Most vertebrates' brains die within minutes when deprived of molecular oxygen (anoxia), mainly due to excitotoxic cell death (ECD). ECD begins with a massive and uncontrolled influx of Ca2+ into neurons by over-stimulation of N-methyl-d-aspartate receptors(NMDARs), resulting in the activation of Ca2+-dependent phospholipases and proteases that cause membrane depolarisation, uncontrolled cellular swelling and, ultimately, cell death. Some ectothermic vertebrates,such as the western painted turtle (Chrysemys picta bellii), are remarkably anoxia tolerant and can survive days to months without oxygen and recover without any apparent brain damage. One mechanism that is believed to enable these animals to accomplish such a feat is their ability to prevent ECD by reducing NMDAR activity during anoxia. However, the mechanisms underlying the attenuation of turtle brain NMDAR activity have not been fully elucidated.
Thomas Buck's team at the University of Toronto was determined to tease out how NMDAR activity is reduced in the turtle brain during anoxia. In order to do so, Buck's team obtained cortical slices from painted turtles' brains and used whole-cell patch clamp techniques to measure NMDAR currents from individual cortical neurons during normoxia and after 0, 20 and 40 min of anoxic exposure. As they expected, the team confirmed that turtle neuronal NMDAR activity is reduced during anoxia; during the 40-min anoxic period they observed a 56% decrease in whole-cell NMDAR currents from normoxic levels.
To discover which intracellular modulators are responsible for this reduction of neuronal NMDAR activity, the team pharmacologically blocked possible modulators of NMDAR activity in the turtles' brains and again measured NMDAR currents from neurons in normoxic and anoxic experiments. Suspecting that protein phosphatases play a role, the team incubated the turtles' cortical slices in inhibitors of serine/threonine protein phosphatases PP1 and 2A and found they could abolish the anoxia-induced reduction in NMDAR currents. To test the role of intracellular Ca2+, they added the calcium chelator BAPTA to the recording electrode solution (which is continuous with the cytoplasm in whole-cell patch clamp experiments) and found that this also abolished the reduction in NMDAR currents. This indication that intracellular Ca2+ modulates NMDAR activity led the team to wonder about the role of calmodulin, an intermediary protein that senses calcium levels and relays signals to various calcium-sensitive enzymes, ion channels and other proteins. Sure enough, when the team pharmacologically blocked calmodulin during anoxia, they did not see a reduction in NMDAR current, indicating that calmodulin also controls NMDAR activity. The team concluded that protein phosphatases PP1 and 2A,intracellular Ca2+ and calmodulin all work in concert to decrease NMDAR activity during anoxia.
Synthesizing their findings with previously published data, the team proposes a novel mechanism of how NMDAR activity is attenuated during anoxia. The team suggests that during anoxia, protein phosphatases PP1 and 2A dephosphorylate the NR1 subunit of the NMDAR receptor. Dephosphorylation of the NMDAR receptor subsequently enables calmodulin, which must first be activated by Ca2+, to bind to the receptor and disrupt NMDAR binding to α-actinin-2, a molecule that normally connects the NMDAR receptor to the cytoskeleton. Disruption of this connection results in the dissociation of the NMDAR from the cytoskeleton, ultimately leading to a decrease in receptor activity and prevention of ECD.