Marwick KFM et al. (2019), The human NMDA receptor GluN2AN615K variant inf...

XB-ART-56077

Pharmacol Res Perspect 2019 Aug 01;74:e00495. doi: 10.1002/prp2.495.

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The human NMDA receptor GluN2AN615K variant influences channel blocker potency.

Marwick KFM , Skehel PA , Hardingham GE , Wyllie DJA .

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N-methyl-D-aspartate (NMDA) receptors are glutamate receptors with key roles in synaptic plasticity, due in part to their Mg2+ mediated voltage-dependence. A large number of genetic variants affecting NMDA receptor subunits have been found in people with a range of neurodevelopmental disorders, including GluN2AN615K (GRIN2AC1845A) in two unrelated individuals with severe epileptic encephalopathy. This missense variant substitutes a lysine in place of an asparagine known to be important for blockade by Mg2+ and other small molecule channel blockers. We therefore measured the impact of GluN2AN615K on a range of NMDA receptor channel blockers using two-electrode voltage clamp recordings made in Xenopus oocytes. We found that GluN2AN615K resulted in block by Mg2+ 1 mmol/L being greatly reduced (89% vs 8%), block by memantine 10 μmol/L (76% vs 27%) and amantadine 100 μmol/L (45% vs 17%) being substantially reduced, block by ketamine 10 μmol/L being modestly reduced (79% vs 73%) and block by dextromethorphan 10 μmol/L being enhanced (45% vs 55%). Coapplying Mg2+ with memantine or amantadine did not reduce the GluN2AN615K block seen with either small molecule. In addition, we measured single-channel conductance of GluN2AN615K-containing NMDA receptors in outside-out patches pulled from Xenopus oocytes, finding a 4-fold reduction in conductance (58 vs 15 pS). In conclusion, the GluN2AN615K variant is associated with substantial changes to important physiological and pharmacological properties of the NMDA receptor. Our findings are consistent with GluN2AN615K having a disease-causing role, and inform potential therapeutic strategies.

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GO keywords: regulation of synaptic plasticity

