The Mechanism of NMDA Receptor Hyperexcitation in High Pressure Helium and Hyperbaric Oxygen

Abstract

Professional divers exposed to pressures greater than 1.1 MPa may suffer from the high pressure neurological syndrome (HPNS). Divers who use closed-circuit breathing apparatus face the risk of CNS hyperbaric oxygen toxicity (HBOTox). Both syndromes are characterized by reversible CNS hyperexcitability, accompanied by cognitive and motor deficits. Previous studies have demonstrated that the hyperexcitability of HPNS is induced mainly by NMDA receptors (NMDARs). In our recent studies, we demonstrated that the response of NMDARs containing GluN1 + GluN2A subunits was increased by up to 50% at high pressure (HP) He, whereas GluN1 + GluN2B NMDARs response was not affected under similar conditions. Our aim was to compare the responses of both types of NMDARs under HBOTox conditions to those of HP He and to reveal their possible underlying molecular mechanism(s). The two combinations of NMDARs were expressed in Xenopus laevis oocytes, placed in a pressure chamber, voltage-clamped, and their currents were tested at 0.1 (control) -0.54 MPa 100% O₂ or 0.1-5.1 MPa He pressures. We show, for the first time, that NMDARs containing the GluN2A subunit exhibit increased responses in 100% O₂ at a pressure of 0.54 MPa, similar to those observed in 5.1 MPa He. In contrast, the GluN1 + GluN2B response is not sensitive to either condition. We discovered that neither condition produced statistically significant changes in the voltage-dependent Mg²⁺ inhibition of the response. The averaged IC₅₀ remained the same, but a higher [Mg²⁺] _o was required to restore the current to its control value. The application of TPEN, a Zn²⁺ chelator, in control, HP He and HBOTox conditions, revealed that the increase in GluN1 + GluN2A current is associated with the removal of the high-affinity voltage-independent Zn²⁺ inhibition of the receptor. We propose that HPNS and HBOTox may share a common mechanism, namely removal of Zn²⁺ from its specific binding site on the N-terminal domain of the GluN2A subunit, which increases the pore input-conductance and produces larger currents and consequently a hyperexcitation.

Keywords: GluN2A; HBO; HPNS; NMDAR; O2 toxicity; Xenopus laevis oocytes; high pressure; zinc.

FIGURE 1

Current amplitudes of the GluN1-1a + GluN2A subtype in HBO at different pressures. (A) An example of HBO effects on GluN1-1a + GluN2A current response. All pressure steps were applied to the same oocyte. The applied agonists were glutamate (100 μM) and glycine (10 μM) with no [Mg²⁺]_o added. The 20 s agonists application time is indicated by horizontal bars. (B) Quantitative analysis of the NMDAR currents. Responses were normalized to the control value at 0.1 MPa (when the recording solution is saturated with 100% O₂) for each tested oocyte (indicated by numbers in the bars). Oocytes were exposed to 2–4 increased pressure steps. ^∗∗p < 0.01.

FIGURE 2

Current amplitudes of the GluN1-1a + GluN2A subtype at different [Mg²⁺]_o. Currents were normalized (%) to the [Mg²⁺]_o = 0 response at 0.1 MPa. (A) Compression with He (n = 8). (B) Compression with 100% O₂ (n = 11).

FIGURE 3

Example of the HP He effect on the Mg²⁺ dose-response curve. There were no significant changes in the dose-response curve and the IC₅₀ for the GluN1-1a + GluN2A subtype (measurements were performed on the same oocyte under control and HP He conditions). Current amplitudes of the receptor were normalized to its [Mg²⁺]_o = 0 response, and dose-response curve fit was performed for each experiment.

FIGURE 4

Quantitative analysis of GluN1-1a + GluN2A current changes in HP He and HBO with and without TPEN. Current amplitude is expressed as mean ± SEM in control (0.1 MPa), 0.54 MPa O₂ and 5.1 MPa He, with 0 μM or 1 μM TPEN. Splice variant GluN1-1a was co-expressed with the GluN2A subunit. Numbers in the bars indicate number of oocytes tested. **p < 0.005, ***p < 0.001.

FIGURE 5

Quantitative analysis of GluN1-1a + GluN2B currents in HBO and with TPEN. (A) Current amplitude expressed as mean ± SEM in control (0.1 MPa) and 0.54 MPa O₂ (n = 7). (B) Current amplitude expressed as mean ± SEM with 0 μM or 1 μM TPEN in control (0.1 MPa) (n = 5). No statistically significant difference was found for either experiment.