4.1 Functional and pharmacological properties of disease-associated de novo mutations
4.1.3 Disease-related mutations affect receptor desensitization and open probability 52
concentrations of glutamate and glycine (1 mM and 30 μM, respectively) to reach maximum desensitization. The degree of desensitization was determined by the analysis of the ratio between the peak and steady-state current responses (see Equation 5). Whereas the WT receptors were desensitized by 16% (Figure 4.4A,D), hGluN1/hGluN2B(V618G) receptors were desensitized by only 4% (Figure 4.4D). In contrast, the degree of desensitization of the GluN1/hGluN2B(V558I) receptors was increased to 73% (Figure 4.4B,D).
Figure 4.4. Disease-associated mutations affect the desensitization and the Po of the receptor.
(A-C) Representative current responses to 1 mM glutamate and their inhibition by 1 μM MK-
53 801 in the WT (A), hGluN1/hGluN2B(V558I) (B), and hGluN1/hGluN2B(L825V) (C) receptors. (Aa-Ca) Demonstrate the glutamate-induced responses on an extended time scale.
(Ab-Cb) Demonstrate the onset of MK-801 block on an extended time scale at the WT (grey traces) and mutated (black traces) receptor responses. Duration of glutamate and MK-801 application is indicated by filled and open bars, respectively. (D-E) Bar graphs represent the desensitization (D) and open probability (E) of WT and mutated receptors. Statistical differences between the groups were tested by Kruskal-Wallis one-way ANOVA on Ranks.
Mann-Whitney Rank Sum Test was used to check the statistical difference between the WT and mutated receptors. Error bars indicate SEM; n = 4-19; * p < 0.05.
To calculate the kinetic constants of desensitization (𝑘𝑑) and resensitization (𝑘𝑟) in WT and mutated receptors, we determined the rate of the desensitization onset (see Equations 6-7). The desensitization of the WT receptor responses was characterized by the 𝑘𝑑 = 0.24 s-1 (Table 4.3). In contrast, the values of the 𝑘𝑑 in hGluN1/hGluN2B(V558I) and hGluN2B(W607C) receptors were 14.8- and 6.6- fold higher than in the WT receptors (Table 4.3). The values of the 𝑘𝑑 in hGluN1/hGluN2B(L825V) receptors were similar to those in the WT receptors (Table 4.3). The responses of the WT receptors were characterized by the 𝑘𝑟 = 1.06 (Table 4.3). The value of the 𝑘𝑟 determined in hGluN1/hGluN2B(W607C) receptors was 4.5- fold higher in comparison to those determined in the WT receptors. No statistically significant difference was found between the 𝑘𝑑 values determined in WT receptors and mutated hGluN1/hGluN2B(V558I) and hGluN1/hGluN2B(L825V) receptors (Table 4.3). Analysis of the kinetic constants for the hGluN1/hGluN2B(V618G) receptors was precluded due to the low amplitude of the current responses.
Table 4.3. Summary of the kinetic constants of desensitization (𝑘𝑑) and resensitization (𝑘𝑟) determined in WT and mutated receptors
Mutation 𝑘𝑑± SEM (s-1) 𝑘𝑟 ± SEM (s-1) n
hGluN2B(WT) 0.24 ± 0.08 1.06 ± 0.17 10
hGluN2B(V558I) 3.55 ± 0.46* 1.29 ± 0.28 7
hGluN2B(W607C) 1.59 ± 0.54* 4.79 ± 1.22* 4
hGluN2B(L825V) 0.43 ± 0.16 1.47 ± 0.20 6
Statistical differences between the groups were tested by Kruskal-Wallis one-way ANOVA on Ranks. Mann-Whitney Rank Sum Test was used to check the statistical difference between WT and mutated receptors; n indicates the number of cells in the group; * p < 0.05.
The Po of the WT and mutated receptors was evaluated by analysing the kinetics of the MK-801 blockade. The receptors were activated by 1 mM glutamate and then the glutamate- induced currents were inhibited by the application of 1 μM MK-801. The rate of MK-801
54 blockade was fitted to the kinetic model (see Equation 8). The results indicated that the Po of the hGluN1/hGluN2B(V558I), hGluN1/hGluN2B(W607C), hGluN1/hGluN2B(V618G), and hGluN1/hGluN2B(L825V) receptors was substantially lower than that in WT receptors (Figure 4D).
4.1.4 hGluN1/hGluN2B (L825V) receptors exhibit increased sensitivity to potentiating neurosteroids
Next, we evaluated the effect of hGluN2B(V558I; W607C; V618G; L825V) mutations on the receptor sensitivity to naturally-occurring potentiating neurosteroid PE-S and its synthetic analogue AND-hSuc. Due to the disuse-dependent effect of potentiating steroids (Horak et al., 2004), a low concentration of glutamate (1 μM) was used in these experiments.
At hGluN1/hGluN2B(L825V) receptors, both PE-S (100 μM) and AND-hSuc (30 μM) induced significantly greater potentiation of glutamate-induced responses than that at WT receptors (p ˂ 0.05, unpaired t-test) (Figure 4.5A-C). In contrast, the responses of hGluN1/hGluN2B(V558I; W607C; V618G) receptors were potentiated to the same extent as the responses of WT receptors (Figure 4.5A-C). Next, performed the dose-response analyses of PE-S (10-100 μM) and AND-hSuc (1-30 μM) potentiation at the WT and hGluN1/hGluN2B(L825V) receptors (Figure 4.5D-E). The dose-response curves for PE-S and AND-hSuc effects indicate that the augmented steroid potentiation in hGluN1/hGluN2B(L825V) receptors is associated with elevated steroid efficacy rather than with increased affinity.
55 Figure 4.5. hGluN1/hGluN2B(L825V) receptors exhibit increased sensitivity to PE-S and AND-hSuc. (A-B) Representative current responses of WT and hGluN1/hGluN2B(L825V) receptors to 1 mM glutamate alone and in the presence of 100 μM PE-S (A) or 30 μM AND- hSuc (B). Duration of glutamate and steroid application is indicated by filled and open bars, respectively. (C) Graph shows the degree of PE-S (100 μM) and AND-hSuc (30 μM) potentiation of glutamate (1 μM) responses in WT and mutated hGluN1/hGluN2B receptors.
The black line shows a linear regression for data points from all mutated receptors (correlation coefficient r2 = 0.928; p = 0.001). (D-E) Dose-response curves for the potentiation of WT (filled) and hGluN1/hGluN2B(L825V) receptor responses by 10-100 μM PE-S (D) and 1-30 μM AND-hSuc (E). In WT receptors, PE-S induced potentiation was characterized by the following parameters: Emax =101 ± 9 %; EC50 = 24.3 ± 1.8 μM;
h = 1.54 ± 0.08 (n = 7); AND-hSuc induced potentiation was characterized by the following parameters: Emax = 570 ± 57 %; EC50 = 9.5 ± 1.0 μM; h = 1.50 ± 0.16 (n = 6). In hGluN1/hGluN2B(L825V) receptors, the PE-S potentiation was characterized by the following parameters: Emax = 185 ± 50 % (p = 0.030 versus WT); EC50 = 24.5 ± 1.2 μM (p = 0.842 versus WT); h = 1.52 ± 0.12 (n = 5); the AND-hSuc potentiation was characterized by the following parameters: Emax = 1492 ± 514% (p = 0.030 versus WT); EC50
= 9.6 ± 2.03 μM (p = 0.961 versus WT); h = 1.58 ± 0.36 (n = 6).
56