???displayArticle.omims??? MACROCEPHALY AND EPILEPTIC ENCEPHALOPATHY

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	Figure 1. GluN2AN615K reduces Mg2+, memantine and amantadine block. A and B, Representative two‐electrode voltage‐clamp recordings made from oocytes expressing GluN2AWT or GluN2AN615K–containing NMDA receptors showing response to glutamate (30 μmol/L) and inhibition by memantine (10 μmol/L), Mg2+ (1 mmol/L) and the two combined, in the continuous presence of glycine (30 μmol/L). Holding potential –60 mV. C, Summary data showing percentage inhibition by memantine (10 μmol/L), Mg2+ (1 mmol/L) and the two combined for oocytes transfected with either GluN2AWT or GluN2AN615K –containing NMDA receptors, voltage‐clamped at −60 mV. A two‐way repeated measures ANOVA (blocker as within subjects factor, subunit as between subjects factor) showed a significant main effect of channel blocker (F 2,39 = 26.1, P = 6.4E‐8) and of subunit (F 1,39 = 588.3, P < 2E‐16) with a significant two‐way interaction (F 2,39 = 7.3, P = 0.002). Planned post ‐hoc Bonferroni corrected independent Welch t‐tests showed that GluN2AN615K was associated with lower blockade by memantine (WT: 76 ± 1%, N615K: 27 ± 2%, t 10.1 = 17.1, P = 4.9E‐8), by Mg2+ (WT: 89 ± 1%, N615K: 8 ± 3%, t 7.4 = 23.2, P = 1.8E‐7) and by the two combined (WT: 94 ± 1%, N615K: 29 ± 4%, t 6.4 = 15.7, P = 1.2E‐5). Planned post‐hoc Bonferroni corrected paired t‐tests showed that combining memantine and Mg2+ led to a higher degree of block in oocytes expressing GluN2AWT subunits t 8 = 17.5, P = 5.8E‐7) but not in those expressing GluN2AN615K subunits (t 6 = 1.4, P > 0.2) (WT: n = 9 oocytes, N615K: n = 7 oocytes). D, Summary data showing percentage inhibition by amantadine (100 μmol/L), Mg2+ (1 mmol/L) and the two combined for oocytes transfected with either GluN2AWT or GluN2AN615K –containing NMDA receptors, voltage‐clamped at –60 mV. A two‐way repeated measures ANOVA (blocker as within subjects factor, subunit as between subjects factor) showed a significant main effect of channel blocker (F 2,39 = 146.5, P < 2E‐16) and of subunit (F 1,39 = 481.8, P < 2E‐16) with a significant two‐way interaction (F 2,39 = 20.0, P = 1E‐6). Planned post‐hoc Bonferroni corrected independent Welch t‐tests showed that GluN2AN615K was associated with lower blockade by amantadine (WT: 45 ± 4%, N615K: 17 ± 1%, t 10.3 = 7.3, P = 2.2E‐5), by Mg2+ (WT: 88 ± 1%, N615K: 6 ± 1%, t 13.7 = 39.9, P = 7.0E‐15) and by the two combined (WT: 91 ± 1%, N615K: 20 ± 4%, t 7.2 = 19.2, P = 1.4E‐15). Planned post‐hoc Bonferroni corrected paired t‐tests showed that combining memantine and Mg2+ led to a higher degree of block in oocytes expressing GluN2AWT subunits (t 8 = 14.5, P = 2.5E‐6) but not in those expressing GluN2AN615K subunits (t 6 = 1.2, P > 0.3) (WT: n = 9 oocytes, N615K: n = 7 oocytes)
	Figure 2. GluN2AN615K reduces ketamine block but increases dextromethorphan block. A and B, Representative two‐electrode voltage‐clamp recordings made from oocytes expressing GluN2AWT or GluN2AN615K–containing NMDA receptors showing response to glutamate (30 μmol/L) and inhibition by ketamine (10 μmol/L) in the continuous presence of glycine (30 μmol/L). Holding potential –60 mV. C, Summary data showing percentage inhibition by ketamine (10 μmol/L) for oocytes transfected with either GluN2AWT or GluN2AN615K –containing NMDA receptors, voltage‐clamped at –60 mV. An independent Welch t‐test showed that GluN2AN615K was associated with a reduction in block (WT: 79 ± 2 (n = 11 oocytes), N615K: 73 ± 1 (n = 6 oocytes), t 14.9 = 2.6, P = 0.019). D and E, Representative two‐electrode voltage‐clamp recordings made from oocytes transfected with GluN2AWT or GluN2AN615K–containing NMDA receptors showing response to glutamate (30 μmol/L) and inhibition by dextromethorphan (10 μmol/L) in the continuous presence of glycine (30 μmol/L). Holding potential –60 mV. F, Summary data showing percentage inhibition by dextromethorphan (10 μmol/L) for oocytes transfected with either GluN2AWT or GluN2AN615K –containing NMDA receptors, voltage‐clamped at –60 mV. An independent Welch t‐test showed that GluN2AN615K was associated with an increase in block (WT: 45 ± 2 (n = 7 oocytes), N615K: 55 ± 3 (n = 8 oocytes), t 12.6 = 2.8, P = 0.015)
	Figure 3. GluN2AN615K reduces single‐channel conductance. A and B, Representative voltage‐clamp recordings made from outside‐out patches from oocytes expressing GluN2AWT or GluN2AN615K –containing NMDA receptors in the presence of glutamate (30 μmol/L) and glycine (30 μmol/L). “C” = closed, “O” = open. Holding potential −60 mV. C, Representative amplitude histograms showing fitted normal distributions, superimposed from two different patches held at –60 mV. The means of the fitted distributions/holding potential were used to calculate conductance. D, Summary data showing conductance, calculated as current amplitude/holding potential. A t‐test showed a reduction in conductance in oocytes expressing GluN2AWT (58 ± 3 pS (n = 4), total events = 2555) and GluN2AN615K (15 ± 1 pS (n = 3), total events = 774, t 3.4 = 14.6, P = 0.0004)

References [+] :

Allen, Unexplained early onset epileptic encephalopathy: Exome screening and phenotype expansion. 2016, Pubmed