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. 2012 Feb 3;287(6):4348-59.
doi: 10.1074/jbc.M111.253740. Epub 2011 Dec 16.

N-methyl-D-aspartate receptor mechanosensitivity is governed by C terminus of NR2B subunit

Pallab Singh  1 , Shachee Doshi, Jennifer M Spaethling, Adam J Hockenberry, Tapan P Patel, Donna M Geddes-Klein, David R Lynch, David F Meaney
Affiliations

N-methyl-D-aspartate receptor mechanosensitivity is governed by C terminus of NR2B subunit

Pallab Singh et al. J Biol Chem. .

Abstract

N-methyl-D-aspartate receptors (NMDARs), critical mediators of both physiologic and pathologic neurological signaling, have previously been shown to be sensitive to mechanical stretch through the loss of its native Mg(2+) block. However, the regulation of this mechanosensitivity has yet to be further explored. Furthermore, as it has become apparent that NMDAR-mediated signaling is dependent on specific NMDAR subtypes, as governed by the identity of the NR2 subunit, a crucial unanswered question is the role of subunit composition in observed NMDAR mechanosensitivity. Here, we used a recombinant system to assess the mechanosensitivity of specific subtypes and demonstrate that the mechanosensitive property is uniquely governed by the NR2B subunit. NR1/NR2B NMDARs displayed significant stretch sensitivity, whereas NR1/NR2A NMDARs did not respond to stretch. Furthermore, NR2B mechanosensitivity was regulated by PKC activity, because PKC inhibition reduced stretch responses in transfected HEK 293 cells and primary cortical neurons. Finally, using NR2B point mutations, we identified a PKC phosphorylation site, Ser-1323 on NR2B, as a unique critical regulator of stretch sensitivity. These data suggest that the selective mechanosensitivity of NR2B can significantly impact neuronal response to traumatic brain injury and illustrate that the mechanical tone of the neuron can be dynamically regulated by PKC activity.

