Calcium is one of the most important ions in the central nervous system, essential for the regulation of neuronal excitability, synaptic transmission, neuronal development and differentiation. Alterations of Ca2+ homeostasis have been shown to be involved in the pathology of various neurological diseases/disorders. Accumulation of intracellular Ca2+ ([Ca2+]i), for example, has been recognized as a central pathological feature in brain ischemia. Along with an increase in [Ca2+]i, ischemia also causes a dramatic decrease in the concentration of extracellular Ca2+ ([Ca2+]e). Although it is well-known that the increase of [Ca2+]j is critical for excitatory neuronal injury, it is not clear whether the alteration of [Ca2+]e plays any role in the pathology of brain ischemia. We have previously demonstrated that lowering [Ca2+]e to the level commonly seen in brain ischemia strongly depolarized and excited cultured hippocampal neurons through activation of a non-selective cation channel. This channel has electrophysiological properties and pharmacological profiles different from known channels, suggesting activation of a novel Ca2+-sensitive ion channel. Our preliminary study also demonstrated that activation of this channel caused an increase in [Ca2+]i and potentiated NMDA-receptor mediated membrane responses as well as neuronal injury. We therefore hypothesize that decreases of [Ca2+]e to the level seen in brain ischemia activates a distinct Ca2+-sensing non-selective cation (csNSC) channel. Activation of these channels induces membrane depolarization and neuronal excitation, which contributes to excitotoxicity either directly, or indirectly through potentiation of NMDA receptor mediated responses. Our objective is to provide additional evidence that csNSC is indeed a distinct new channel and to investigate its pathological role in hypoxic/ischemic neuronal injury. In addition to cultured neurons, we will characterize detailed electrophysiological properties of the csNSC channel in acutely dissociated mature neurons and the neurons in brain slices, develop a pharmacological profile and search for a specific channel blocker. We will define the Ca2+-permeability of the csNSC channel, and determine whether ischemic treatment enhances the Ca2+-permeability. Since NMDA channels play a critical role in excitatory neuronal injury, we will characterize detailed interaction of csNSC channels with NMDA receptor-mediated membrane responses and neuronal injury. Using in vitro ischemic models, we will determine whether preventing the activation of csNSC channels protects neurons from ischemic injury.
Specific Aims are: 1. Provide further evidence that lowering [Ca2+]e to the level seen in brain ischemia activates a distinct nonselective cation channel in the central nervous system. 2. Demonstrate that csNSC channels are Ca2+-permeable. 3. Demonstrate potentiation of NMDA channel function by csNSC activation. 4; Determine the potential role of csNSC channels in ischemic neuronal injury.
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