Calmodulin-dependent Inactivation Research Articles

The molecular basis of the inhibition of CaV1 calcium-dependent inactivation by the distal carboxy tail

Ca2+/calmodulin-dependent inactivation (CDI) of CaV channels is a critical regulatory process that tunes the kinetics of Ca2+ entry for different cell types and physiologic responses. CDI is mediated by calmodulin (CaM), which is bound to the IQ domain of the CaV carboxy tail. This modulatory process is tailored by alternative splicing such that select splice variants of CaV1.3 and CaV1.4 contain a long distal carboxy tail (DCT). The DCT harbors an inhibitor of CDI (ICDI) module that competitively displaces CaM from the IQ domain, thereby diminishing CDI. While this overall mechanism is now well described, the detailed interactions required for ICDI binding to the IQ domain are yet to be elucidated. Here, we perform alanine-scanning mutagenesis of the IQ and ICDI domains and evaluate the contribution of neighboring regions to CDI inhibition. Through FRET binding analysis, we identify functionally relevant residues within the CaV1.3 IQ domain and the CaV1.4 ICDI and nearby A region, which are required for high-affinity IQ/ICDI binding. Importantly, patch-clamp recordings demonstrate that disruption of this interaction commensurately diminishes ICDI function resulting in the re-emergence of CDI in mutant channels. Furthermore, CaV1.2 channels harbor a homologous DCT; however, the ICDI region of this channel does not appear to appreciably modulate CaV1.2 CDI. Yet coexpression of CaV1.2 ICDI with select CaV1.3 splice variants significantly disrupts CDI, implicating a cross-channel modulatory scheme in cells expressing both channel subtypes. In all, these findings provide new insights into a molecular rheostat that fine-tunes Ca2+-entry and supports normal neuronal and cardiac function.

Ca2+-CaM Dependent Inactivation of RyR2 Underlies Ca2+ Alternans in Intact Heart.

Ca2+ alternans plays an essential role in cardiac alternans that can lead to ventricular fibrillation, but the mechanism underlying Ca2+ alternans remains undefined. Increasing evidence suggests that Ca2+ alternans results from alternations in the inactivation of cardiac RyR2 (ryanodine receptor 2). However, what inactivates RyR2 and how RyR2 inactivation leads to Ca2+ alternans are unknown. To determine the role of CaM (calmodulin) on Ca2+ alternans in intact working mouse hearts. We used an in vivo local gene delivery approach to alter CaM function by directly injecting adenoviruses expressing CaM-wild type, a loss-of-function CaM mutation, CaM (1-4), and a gain-of-function mutation, CaM-M37Q, into the anterior wall of the left ventricle of RyR2 wild type or mutant mouse hearts. We monitored Ca2+ transients in ventricular myocytes near the adenovirus-injection sites in Langendorff-perfused intact working hearts using confocal Ca2+ imaging. We found that CaM-wild type and CaM-M37Q promoted Ca2+ alternans and prolonged Ca2+ transient recovery in intact RyR2 wild type and mutant hearts, whereas CaM (1-4) exerted opposite effects. Altered CaM function also affected the recovery from inactivation of the L-type Ca2+ current but had no significant impact on sarcoplasmic reticulum Ca2+ content. Furthermore, we developed a novel numerical myocyte model of Ca2+ alternans that incorporates Ca2+-CaM-dependent regulation of RyR2 and the L-type Ca2+ channel. Remarkably, the new model recapitulates the impact on Ca2+ alternans of altered CaM and RyR2 functions under 9 different experimental conditions. Our simulations reveal that diastolic cytosolic Ca2+ elevation as a result of rapid pacing triggers Ca2+-CaM dependent inactivation of RyR2. The resultant RyR2 inactivation diminishes sarcoplasmic reticulum Ca2+ release, which, in turn, reduces diastolic cytosolic Ca2+, leading to alternations in diastolic cytosolic Ca2+, RyR2 inactivation, and sarcoplasmic reticulum Ca2+ release (ie, Ca2+ alternans). Our results demonstrate that inactivation of RyR2 by Ca2+-CaM is a major determinant of Ca2+ alternans, making Ca2+-CaM dependent regulation of RyR2 an important therapeutic target for cardiac alternans.

Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes.

TRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved. We performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes. Both lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton. The gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC's coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

ATP hydrolysis is critically required for function of CaV1.3 channels in cochlear inner hair cells via fueling Ca2+ clearance.

Sound encoding is mediated by Ca(2+) influx-evoked release of glutamate at the ribbon synapse of inner hair cells. Here we studied the role of ATP in this process focusing on Ca(2+) current through CaV1.3 channels and Ca(2+) homeostasis in mouse inner hair cells. Patch-clamp recordings and Ca(2+) imaging demonstrate that hydrolyzable ATP is essential to maintain synaptic Ca(2+) influx in inner hair cells via fueling Ca(2+)-ATPases to avoid an increase in cytosolic [Ca(2+)] and subsequent Ca(2+)/calmodulin-dependent inactivation of CaV1.3 channels.

Calmodulin-dependent activation and inactivation of anoctamin calcium-gated chloride channels

Calcium-dependent chloride channels serve critical functions in diverse biological systems. Driven by cellular calcium signals, the channels codetermine excitatory processes and promote solute transport. The anoctamin (ANO) family of membrane proteins encodes three calcium-activated chloride channels, named ANO 1 (also TMEM16A), ANO 2 (also TMEM16B), and ANO 6 (also TMEM16F). Here we examined how ANO 1 and ANO 2 interact with Ca2+/calmodulin using nonstationary current analysis during channel activation. We identified a putative calmodulin-binding domain in the N-terminal region of the channel proteins that is involved in channel activation. Binding studies with peptides indicated that this domain, a regulatory calmodulin-binding motif (RCBM), provides two distinct modes of interaction with Ca2+/calmodulin, one at submicromolar Ca2+ concentrations and one in the micromolar Ca2+ range. Functional, structural, and pharmacological data support the concept that calmodulin serves as a calcium sensor that is stably associated with the RCBM domain and regulates the activation of ANO 1 and ANO 2 channels. Moreover, the predominant splice variant of ANO 2 in the brain exhibits Ca2+/calmodulin-dependent inactivation, a loss of channel activity within 30 s. This property may curtail ANO 2 activity during persistent Ca2+ signals in neurons. Mutagenesis data indicated that the RCBM domain is also involved in ANO 2 inactivation, and that inactivation is suppressed in the retinal ANO 2 splice variant. These results advance the understanding of Ca2+ regulation in anoctamin Cl− channels and its significance for the physiological function that anoctamin channels subserve in neurons and other cell types.

Resolving the Grip of the Distal Carboxy Tail on the Proximal Calmodulatory Region of CaV Channels

Ca2+/calmodulin-dependent inactivation (CDI) of CaV channels plays a crucial role in the homeostasis of intracellular Ca2+, and serves as an ideal prototype for investigating Ca2+ feedback regulation within biological systems. The prevailing view of CDI mechanism is that Ca2+-free calmodulin (apoCaM) preassociates with the IQ domain of the proximal carboxy terminus of channels, and this ‘resident’ calmodulin (CaM) acts as Ca2+ sensor to somehow trigger CDI upon Ca2+ binding. The downstream CaM/channel configurations leading to CDI are complex and under investigation at present. Contrasting with this complexity, CaV1.3 and CaV1.4 feature a long-carboxy-tail splice variant that minimizes CDI by a beautifully simple mechanism. We recently combined electrophysiology and a genetically encoded fluorescent sensor to record CDI and CaM concentration simultaneously (Nature463:968), thereby demonstrating that the distal-carboxy-tail (DCT) harbors an ICDI module that competes with apoCaM for binding to the IQ domain. By kicking off apoCaM, ICDI diminishes CDI in an eminently straightforward manner; thus, the IQ/ICDI complex now looms as a simplified prototype for novel channel modulators, and as an easy entrez into complex CaM/channel configurations underlying CDI. Here, we perform alanine-scanning mutagenesis of the entire IQ domain and ICDI domain. Hotspot mutations which reduce IQ/ICDI binding affinity also commensurately diminish ICDI function (i.e., permit restoration of CDI). Furthermore, the ensemble of effects can be well fit with a Michaelis-Menten equation incorporating the presence of a competitive inhibitor. Thus, the collection of hotspots can be used to identify the minimal functional segments of IQ/ICDI interaction to facilitate structure building, and to provide clues for designing small peptides and/or molecules that regulate CDI of specific CaV channels.

