No abstract

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.

Protein kinase C (PKC) phosphorylates the NR1 and NR2A subunits of NMDARs at consensus sites located within their intracellular C-terminal tails. However, the functional consequences of these biochemical events are not well understood. In HEK293 cells expressing NR1/NR2A, activation of endogenous PKC by 4b-phorbol 12-myristate 13-acetate (PMA) increased NMDAR desensitization as evidenced by a reduced steady-state current without any change in peak. The effects of PMA on NMDAR-mediated responses were prevented by specific PKC inhibitors and were not mimicked by an inactive enantiomer of PMA. The effects of PMA were preserved despite mutagenesis of the major PKC sites on the NR1 subunit (S889A, S890A, S896A and S897A) or removal of the entire NR1 C-terminal tail (NR1 stop838 ). When co-expressing NR1 stop838 /NR2A the effects of PMA could only be observed with agonist concentrations sufficient to induce glycineinsensitive desensitization. Moreover, the effects of PMA were observed in receptors composed of NR1/NR2A and NR1/ NR2B, but not NR1/NR2C, a subunit combination in which desensitization is absent. The NR2 subunit dependence suggested that the actions of PMA might require specific PKC sites previously identified within NR2A. However, a C-terminal truncated form of NR2A (NR2A stop905 ) remained responsive to PMA. We conclude that activation of PKC increases NMDAR glycine-insensitive desensitization independently of previously identified sites located within the NR1 C-terminus and distal segment of the NR2A C-terminus. Keywords: desensitization, HEK293, NMDA, protein kinase C, recombinant receptors, whole-cell recordings. The modulation of synaptic responses through activitydependent postsynaptic processes is thought to form the basis for learning and memory. In this respect, the characteristic biophysical properties of the NMDA receptor (NMDAR), namely high permeability to Ca 2+ and voltage-dependent block by Mg 2+ (Nowak et al. 1984), allow it to function as a gate for the induction of synaptic plasticity at most excitatory synapses. Conversely, these properties can also underlie the pathophysiological consequences (e.g. seizures and excitotoxicity) resulting from the over-stimulation of NMDARs. Consequently, a number of activity-dependent processes operate on various time scales to limit NMDAR function.Among ligand-gated receptors, NMDARs are unique in that they require the binding of two agonists in order to be activated; glutamate binds to the NR2 subunit (Laube et al. 1997), whereas the essential co-agonist, glycine, binds to the smaller NR1 subunit (Kuryatov et al. 1994). These agonists produce three forms of desensitization that act concurrently to limit the time course of NMDA receptor-mediated responses. Glycineindependent desensitization is determined by the N-terminal region of the NR2 subunit (Krupp et al. 1998;Villarroel et al. 1998), while glycine-dependent desensitization results from negative allosterism between glutamate and glycine binding sites (Benveniste et al. 1990). A third form ...