Figure 6. Reduced inhibitory effect for extracellular Cd²⁺ binding to the activated NMDA channel with neutralizing mutations in the DRPEER motif.

(a) to (d) NMDA receptor currents are elicited by the same protocols as those in Fig. 1c. The effect of 30 μM Cd²⁺ is less pronounced in the D658A and in the E661A mutant channels, and even so in the E661AE662A double and D658AE661AE662A triple mutant channels. (e) The relative current is defined by the ratio between the sustained currents in 30 μM Cd²⁺ and in control (n = 3–7). Note the tendency of reduced Cd²⁺ effect with decreased number of negative charges in the motif. P = 0.84, 0.0019, 0.0037, 9.1 * 10⁻⁶, and 0.00031 for D658A, E661A, E662A, E661AE662A double, and D658AE661AE662A triple mutant channels compared with the wild-type (WT) channel, respectively. (Inset) The apparent dissociation constants between Cd²⁺ and the activated wild-type (WT), E661A, E662A, and E661AE662A mutant channels are simplistically derived with the Hill equation (assuming a Hill coefficient of 1, see Fig. 2) and the relative sustained currents in 30 μM Cd²⁺, and are 3.2, 7.7, 5.6, and 13.9 μM, respectively. Double mutant cycle analysis shows a coupling coefficient ((Kd_WT × Kd_E661AE662A)/(Kd_E661A × Kd_E662A)) of 1.03 for the two point mutations E661A and E662A in terms of Cd²⁺ binding to the activated NMDA channel. (f) In the E661AE662A double mutant channels, NMDA currents are elicited by the same protocols as those in Fig. 1a (see inset currents). The relative current is defined by the ratio between the sustained currents in 2 mM extracellular Ca²⁺ and in control. The effect of Ca²⁺ is significantly decreased by the E661AE662A double mutation (n = 6; WT: n = 9). ***P = 5.1*10⁻⁵.