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Abstract
Homocysteine (HCY) is an endogenous redox active amino acid, best known as contributor to various neurodegenerative disorders. Although it is known that HCY can activate NMDA receptors (NMDARs), the mechanisms of its action on receptors composed of different NMDA receptor subunits remains almost unknown. In this study, using imaging and patch clamp technique in cultured cortical neurons and heterologous expression in HEK293T cells we tested the agonist activity of HCY on NMDARs composed of GluN1 and GluN2A subunits (GluN1/2A receptors) and GluN1 and GluN2B subunits (GluN1/2B receptors). We demonstrate that the time courses of Ca2+ transients and membrane currents activated by HCY and NMDA in cortical neurons are drastically different. Application of HCY to cortical neurons induced responses, which in contrast to currents induced by NMDA (both in the presence of glycine) considerably decreased to steady state of small amplitude. In contrast to NMDA, HCY-activated currents at steady state were resistant to the selective GluN2B subunit inhibitor ifenprodil. In calcium-free external solution the decrease of NMDA evoked currents was abolished, suggesting the Ca2+-dependent NMDAR desensitization. Under these conditions HCY evoked currents still declined almost to the baseline suggesting Ca2+-independent desensitization. In HEK293T cells HCY activated NMDARs of GluN1/2A and GluN1/2B subunit compositions with EC50s of 9.7 ± 1.8 and 61.8 ± 8.9 μM, respectively. Recombinant GluN1/2A receptors, however, did not desensitize by HCY, whereas GluN1/2B receptors were almost fully desensitized by HCY. Thus, HCY is a high affinity agonist of NMDARs preferring the GluN1/2A subunit composition. Our data suggest that HCY induced native NMDAR currents in neurons are mainly mediated by the “synaptic type” GluN1/2A NMDARs. This implies that in hyperhomocysteinemia, a disorder with enlarged level of HCY in plasma, HCY may persistently contribute to post-synaptic responses mediated by GluN2A-containing NMDA receptors. On the other hand, HCY toxicity may be limited by desensitization typical for HCY-induced activation of GluN2B-containing extrasynaptic receptors. Our findings, therefore, provide an evidence for the physiological relevance of endogenous HCY, which may represent an effective endogenous modulator of the central excitatory neurotransmission.
Highlights
L-Homocysteine [2-amino-4-sulfanylbutanoic acid (HCY)] is an endogenous sulfur-containing amino acid involved in synthesis of methionine and cysteine
As the [Ca2+]i increase could be determined by Ca2+ entry through the NMDA receptors (NMDARs), voltage gated calcium channels, and/or Ca2+ release from intracellular stores, we explored the action of HCY using patch clamp technique
The main finding of this study is that the endogenous amino acid, HCY, implicated in various brain diseases preferentially activates “synaptic type” GluN2A subunit-containing NMDA receptors with specific kinetics properties including limited receptor desensitization
Summary
L-Homocysteine [2-amino-4-sulfanylbutanoic acid (HCY)] is an endogenous sulfur-containing amino acid involved in synthesis of methionine and cysteine. The normal HCY level in plasma is generally below 16 μM (Shi et al, 2003). A deficit of folic acid and vitamins B or the C677T polymorphism of the 5 -10 -methylenetetrahydrofolate reductase gene (a substitution of cytosine for thymine at position 677, C677T) can cause an elevation of HCY level (a condition known as hyperhomocysteinemia, Kowa et al, 2000). High level of HCY have been proposed to contribute to a variety of cardiovascular and neurodegenerative disorders, such as Alzheimer’s and Parkinson’s disease (Kuhn et al, 2001; Kruman et al, 2002; Sachdev, 2005) as well as amyotrophic lateral sclerosis (Zoccolella et al, 2010). We previously showed that the high level of HCY led to the neuronal death through activation of NMDA receptors (NMDARs) and mGluR5 (Abushik et al, 2014)
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