NMDA receptors have received much attention over the last few decades, due to their role in many types of neural plasticity on the one hand, and their involvement in excitotoxicity on the other hand. There is great interest in developing clinically relevant NMDA receptor antagonists that would block excitotoxic NMDA receptor activation, without interfering with NMDA receptor function needed for normal synaptic transmission and plasticity. This review summarizes current understanding of the structure of NMDA receptors and the mechanisms of NMDA receptor activation and modulation, with special attention given to data describing the properties of various types of NMDA receptor inhibition. Our recent analyses point to certain neurosteroids as NMDA receptor inhibitors with desirable properties. Specifically, these compounds show use-dependent but voltage-independent block, that is predicted to preferentially target excessive tonic NMDA receptor activation. Importantly, neurosteroids are also characterized by use-independent unblock, compatible with minimal disruption of normal synaptic transmission. Thus, neurosteroids are a promising class of NMDA receptor modulators that may lead to the development of neuroprotective drugs with optimal therapeutic profiles.

Key pointsr NMDA receptors (NMDARs) are tetrameric cation channels permeable to calcium; they mediate excitatory synaptic transmission in the CNS and their excessive activation can lead to neurodegeneration.r Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood.r Using cultured rat cerebellar granule cells, we show that acute and chronic pretreatments resulting in cell cholesterol depletion profoundly diminish NMDAR responses and increase NMDAR desensitization, and also that cholesterol enrichment potentiates NMDAR responses; however, cholesterol manipulation has no effect on the amplitude of AMPA/kainate receptor responses.r Diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the ion channel open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state.r These results demonstrate the physiological role of membrane lipids in the modulation of NMDAR activity.Abstract NMDA receptors (NMDARs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the CNS. Although these receptors are in direct contact with plasma membrane, lipid-NMDAR interactions are little understood. In the present study, we aimed at characterizing the effect of cholesterol on the ionotropic glutamate receptors. Whole-cell current responses induced by fast application of NMDA in cultured rat cerebellar granule cells (CGCs) were almost abolished (reduced to 3%) and the relative degree of receptor desensitization was increased (by seven-fold) after acute cholesterol depletion by methyl-β-cyclodextrin. Both of these effects were fully reversible by cholesterol repletion. By contrast, the responses mediated by AMPA/kainate receptors were not affected by cholesterol depletion. Similar results were obtained in CGCs after chronic inhibition of cholesterol biosynthesis by simvastatin and acute enzymatic cholesterol degradation to 4-cholesten-3-one by cholesterol oxidase. Fluorescence anisotropy measurements showed that membrane fluidity increased after methyl-β-cyclodextrin pretreatment. However, no change in fluidity was observed after cholesterol enzymatic degradation, suggesting that the effect of cholesterol on NMDARs is not mediated by changes in membrane fluidity. Our data show that diminution of NMDAR responses by cholesterol depletion is the result of a reduction of the open probability, whereas the increase in receptor desensitization is the result of an increase in the rate constant of entry into the desensitized state. Surface NMDAR population, agonist affinity, single-channel conductance and open time were not altered in cholesterol-depleted CGCs. The results of our experiments show that cholesterol is a strong endogenous modulator of NMDARs. N,N,3,phenylammonium p-toluenesulphonate.

SUMMARY1. The whole-cell and outside-out configurations of the patch-clamp technique were used to record responses to excitatory amino acids in mouse hippocampal neurones in cell culture at different pH. The amino acids kainate, quisqualate, Nmethyl-D-aspartate (NMDA) and L-glutamate were applied by a rapid perfusion system.2. In the whole-cell recording mode the responses to NMDA or to low concentrations of glutamate, recorded in the absence of Mg2+ and with glycine in the extracellular superfusion solution, were antagonized by acidic pH and potentiated by an alkaline extracellular solution. Decrease in pH from 7-3 to 6-0 reduced NMDA responses to 33 + 2 % and an increase in pH from 7-3 to 8-0 potentiated it to 141 + 6 %. The responses to quisqualate and kainate were only slightly changed by altering the pH from 7-3 to 6-3 or 8-3.3. The equilibrium dissociation constant (Kd) for H+ antagonism of responses to NMDA, estimated from the fit of a single-binding-site adsorption isotherm, was calculated to be 0-25 + 0'06 /SM, corresponding to pH 6X6 + 0-1. The H+ attenuation of NMDA current was voltage independent at membrane potentials -60 to + 30 mV.4. H+ antagonism of responses to NMDA was reduced when the NMDA concentration was lowered. In the pH range 6-3-8X3 the H+-induced reduction did not vary with the concentration of glycine or Mg2+. The sensitivity of NMDA current to Zn2+ was unchanged in the pH range 6-3 + 8-0. These results suggest that H+ ions do not directly interfere with the binding of NMDA to its agonist recognition site or with the binding of glycine, Mg2+ and Zn2+ to the specific allosteric sites on the NMDA receptor-channel complex.5. In outside-out patches held at -60 mV, unitary NMDA-activated currents were recorded at pH 7-3 and 6-3. The mean NMDA single-channel conductance (y) obtained for the largest and most frequent openings were: Y7.3 = 52-5 + 0-8 pS and Y6-3 = 51'8 + 0 9 pS. The duration of the mean channel open time, rO, decreased from 4.75 + 0-25 ms in the control at pH 7-3 to 3-59 + 0-21 ms at pH 6-3. The mean burst duration, Tb' was reduced from 8-51 +0-78 ms at control pH 7-3 to 5*1 +0-34 ms at pH 6-3. The frequency of NMDA channel bursts was reduced by 31%.6. Our results indicate that NMDA-activated ion channels on mouse hippocampal neurones are regulated by extracellular pH at an allosteric site distinct from those MS 8056 L. VYKLICKY JR, V. VLACHOVA AND J. KRUSEK which have been identified previously. We suggest that the interaction of H+ with a specific binding site, tentatively identified as histidine, results in a change of NMDA channel kinetics.

SummaryThe adenylate cyclase toxin-haemolysin of Bordetella (CyaA) targets CD11b+ myeloid phagocytes and translocates across their cytoplasmic membrane an adenylate cyclase (AC) enzyme that catalyses conversion of cytosolic ATP into cAMP. In parallel, CyaA acts as a cytolysin forming cation-selective pores, which permeabilize cell membrane and eventually provoke cell lysis. Using cytolytic activity, potassium efflux and patch-clamp assays, we show that a combination of substitutions within the pore-forming (E570Q) and acylation-bearing domain (K860R) ablates selectively the cell-permeabilizing activity of CyaA. At the same time, however, the capacity of such mutant CyaA to translocate the AC domain across cytoplasmic membrane into cytosol of macrophage cells and to elevate cellular cAMP concentrations remained intact. Moreover, the combination of E570Q+K860R substitutions suppressed the residual cytolytic activity of the enzymatically inactive CyaA/OVA/AC -toxoid on CD11b-expressing monocytes, while leaving unaffected the capacity of the mutant toxoid to deliver in vitro a reporter CD8 + T cell epitope from ovalbumin (OVA) to the cytosolic pathway of dendritic cells for MHC class I-restricted presentation and induce in vivo an OVA-specific cytotoxic T cell response. CyaA, hence, employs a mechanism of AC enzyme domain translocation across cellular membrane that avoids passage across the cytolytic pore formed by toxin oligomers.