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
The postsynaptic density (PSD) at excitatory dendritic synapses comprises a protein complex of glutamate receptors, scaffolding elements, and signaling enzymes. For example, NMDA receptors (NMDARs) are linked to several proteins in the PSD, such as PSD-95, and are also tethered via binding proteins such as alpha-actinin directly to filamentous actin of the cytoskeleton. Depolymerization of the cytoskeleton modulates the activity of NMDARs, and, in turn, strong activation of NMDARs can trigger depolymerization of actin. Myosin, the motor protein of muscular contraction and nonmuscle motility, is also associated with NMDARs and the PSD. We show here that constitutively active myosin light chain kinase (MLCK) enhances NMDAR-mediated whole-cell and synaptic currents in acutely isolated CA1 pyramidal and cultured hippocampal neurons, whereas inhibitors of MLCK depress these currents. This MLCK-dependent regulation was observed in cell-attached patches but was lost after excision to inside-out patches. Furthermore, the enhancement induced by constitutively active MLCK and the depression of MLCK inhibitors were eliminated after depolymerization of the cytoskeleton. NMDARs and MLCK did not colocalize in clusters on the dendrites of cultured hippocampal neurons, further indicating that the effects of MLCK are mediated indirectly via actomyosin. Our results suggest that MLCK enhances actomyosin contractility to either increase the membrane tension on NMDARs or to alter physical relationships between the actin cytoskeleton and the linker proteins of NMDARs.
The mechanisms by which insulin modulates neuronal plasticity and pain processes remain poorly understood. Here we report that insulin rapidly increases the function of glycine receptors in murine spinal neurons and recombinant human glycine receptors expressed in human embryonic kidney cells. Whole-cell patch-clamp recordings showed that insulin reversibly enhanced current evoked by exogenous glycine and increased the amplitude of spontaneous glycinergic miniature inhibitory postsynaptic currents recorded in cultured spinal neurons. Insulin (1 M) also shifted the glycine concentration-response plot to the left and reduced the glycine EC 50 value from 52 to 31 M. Currents evoked by a submaximal concentration of glycine were increased to approximately 140% of control. The glycine receptor ␣ subunit was sufficient for the enhancement by insulin because currents from recombinant homomeric ␣ 1 receptors and heteromeric ␣ 1  receptors were both increased. Insulin acted at the insulin receptor via pathways dependent on tyrosine kinase and phosphatidylinositol 3 kinase because the insulin effect was eliminated by the insulin receptor antagonist, hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol 3 kinase antagonist wortmannin. Together, these results show that insulin has a novel regulatory action on the potency of glycine for ionotropic glycine receptors.
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