Nanometer Resolution of Cav-Generated Ca2+ Gradients

Diffusion theory predicts that each pA of Ca2+ flux through a voltage-gated Ca2+ channel (CaV) should generate ∼100 μM free [Ca2+] in the channel nanodomain, with steep spatial gradient and little sensitivity to intracellular Ca2+ buffers. This is widely quoted, and bears on synaptic transmission, activity-dependent plasticity, and excitation-contraction coupling. Yet, experimental verification has been difficult because diffusible Ca2+ indicators report space-averaged [Ca2+], and CaV-tethered fluorescent indicators cannot distinguish between active and silent channels. Here, we used Ca2+/calmodulin-dependent inactivation (CDI) of CaV1.3 channels themselves as a nearfield indicator of nanodomain [Ca2+], noting that CDI provides an ionic readout based only on active channels. We calibrated the [Ca2+]-sensitivity of CDI by monitoring the decay of Li+ currents following spatially-uniform Ca2+ uncaging steps. We then ran Ca2+ through the same channels to generate local [Ca2+] gradients, and titrated the external Ca2+ concentration until CDI diminished ∼50%. The corresponding unitary flux was resolved through a combination of single-channel and whole-cell recordings, enabling determination of the relationship between unitary Ca2+ flux and nanodomain [Ca2+]. Contrary to theory, nanodomain [Ca2+] signals are larger and more buffer-sensitive than expected: ∼800 μM/pA in 10 mM intracellular BAPTA, and ∼200 μM/pA in 60 mM BAPTA. These findings indicate that diffusion of free Ca2+ ions is ∼10-fold slower in the nanodomain versus other regions of the cell, and that calmodulin (the Ca2+-sensing subunit for CDI) is ∼7 nm from the channel pore. Our results suggest that the nanodomain is a tortuous environment, where a Ca2+ ion would collide with an obstacle in 9 out of every 10 of its Brownian movements. Under physiological buffering, we predict that CaVs, by virtue of their crowded nanodomains, convert small Ca2+ fluxes into enormous Ca2+ concentrations, ∼1 mM/pA over distances of 10-20 nm.

A schistosome [beta] subunit remodels inactivation of a calcium channel via an N-terminal polyacidic motif

AbstractThe beta subunit of high voltage-activated Ca (Cav) channels targets the pore forming [alpha]~1~ subunit to the plasma membrane and defines the biophysical phenotype of the Cav channel complex. Cav channel inactivation following activation and opening is tightly regulated and is an essential property that not only prevents excessive entry of Ca^2+^ into the cell but may also have functions in signal transduction. The [beta] subunit modulates Ca^2+^-dependent and voltage-dependent components of Cav channel inactivation via its interaction with the I-II linker of the [alpha]~1~ subunit. Here, using Cav2.3 and whole-cell patch-clamp, we show that a [beta] subunit from the human parasite Schistosoma mansoni ([beta]~Sm~) accelerates inactivation via a unique, long N-terminal polyacidic motif (NPAM). The accelerating effect of NPAM-containing subunits, both native ([beta]~Sm~)and chimeric mammalian [beta]~1b~, [beta]~2a~ and [beta]~3~ subunits to which NPAM had been attached, was only apparent when Ca^2+^ was internally buffered with BAPTA (5 mM) or when Ba^2+^ was used as the charge carrier, two commonly used strategies to eliminate Ca^2+^/calmodulin dependent inactivation. These results indicate that calmodulin is not involved. In addition to accelerating inactivation, NPAM-containing [beta] subunits significantly reduced current density with respect to their non NPAM-bearing counterparts. Interestingly, when the amino acids N terminal to NPAM were deleted, inactivation of Cav2.3 currents was faster than in the presence of the entire N-terminal portion of the [beta]~Sm~ subunit, as if the pre-NPAM region counteracts the effect of NPAM. Presence of NPAM also resulted in currents that activated faster, suggesting that NPAM increases open channel probability. However, NPAM does not modulate inactivation gating. In summary, this study identifies a structural determinant of Cav channel inactivation that is entirely unlike those previously known.

