Multiple structural elements determine subunit specificity of Mg2+ block in NMDA receptor channels

Abstract

In NMDA receptor channels, subtype-specific differences of Mg2+ block are determined by the NR2 subunits. Channels assembled from the NR1-NR2A or NR1-NR2B subunits are blocked more strongly than channels formed by the NR1-NR2C or NR1-NR2D subunits, predominantly reflecting a difference in voltage dependence. A determinant of Mg2+ block common to the NR2 subunits is located in the M2 domain (N-site or Q/R/N-site). However, subunit-specific differences of block suggested that additional structural elements exist. Chimeric NR2 subunits were constructed by replacing segments of the least sensitive NR2C subunit with homologous segments of the most sensitive NR2B subunit. Mutant NR2 subunits were coexpressed with wild-type NR1 in Xenopus oocytes, and Mg2+ block was quantified. Replacement of the entire M1-M4 region resulted in a chimera with a sensitivity of Mg2+ block similar to that of the NR2B wild type. Replacing smaller segments or introducing point mutations did not generate channels with Mg2+ block characteristic of NR2B wild type. However, combining in a single chimera three small segments (M1, M2-M3 linker, M4), each independently mediating an increase in Mg2+ block, produced channels close to NR2B wild type. Thus, differences in Mg2+ block as controlled by the NR2 subunits cannot be explained by a single structural determinant in addition to the N-site. Moreover, three elements of the NR2 subunit are the major determinants of subtype-specific differences of Mg2+ block in heteromeric NMDA receptor channels.

Fig. 5.

Schematic diagram of wild-type and chimeric subunits. In all subunits, the core region containing M1–M4 is 281 amino acids long and is shown enlarged relative to the N and C termini.Numbers without units denote number of amino acids. Thesmall numbers next to the schematic representation of the chimeras indicate the number of differing amino acids that are replaced by the given chimera. The middle panel shows log(IC_50,−100mV) values of Mg²⁺ block; δ is shown on the right panel. Bars represent mean ± SEMs. Indication of significance levels determined by the Tukey–Kramer test: cor b = p < 0.05, cc or bb = p < 0.01 (comparison to NR2C or NR2B, respectively).

Fig. 3.

Alignment of the core region of rat NR2 subunit amino acid sequences including the four hydrophobic domains (M1–M4). An asterisk indicates the location of a determinant of Mg²⁺ block in the M2 domain (N-site). Residues printed in boldface were exchanged in a given chimera, and underlined positions emphasize group-specific differences ([NR2A = NR2B] ≠ [NR2C = NR2D]) in amino acid sequence. Numbers given on the right side indicate the position of the rightmost amino acid in each row (72 AA) within the given subunit. Fusion positions of chimeras are denoted bycapital letters in the order of their appearance:M1 = A/B (only for this chimera, the sequence of NR2A was inserted, printed in bold italic letters); M12 = A/D; L1 = B/C; M13 = A/E; M2a = SV..A replaced by AI..G; L2 = D/E; M14 = A/I; M34 = E/I; M34a = F/I; M4 = G/I; M4a = G/H.

Fig. 1.

Differential Mg²⁺ block of four NR1-NR2 subtypes expressed in Xenopus oocytes. Representative I–V curves recorded in the presence of different Mg²⁺ concentrations in low Ca²⁺ Ringer’s. I–V curves were normalized to the current at −100 mV in nominally Mg²⁺-free solution. IC_50,−100mV values are shown in Table 1. The control I–V curves of NR1-NR2A and NR1-NR2B were slightly blocked by residual Mg²⁺ in the Ringer’s. However, the influence on calculation of the IC_50,−100mV was negligible, and the values given in Table 1 might be slight underestimates of the true IC_50,−100mV. Variations of the reversal potential in the range of −10 to 0 mV were observed between different oocytes. In some instances, the outward current passed by NR1-NR2B channels was potentiated in the presence of 1 mmMg²⁺.

Fig. 4.

Selected I–V curves of four chimeras representing different levels of Mg²⁺ block. See also legend to Figure 1. IC_50,−100mV values are shown in Table 1.

Fig. 2.

Voltage dependence of Mg²⁺block. A, Dose–response curves for Mg²⁺ at different potentials (−100, −80, −60, −40 mV) derived from the I–V curve of NR1-NR2C shown in Figure 1. Values of the fractional block are shown assquares. The fit used to determine the IC_50,−100mV (in this example, 14.7 μm Mg²⁺) is shown as athick line. B, Same as in A for NR1-NR2B; values of the fractional block derived from theI–V curve shown in Figure 1 are shown astriangles. The IC_50,−100mV is 2.4 μm Mg²⁺. C, Difference in voltage dependence of the two representative Mg²⁺ series shown in A andB and Figure 1 (printed in black). The data were fitted in the range from −80 to −30 mV, as indicated by the two lines. Examples for the NR1-NR2A and NR1-NR2D are printed ingray.