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Figures

FIGURE 1.
FIGURE 1.
NR1/NR2B NMDARs are more sensitive to stretch than NR1/NR2A. HEK 293 cells, plated on flexible membranes, were transfected with GFP-PSD95 alone, or along with NR1 and either NR2A or NR2B. A, representative images of transfected cells, pre- and poststimulation, that were stimulated by either 40% stretch (left) or 100 μm NMDA (right). B and C, average Fura ratio, representing intracellular calcium, following stretch (B) or NMDA stimulation (C), demonstrates that although NR1/NR2B- and NR1/NR2A-expressing cells have a similar response to NMDA, stretch-induced calcium influx is greater in NR1/NR2B-expressing cells. D, the average peak fractional change in Fura ratio, normalized to the NR1/NR2B response, following stretch demonstrates significant stretch sensitivity in NR1/NR2B-transfected cells (*, p < 0.05 compared with GFP-PSD95), whereas NR1/NR2A stretch responses are not different from control GFP-PSD95 responses. E, normalized response to NMDA stimulation was similar among NR1/NR2A and NR1/NR2B-transfected cells (*, p < 0.05 compared with GFP-PSD95).
FIGURE 2.
FIGURE 2.
Mechanoactivation of NMDARs in cortical neurons. NR1/NR2B NMDARs in primary cortical neurons were isolated by pretreating cultures with bicuculline methiodide (10 μm) in the presence of MK-801 (50 μm), a technique to block NR1/NR2B, NR1/NR2A, and NR1/NR2A/NR2B NMDARs at the synapse. A, calcium response following 100 μm NMDA stimulation (inset, Fura traces immediately following NMDA stimulation (arrow)) in untreated neurons (black) and neurons pretreated with synaptic block protocol (dark gray) or synaptic block and Ro-256981 (1 μm) (light gray). The response was significantly reduced in cultures with synaptic block and abolished in synaptically blocked cultures that were concurrently treated with Ro 25-6981 (*, p < 0.05 compared with untreated; #, p < 0.05 compared with synaptic block alone), demonstrating that nearly all extrasynaptic NMDARs were of the NR1/NR2B subtype. B, calcium response following stretch (inset, Fura traces immediately following stretch (arrow)) was measured in untreated neurons (black) and neurons pretreated with synaptic block protocol (dark gray) or APV (25 μm) (light gray). The response was reduced, but not eliminated, in cultures with synaptic block (*, p < 0.05 compared with untreated) and was abolished in APV treated cultures (*, p < 0.05 compared with untreated; #, p < 0.05 compared with synaptic block), demonstrating that extrasynaptic NR1/NR2B NMDARs significantly contribute to the stretch response in neurons. C, cultures pretreated with bafilomycin A1 (500 nm) to block vesicular refilling showed no significant difference in calcium influx when compared with cultures pretreated with APV. Both APV- and bafilomycin-treated cultures were significantly different from untreated controls (*, p < 0.05).
FIGURE 3.
FIGURE 3.
Intermediate mechanosensitivity in triheteromeric NR1/NR2A/NR2B receptors. The cells were transfected with GFP-PSD95, with either NR1 and NR2B, or with NR1, NR2A and NR2B. Cells transfected with all subunits were either left untreated or treated with NR1/NR2B-specific antagonist, Ro 25-6981. A, normalized response to stretch was significantly decreased, but not eliminated, in cells expressing NR1, NR2A, and NR2B, demonstrating that these cells exhibit intermediate mechanosensitivity (*, p < 0.05 compared with NR1/NR2B). B, response to 100 μm NMDA application was not different. Treatment with Ro 25-6981 did not alter the response of NR1/NR2A/NR2B-transfected cells, suggesting that expressed NMDARs in these cells are primarily triheteromeric.
FIGURE 4.
FIGURE 4.
NR1/NR2B stretch response is dependent on glutamate activity. Cells transfected with GFP-PSD95, NR1, and NR2B were left untreated or pretreated with NMDAR competitive antagonist, APV, or NMDAR pore blocker, MK801. A, normalized peak change in Fura ratio after stretch shows that treatment with either antagonist significantly reduced stretch response (* p < 0.05 compared with NR1/NR2B). The response in NR1/NR2B-transfected cells treated with MK801 was significantly greater than those treated with APV (#, p < 0.05 compared with NR1/NR2B + APV), suggesting that the receptor pore cannot be fully blocked by MK801 after stretch. B, normalized response following 100 μm NMDA stimulation was completely blocked by both types of NMDAR antagonists (*, p < 0.05). Both APV and MK801 completely eliminated the response to NMDA treatment (D) and significantly reduced the response to stretch (C) across the population of NRI/NR2B-transfected cells.
FIGURE 5.
FIGURE 5.
NR2B C-terminal tail confers mechanosensitivity. The cells were transfected with NR2B or with truncation mutants of NR2B, NR2B-1036X, or NR1433X, which truncate the C-terminal tail distal to amino acid 1036 and 1433, respectively. A and B, normalized stretch response was significantly decreased in cells expressing NR2B-1036X (A), whereas response to 100 μm was not different (B). C and D, response to stretch was slightly increased in cells expressing NR2B-1433X (C), whereas response to NMDA stimulation was not different (D) among NR2B and NR2B-1433X. E and F, differential mechanosensitivity was also seen in cultures where PSD-95 was absent, where NR2B-1036X again demonstrated a reduced stretch response (E) and similar NMDA response (F), compared with wild type. This suggests that NR2B mechanosensitivity is conferred by the intracellular domain within amino acids 1036–1433 (*, p < 0.05 compared with NR1/NR2B response).
FIGURE 6.
FIGURE 6.
PKC inhibition in recombinant NMDARs and primary neurons reduces receptor mechanosensitivity. Primary cortical cultures (day 15 in vitro) were treated left untreated or treated with tamoxifen or PMA. A, representative images of treated and untreated cortical cultures before and after stretch (left panels) or NMDA (right panels) stimulation. B, stretch response, normalized to response of untreated cultures, was significantly reduced in tamoxifen-treated cultures, whereas PMA had no effect (*, p < 0.05 compared with untreated). C, response to 100 μm NMDA stimulation was unchanged in tamoxifen-treated cells but increased in PMA-treated cells (*, p < 0.05 compared with untreated). HEK 293 cells transfected with GFP-PSD95, NR1, and NR2B were left untreated or treated with PKC inhibitor, tamoxifen, or PKC activator, PMA. D, tamoxifen treatment significantly decreased stretch response, whereas PMA had no effect (* p < 0.05 compared with NR1/NR2B). E, neither treatment produced significant change following NMDA stimulation in transfected HEKs.
FIGURE 7.
FIGURE 7.
NR2B mechanosensitivity is critically regulated by a single PKC phosphorylation site. The cells were transfected with NR2B or with NR2B point mutations, NR2B-S1303A or NR2B-S1323A, which contain serine to alanine point mutations at PKC phosphorylation sites Ser-1303 or Ser-1323, respectively. A and B, normalized stretch response (A) and NMDA response (B) was unchanged with the expression of NR2B-S1303A. C and D, response to stretch was significantly decreased in cells expressing NR2B-1433X (C), whereas response to NMDA stimulation was not different among NR2B and NR2B-S1323A (D). This suggests that NR2B mechanosensitivity is regulated by the PKC phosphorylation site, Ser-1323, on the NR2B C-terminal tail (*, p < 0.05 compared with NR1/NR2B response).
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