Calcium (Ca2+) influx through N-methyl-D-asparate receptors (NMDARs) is widely held to be the requisite step initiating delayed neuronal death following ischemic stroke. However, blocking NMDARs fails to prevent the accumulation of intracellular Ca2+ ([Ca2+]i) and subsequent neurotoxicity. This suggests that alternate, as yet uncharacterized Ca2+-influx pathways exist in neurons. Transient receptor melastatin 2 (TRPM2) is a Ca2+-permeable member of the transient receptor potential melastatin family of cation channels whose activation by reactive oxygen/nitrogen species (ROS/RNS) and ADP-ribose (ADPR) is linked to cell death. While these channels are broadly expressed in the central nervous system (CNS), the presence of TRPM2 in neurons remains controversial and more specifically, whether they are expressed in neurons of the hippocampus is an open question. Here, I employ a combination of molecular, biochemical and electrophysiological approaches to demonstrate that functional TRPM2 channels are expressed in pyramidal neurons of the hippocampus. Unlike in heterologous expression systems, the ADPR-dependent activation of TRPM2 in neurons required a concomitant rise in [Ca2+]i via either voltage-dependent Ca2+ channels or NMDARs. While short, repeated NMDA applications activated a TRPM2-like current in the absence of exogenous ADPR, sustained NMDA application to hippocampal neurons resulted in the activation of a pannexin1 (Px1) hemichannel. Px1 hemichannels are large conductance, nonjunctional gap junction channels that can be activated following periods of oxygen-glucose deprivation (OGD) in neurons. Activation of Px1 required the influx of Ca2+ through NMDARs. Supplementing the intracellular milieu with adenosine triphosphate (ATP) prevented Px1 activation, suggesting that hemichannels may be activated during periods of mitochondrial dysfunction and metabolic failure. Our findings have potential implications for the treatment of diseases such as cerebral ischemia and Alzheimer’s disease (AD) as they implicate two novel ion channels in the excitotoxic signaling cascade activated downstream of NMDARs.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/24324 |
| Date | 13 April 2010 |
| Creators | Olah, Michelle Elizabeth |
| Contributors | MacDonald, John F. |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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