Distinctive Inactivation Defects of Differing Mutant Calcium Channels Underlying Timothy Syndrome

Timothy Syndrome (TS) is a multisystem disorder characterized by autism, immune deficiencies, and cardiac arrhythmias. Intriguingly, the underlying defect comes down to a single point mutation (either G402S or G406R) in the IS6 region of CaV1.2 channels. These channels are critical conduits of Ca2+ entry into the heart, smooth muscle and brain. As such, these channels employ two forms of feedback regulation−voltage-dependent inactivation (VDI) and Ca2+/calmodulin-dependent inactivation (CDI). In TS, these regulatory mechanisms are disrupted, resulting in inappropriate Ca2+ feedback. Given that the pattern of multisystem pathology differs for the two types of mutant channels, we here undertook in-depth biophysical analysis of the altered inactivation in each of these constructs. As reported, both mutants exhibited strongly attenuated VDI. Rather surprisingly, however, both constructs also demonstrated a clear reduction of CDI, in contrast to a previous study reporting selective weakening of VDI (PNAS105:11987). Further analysis revealed that the CDI deficits in the two mutants may arise from very different mechanisms. For G406R, voltage-dependent activation is strongly shifted to more negative potentials, while estimated maximal open probability (PO/max) at saturating depolarization was only slightly altered. According to an allosteric mechanism of CDI (Biophys J96:222a), this favoring of channel activation would reduce CDI, because opening would be enhanced even within inactivated channels (i.e., the current decrease seen upon channel inactivation would be lessened). By contrast, the G402S mutation caused a marked depolarizing shift in voltage-dependent activation, with largely unchanged PO/max. This outcome would sharply diminish channel opening at physiological voltages, yielding attenuated CDI via decreased entry into inactivated states. Recognizing these divergent mechanisms of CDI disruption may shed light on the differing disease phenotypes elaborated by the two mutations, and ultimately prove beneficial in tailoring treatments for each TS population.

End-stage Mechanisms Underlying Voltage and Ca2+/Calmodulin-Dependent Inactivation (VDI and CDI) of CaV1.3 Channels

The past decade has witnessed many discoveries about the early events that underlie calmodulin (CaM) regulation of Ca2+ channels. Much is known about the positioning of apoCaM (Ca2+-free) on channels, and the initial Ca2+/CaM interaction sites. Beyond this, precious little is clear about the eventual actions of this central genre of Ca2+ channel modulation. Does CDI involve hinged-lid occlusion, selectivity filter collapse, or allosteric inhibition of activation gating? Do CDI and VDI reach the same ultimate conformation? All these proposals remain in flux. Here, we deduce that mutations within the S6 activation gates would produce discriminating effects on activation and inactivation, depending on which mechanism holds true. For the first two end-stage mechanisms (hinged-lid occlusion and selectivity filter collapse), S6 mutations that enhance channel opening are predicted to strengthen channel inactivation. By contrast, for an allosteric mechanism, such mutations would actually weaken inactivation. These predictions motivated exhaustive mutagenesis of the S6 segments of all four homologous domains of CaV1.3. We find that S6 mutations affect VDI and CDI in strikingly different ways, indicating a fundamental divergence of their end-stage mechanisms. The pattern seen for CDI agrees remarkably well with that predicted for an allosteric mechanism. By contrast, VDI effects cannot be fully explained by any previously described end-stage mechanism. Instead, mapping the functional VDI effects onto a structural homology model of CaV1.3 reveals a telling structural pattern, suggestive of a novel ‘hinged-lid-shield’ mechanism. In this scheme, CaV1.3 channels feature a specialized distal S6 ‘shield’ that repels lid closure. We validate this proposal with experiments in which the integrity of the shield and the mobility of the hinge-lid are independently modified. In all, these advances furnish a rich mechanistic backdrop for the many Ca2+ channelopathies involving S6 domains.

