Ca2+ toxicity remains the central focus of ischemic brain injury. The mechanism by which toxic Ca2+ loading of cells occurs in the ischemic brain has become less clear as multiple human trials of glutamate antagonists have failed to show effective neuroprotection in stroke. Acidosis is a common feature of ischemia and is assumed to play a critical role in brain injury; however, the mechanism(s) remain ill defined. Here, we show that acidosis activates Ca2+ -permeable acid-sensing ion channels (ASICs), inducing glutamate receptor-independent, Ca2+ -dependent, neuronal injury inhibited by ASIC blockers. Cells lacking endogenous ASICs are resistant to acid injury, while transfection of Ca2+ -permeable ASIC1a establishes sensitivity. In focal ischemia, intracerebroventricular injection of ASIC1a blockers or knockout of the ASIC1a gene protects the brain from ischemic injury and does so more potently than glutamate antagonism. Thus, acidosis injures the brain via membrane receptor-based mechanisms with resultant toxicity of [Ca2+]i, disclosing new potential therapeutic targets for stroke.

Exposure to low Ca 2؉ and/or Mg 2؉ is tolerated by cardiac myocytes, astrocytes, and neurons, but restoration to normal divalent cation levels paradoxically causes Ca 2؉ overload and cell death. This phenomenon has been called the ''Ca 2؉ paradox'' of ischemiareperfusion. The mechanism by which a decrease in extracellular Ca 2؉ and Mg 2؉ is ''detected'' and triggers subsequent cell death is unknown. Transient periods of brain ischemia are characterized by substantial decreases in extracellular Ca 2؉ and Mg 2؉ that mimic the initial condition of the Ca 2؉ paradox. In CA1 hippocampal neurons, lowering extracellular divalents stimulates a nonselective cation current. We show that this current resembles TRPM7 currents in several ways. Both (i) respond to transient decreases in extracellular divalents with inward currents and cell excitation, (ii) demonstrate outward rectification that depends on the presence of extracellular divalents, (iii) are inhibited by physiological concentrations of intracellular Mg 2؉ , (iv) are enhanced by intracellular phosphatidylinositol 4,5-bisphosphate (PIP 2), and (v) can be inhibited by G␣q-linked G protein-coupled receptors linked to phospholipase C ␤1-induced hydrolysis of PIP2. Furthermore, suppression of TRPM7 expression in hippocampal neurons strongly depressed the inward currents evoked by lowering extracellular divalents. Finally, we show that activation of TRPM7 channels by lowering divalents significantly contributes to cell death. Together, the results demonstrate that TRPM7 contributes to the mechanism by which hippocampal neurons ''detect'' reductions in extracellular divalents and provide a means by which TRPM7 contributes to neuronal death during transient brain ischemia.calcium paradox ͉ divalent cation sensing ͉ siRNA ͉ ischemia

No abstract

The effects of extracellular pH (pHo) on calcium‐sensing non‐selective cation (csNSC) channels in cultured mouse hippocampal neurons were investigated using whole‐cell voltage‐clamp and current‐clamp recordings. Decreasing extracellular Ca2+ concentrations ([Ca2+]o) activated slow and sustained inward currents through the csNSC channels. Decreasing pHo activated amiloride‐sensitive transient proton‐gated currents which decayed to baseline in several seconds. With proton‐gated channels inactivated by pre‐perfusion with low pH solution or blocked by amiloride, decreasing pHo to 6.5 inhibited the csNSC currents with a leftward shift of the Ca2+ dose–inhibition curve. Increasing pH to 8.5, on the other hand, caused a rightward shift of the Ca2+ dose–inhibition curve and potentiated the csNSC currents. Intracellular alkalinization following bath perfusion of quinine mimicked the potentiation of the csNSC currents by increasing pHo, while intracellular acidification by addition and subsequent withdrawal of NH4Cl mimicked the inhibition of the csNSC currents by decreasing pHo. Intracellular pH (pHi) imaging demonstrated that decreasing pHo induced a corresponding decrease in pHi. Including 30 mM Hepes in the pipette solution eliminated the effects of quinine and NH4Cl on the csNSC currents, but only partially reduced the effect of lowering pHo. In current‐clamp recordings, decreasing [Ca2+]o induced sustained membrane depolarization and excitation of hippocampal neurons. Decreasing pHo to 6.5 inhibited the low [Ca2+]o‐induced csNSC channel‐mediated membrane depolarization and the excitation of neurons. Our results indicate that acidosis may inhibit low [Ca2+]o‐induced neuronal excitation by inhibiting the activity of the csNSC channels. Both the extracellular and the intracellular sites are involved in the proton modulation of the csNSC channels.

Considering the national proportion of children, a high proportion of hospitalized patients with burn injury were children; those who were male, aged