Membrane Stretch Gates NMDA Receptors

Abstract

NMDARs are ionotropic glutamate receptors widely expressed in the CNS, where they mediate phenomena as diverse as neurotransmission, information processing, synaptogenesis, and cellular toxicity. They function as glutamate-gated Ca²⁺-permeable channels, which require glycine as coagonist, and can be modulated by many diffusible ligands and cellular cues, including mechanical stimuli. Previously, we found that, in cultured astrocytes, shear stress initiates NMDAR-mediated Ca²⁺ entry in the absence of added agonists, suggesting that more than being mechanosensitive, NMDARs may be mechanically activated. Here, we used controlled expression of rat recombinant receptors and noninvasive on-cell single-channel current recordings to show that mild membrane stretch can substitute for the neurotransmitter glutamate in gating NMDAR currents. Notably, stretch-activated currents maintained the hallmark features of the glutamate-gated currents, including glycine-requirement, large unitary conductance, high Ca²⁺ permeability, and voltage-dependent Mg²⁺ blockade. Further, we found that the stretch-gated current required the receptor's intracellular domain. Our results are consistent with the hypothesis that mechanical forces can gate endogenous NMDAR currents even in the absence of synaptic glutamate release, which has important implications for understanding mechanotransduction and the physiological and pathologic effects of mechanical forces on cells of the CNS.SIGNIFICANCE STATEMENT We show that, in addition to enhancing currents elicited with low agonist concentrations, membrane stretch can gate NMDARs in the absence of the neurotransmitter glutamate. Stretch-gated currents have the principal hallmarks of the glutamate-gated currents, including requirement for glycine, large Na⁺ conductance, high Ca²⁺ permeability, and voltage-dependent Mg²⁺ block. Therefore, results suggest that mechanical forces can initiate cellular processes presently attributed to glutamatergic neurotransmission, such as synaptic plasticity and cytotoxicity. Given the ubiquitous presence of mechanical forces in the CNS, this discovery identifies NMDARs as possibly important mechanotransducers during development and across the lifespan, and during pathologic processes, such as those associated with traumatic brain injuries, shaken infant syndrome, and chronic traumatic encephalopathy.

Keywords: NMDARs; ionotropic glutamate receptors; mechanotransduction; patch-clamp; signal transduction; single-molecule.

Figure 1.

Mild suction gates NMDARs in the presence of glycine. A, Current traces recorded from cell-attached patches expressing GluN1/GluN2A receptors with 100 mV applied through the recording pipette. Downward traces represent inward Na⁺ currents at the indicated pressure levels, in the presence (+) or absence (–) of glutamate (Glu, 1 mm) and/or glycine (Gly, 0.1 mm). B, Summary of response dependency on pressure level for each GluN2 subtype in the presence of glycine (0.1 mm) with no glutamate added. *p < 0.05; **p < 0.01; one-way ANOVA, with Bonferroni correction.

Figure 2.

Negative hydrostatic pressure agonizes NMDARs. A, Effect of suction on GluN1/GluN2A receptors, with the indicated agonists (Glu 1 mm, Gly 0.1 mm) and 100 mV in the cell-attached recording pipette. *p < 0.05; **p < 0.01; one-way ANOVA with Bonferroni correction. B, Summary of the effects of suction on receptor activity. *p < 0.05 (unpaired Student's t test).

Figure 3.

Biophysical properties of stretch-gated currents from recombinant NMDARs. A, On-cell patch-clamp current traces recorded from cells expressing GluN1/GluN2A receptors in response to saturating concentrations of Glu (1 mm) (left) and gentle stretch (−40 mmHg). B, Voltage dependency of unitary current amplitude, and summary of Ca²⁺-dependent reduction in unitary conductance. *p < 0.05; **p < 0.01; two-way ANOVA, with Bonferroni correction. C, Current traces recorded with external Mg²⁺ (10 µm) and summary of voltage-dependent reduction in mean open durations.

Figure 4.

Mechanical activation of NMDARs by suction requires their intracellular CTD. Cell-attached Na⁺-current traces recorded from GluN1/GluN2A receptors lacking the intracellular CTD (ΔCTD) (left) and summary of results compared with WT receptors. *p < 0.05 (unpaired Student's t test).

Figure 5.

Stretch gates native NMDARs. A, Suction potentiates currents elicited from recombinant GluN1/GluN2A receptors with low concentration of NMDA (0.1 mm). B, In neurons, suction potentiates NMDA-elicited currents and gates currents of similar unitary amplitude. **p < 0.01; ***p < 0.001; ****p < 0.0001; one-way ANOVA test with Bonferroni correction.