Involvement of astrocytic two-pore domain K+ channels in ischemic pathology

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DESCRIPTION (provided by applicant): Astrocytes are the most numerous cell types in the brain and are known to provide structural, metabolic and homeostatic support to the central nervous system (CNS). Although astrocytes can better survive than neurons in cerebral ischemia, the mechanisms accounting for such a different susceptibility among different brain cells are not clear. Predominant expression of a voltage-independent K+ channel conductance, or passive conductance, is a hallmark of mature astrocytes and essential for the homeostatic support of astrocytes to the CNS. Now we know that two members of the two-pore domain K+ channels (K2Ps) K+ channels, TWIK-1 and TREK-1, are among the long-sought for K+ channels accounting for astrocyte passive conductance. K2Ps can be dynamically modulated by a variety of physiochemical and pathological stimuli, including cerebral-ischemia-produced-neuronal-injury-factors (CIPNJFs), such as hypoxia, hypoglycemia, acidosis and pathological release of neurotransmitters. Pathological induction of K2P expression also contributes to the necrotic and apoptotic cell death and cell proliferation that are the two prominent pathological events occurring in the ischemic infarct and penumbra regions. To understand how the physiological expression of astrocyte K2Ps offers protection to astrocytes against early ischemic insults, and how the long-term ischemic conditions induce altered K2P expression in reactive astrocytes and its consequence on the post-stroke outcomes, we hypothesize that the activity of astrocytic K2Ps can be modulated by CIPNJFs in a manner protecting astrocytes against early ischemic insults, and the altered expression of K2P in reactive astrocytes contributes to the compromised homeostatic function in the peri- infarct penumbra region. Five specific aims are proposed to explore these completely unknown areas. 1) Modulation of astrocyte membrane potential and passive conductance by CIPNJFs. This will be done in rat hippocampal slices with gramicidin perforated patch recording to monitor K2Ps modulation without interfering with the CIPNJFs mediated intracellular energy failure and altered signal transduction; 2) Modulation of electrophysiological response of astrocytes to CIPNJFs by neuroprotectant and TREK-1 channel modulator riluzole and sipatrigine; 3) Identify specific K2P-CIPNJF interaction mechanisms by selective silencing of astrocytic K2Ps with siRNAs in organotypic hippocampal slice cultures; 4) Identify K2P expression in rat hippocampal reactive astrocytes in slices prepared from the penumbra region after reversible middle carotid artery occlusion (rMCAO) by confocal immunocytochemistry. 5) Identify functional K2P in reactive astrocytes in rat focal ischemia penumbra region using electrophysiology in acutely prepared hippocampal slices from the rat rMCAO penumbra region. The proposed studies should provide novel insights into the physiological roles and pathological involvement of astrocytic K2P in cerebral ischemia and whether these predominant astrocytic K+ channels could be potential targets for stroke therapeutic strategy.