Calcium- and Calmodulin-Dependent Inactivation of Calcium Channels in Inner Hair Cells of the Rat Cochlea

Modulation of voltage-gated calcium channels was studied in inner hair cells (IHCs) in an ex vivo preparation of the apical turn of the rat organ of Corti. Whole cell voltage clamp in the presence of potassium channel blockers showed inward calcium currents with millisecond activation and deactivation kinetics. When temperature was raised from 22 to 37 degrees C, the calcium currents of immature IHCs [<12 days postnatal (P12)] increased threefold in amplitude, and developed more pronounced inactivation. This was determined to be calcium-dependent inactivation (CDI) on the basis of its reliance on external calcium (substitution with barium), sensitivity to internal calcium-buffering, and voltage dependence (reflecting the calcium driving force). After the onset of hearing at P12, IHC calcium current amplitude and the extent of inactivation were greatly reduced. Although smaller than in prehearing IHCs, CDI remained significant in the mature IHC near the resting membrane potential. CDI in mature IHCs was enhanced by application of the endoplasmic calcium pump blocker, benzo-hydroquinone. Conversely, CDI in immature IHCs was reduced by calmodulin inhibitors. Thus voltage-gated calcium channels in mammalian IHCs are subject to a calmodulin-mediated process of CDI. The extent of CDI depends on the balance of calcium buffering mechanisms and may be regulated by calmodulin-specific processes. CDI provides a means for the rate of spontaneous transmitter release to be adjusted to variations in hair cell resting potential and steady state calcium influx.

Calmodulin-dependent inactivation of presynaptic calcium currents and its contribution to synaptic depression at developing calyceal synapses

Calmodulin-dependent inactivation of presynaptic calcium currents and its contribution to synaptic depression at developing calyceal synapses

Switching off calcium-dependent inactivation in l -type calcium channels by an autoinhibitory domain

The retinal L-type Ca2+ channel Cav1.4 is distinguished from all other members of the high voltage-activated (HVA) Ca2+ channel family by lacking Ca2+-calmodulin-dependent inactivation. In synaptic terminals of photoreceptors and bipolar cells, this feature is essential to translate graded membrane depolarizations into sustained Ca2+ influx and tonic glutamate release. The sequences conferring Ca2+-dependent inactivation (CDI) are conserved throughout the HVA calcium channel family, raising the question of how Cav1.4 manages to switch off CDI. Here, we identify an autoinhibitory domain in the distal C terminus of Cav1.4 that serves to abolish CDI. We show that this domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C terminus. Deletion of ICDI completely restores Ca2+-calmodulin-mediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI, indicating that ICDI works as an autonomous unit. Furthermore, we show that in the Cav1.2 l-type Ca2+-channel replacement of the distal C terminus by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be introduced principally into other Ca2+ channel types. Our data provide a previously undescribed perspective on the regulation of HVA calcium channels by Ca2+.

Metabolism-based inactivation of neuronal nitric-oxide synthase by components of cigarette and cigarette smoke.

