Ion channel role-glutamate-independent neuronal injury

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DESCRIPTION (provided by applicant): Stroke and global brain ischemia are leading causes of morbidity and mortality. Ischemia causes progressive neuronal death, even after re-perfusion. This delayed neuronal death is largely due to excessive calcium entry into neurons. For many years, therapeutic strategies have focused on the Ca2+-permeable glutamate receptor gated channels as the main target. However, efforts to prevent cell death through the therapeutic use of glutamate receptor antagonists have been disappointing. Although intolerance of glutamate receptor antagonists and timing issues largely contribute to the failure of clinical trials, an emerging new concept also favors the idea that other sources of Ca2+ entry might be equally important in determining the ischemic neuronal death. We have now strong evidence that oxidative stress and oxygen free radicals, produced in ischemia, activate a novel Ca2*-permeable cation channel (the free radical activated channel: FRAC) in cortical neurons and our preliminary data demonstrated that this channel is likely responsible for a glutamate-independent delayed Ca2+ overload in ischemic neuronal death. Our objective is to fully characterize the ionic properties, pharmacology profile and the regulation of this channel. We will attempt to determine the specific form of free radicals responsible for the channel activation and the mechanism of how free radicals activate the FRAC. Simultaneous Ca2+imaging and patch-clamp recording will be used to quantify the Ca2+ entry through FRACs. We will also determine how free radicals alter the excitability of acute! dissociated neurons and the neurons in hippocampal slices. Finally, using in vitro and eventually in vivo ischemic models, we will determine if preventing the activation of FRACs protects neurons from ischemic death.General Hypothesis: Activation of a Ca2+-permeable cation channel by oxygen free radicals (the FRAC) is responsible, at least partially, for a glutamate-independent, delayed Ca2+ overload in ischemic neuronal death.Specific Aims:(1). Electrophysiological Characterization of FRACs.(2). Pharmacological Characterization and intracellular Regulation of FRA Cs.(3). Fluorescent imaging Study of Ca2+ Response Induced by the FRACActivation.(4). Potential Role of FRACActivation in Glutamate-independent Ischemic Neuronal InjuryOur long-term objective is to identify new targets, in addition to glutamate receptors, and novel strategies to protect brain cells from the damage that accompanies the stroke.