o²⁺. Symbol meanings are as in the legend to Fig. 1 c and d. Data for 140 mM Na_o⁺/25 mM Cs_i⁺ are plotted in b as well as a for comparison with other data. Lines are drawn by using Eq. 2, with parameter values given in Table 1.

Figure 3

τ_b and k_−,app depend on [Na⁺]_o. (a) Single-channel currents activated by 5 μM NMDA + 10 μM glycine during block by 30 μM Mg_o²⁺ at −90 mV with [Cs⁺]_i = 130 mM and the indicated [Na⁺]_o. In each pair of current traces, the section of the upper single-channel record indicated by the horizontal bar is replotted below the bar at higher time resolution. (b) Closed-time histograms corresponding to the currents shown in a to the left of each histogram. Lines are four-exponential fits to the histograms. Number of events in each histogram is more than 1,000. (c) Voltage dependence of k_−,app in three different [Cs⁺]_i. Symbol meanings are as in legend to Fig. 1 c and d. (d) Voltage dependence of k_−,app in three different [Na⁺]_o. Because k_−,app does not depend on [Cs⁺]_i (c), points are averages of data at a single [Na⁺]_o and one or more [Cs⁺]_i. ⋄, 140 mM Na_o⁺; ■, 105 mM Na_o⁺; ⧫, 70 mM Na_o⁺. Lines are drawn by using Eq. 3, with parameter values given in Table 1.

Figure 4

Schematic diagram of the NMDA receptor (purple) during block and unblock by Mg_o²⁺. (a) When one or both of the external cation-binding sites are occupied, Mg_o²⁺ cannot enter the channel. (b) When the external cation-binding sites are unoccupied, Mg_o²⁺ can enter and block the channel (rate constant = k₊). (c) Block by Mg_o²⁺ can be terminated by dissociation to the external solution (rate constant = k_−,o) or by permeation of the channel (rate constant = k_−,i). (d) When one or both of the external cation-binding sites is occupied by Na_o⁺, Mg²⁺ blocking the channel cannot dissociate to the external solution but still can permeate the channel (rate constant = k_−,i).