It has been shown that administration of cigarette smoke to rats leads to loss of neuronal nitric-oxide synthase (nNOS) activity and nNOS protein in penile tissue. The exact mechanism for this loss of activity and protein is not known. In the current study, we investigated whether extracts prepared from cigarette smoke or from the cigarette itself could directly inhibit nNOS activity. We discovered that the cigarette smoke extract and the cigarette extract cause a time-, concentration-, and calmodulin-dependent inactivation of nNOS in an in vitro system containing the purified enzyme. L-Arginine, but not D-arginine, protects nNOS from this time-dependent inactivation, suggesting an active site directed event. The kinetics of inactivation are consistent with the metabolism-based or suicide inactivation of nNOS. Based on studies with other metabolism-based inactivators, this cigarette-mediated inactivation may render nNOS more susceptible to proteasomal degradation and thereby may explain the loss of nNOS protein in vivo. The component(s) responsible for nNOS inactivation is not volatile, is not retained by a 3,000 molecular weight cut-off membrane, binds to activated charcoal, and is highly water-soluble under both acidic and basic conditions. The discovery of a direct inactivation of nNOS by an organic, cationic compound(s) present in tobacco and tobacco smoke provides a basis for further study of not only the mechanisms responsible for the biological effects of tobacco but also a search for a potentially novel inactivator of nNOS.

Modulation of L-type Ca2+ Channels by Gβγ and Calmodulin via Interactions with N and C Termini of α1C

Neuronal voltage-dependent Ca(2+) channels of the N (alpha(1B)) and P/Q (alpha(1A)) type are inhibited by neurotransmitters that activate G(i/o) G proteins; a major part of the inhibition is voltage-dependent, relieved by depolarization, and results from a direct binding of Gbetagamma subunit of G proteins to the channel. Since cardiac and neuronal L-type (alpha(1C)) voltage-dependent Ca(2+) channels are not modulated in this way, they are presumed to lack interaction with Gbetagamma. However, here we demonstrate that both Gbetagamma and calmodulin directly bind to cytosolic N and C termini of the alpha(1C) subunit. Coexpression of Gbetagamma reduces the current via the L-type channels. The inhibition depends on the presence of calmodulin, occurs at basal cellular levels of Ca(2+), and is eliminated by EGTA. The N and C termini of alpha(1C) appear to serve as partially independent but interacting inhibitory gates. Deletion of the N terminus or of the distal half of the C terminus eliminates the inhibitory effect of Gbetagamma. Deletion of the N terminus profoundly impairs the Ca(2+)/calmodulin-dependent inactivation. We propose that Gbetagamma and calmodulin regulate the L-type Ca(2+) channel in a concerted manner via a molecular inhibitory scaffold formed by N and C termini of alpha(1C).

Competitive inhibition of NMDA receptor-mediated currents by extracellular calcium chelators.

Calcium chelators have been widely used in electrophysiological recordings of N-methyl-D-aspartate (NMDA) receptor-mediated currents, as well as in studies of excitotoxicity. Intracellularly applied calcium chelators are known to inhibit, at least in part, such calcium-dependent processes as calmodulin-dependent inactivation, calcineurin-dependent desensitization, and rundown of NMDA receptors. On the other hand, the functional consequences and potential nonspecific effects of extracellularly applied chelators have not been extensively investigated. In whole-cell patch-clamp recordings from human embryonic kidney (HEK) 293 cells transiently transfected with recombinant NMDA receptors, we found that addition of calcium chelators such as EGTA shifted the glutamate dose-response curve to the right, from an EC(50) for NR1A/NR2A of 8 microM in 1.8 mM Ca(2+) to approximately 24 microM in a solution containing nominal 0 Ca(2+)/5 mM EGTA and further to approximately 80 microM in 20 mM EGTA. A similar shift in glutamate dose-response was observed for NR1A/NR2B currents. This dose-response shift was not due to a decrease in extracellular Ca(2+) concentration because there was no change in the glutamate EC(50) at Ca(2+) concentrations ranging from 10 mM to nominal 0/200 microM EGTA. Moreover, addition of 5 mM EGTA fully chelated with 6.8 mM Ca(2+) did not produce any shift in the glutamate dose-response curve. We propose that calcium chelators, containing four free carboxyl moieties, competitively inhibit glutamate binding to NMDA receptors.