Non-Ionotropic Activation of the NMDAR, Leading to ERK 1/2 Phosphorylation
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Authors
Slocumb, Melissa
Issue Date
2013
Type
Thesis
Language
en_US
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Abstract
N-methyl-D-aspartate receptors (NMDARs) are important to neuron function. NMDARs are transmembrane ligand-gated and voltage-gated ion channels that pass sodium, potassium, and calcium (MacDermott et al., 1986). They are composed of a tetramer of proteins in the postsynaptic cell membrane of neurons. The NMDAR is unique in the sense that it requires two agonists to stimulate its activation: the excitatory transmitter glutamate, and the co-agonist glycine. It is also unique in voltage-dependent regulation via a magnesium block in the ion channel. When the neuron is depolarized, this block is removed and ions can pass freely through the channel (Nowak et al., 1984). These three properties of passing calcium (MacDermott et al., 1986), being ligand-gated by glutamate, and being voltage-gated with a magnesium plug (Nowak et al., 1984), make the NMDAR important for regulating activity-dependent postsynaptic plasticity, a mechanism believed to underlie learning and memory (Nicoll, 2003). Ionotropic activation of NMDARs by ligands has been implicated in extracellular signal-regulated kinase (ERK) signaling (Martel et al., 2009). ERK is a protein that promotes synaptic plasticity by regulating the membrane trafficking of ?-amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA) receptors, which is closely linked to learning and memory formation (Sweatt, 2004). It is unknown whether NMDARs have a non-ionotropic capacity. My hypothesis is that the single NMDAR agonist glycine is capable of regulating ERK activity in the absence of ion channel activity. NMDAR activation and coupling to intracellular signaling cascades were probed using a pharmacological and molecular biology approach. N-methyl-d-aspartate (NMDA) was used to stimulate the receptor at the glutamate binding site, while the ionotropic pore was pharmacologically and physically blocked. Cultured mouse neurons and transfected Human Embryonic Kidney (HEK) 293 cell cultures were used to determine a subunit-specific role of the NMDAR. ERK1 and ERK2 phosphorylated protein and total ERK protein were measured using Western blot standard procedures (Sambrook and Maniatis, 1989). This research is significant because learning how to regulate NMDAR signaling cascades independent of ionotropic activity with a single ligand could lead to the development of treatments that could promote neuron survival and plasticity in patients with ischemia or neuronal insult. The results presented here must be considered inconclusive since there are several issues with the experimental protocols, only discovered late in the production of this work. While the results cannot be reliably used for any definitive conclusions, they are useful in troubleshooting and in refining these procedures. The understanding that these experiments have brought can be used to create new experiments that will produce results that can be reliably assessed, and these new results may be used to address the hypothesis.
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