Bidirectional modulation of basal forebrain N-methyl-D-aspartate receptor function differentially affects visual attention but not visual discrimination performance

Basal forebrain neuronal circuits, specifically the corticopetal cholinergic system, mediate attentional abilities. The effects of infusions of N-methyl-D-aspartate (NMDA) and the competitive NMDA receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV) into the basal forebrain were assessed in rats trained in an operant task designed to generate measures of sustained attention performance. Control animals were trained in a cued visual discrimination task devoid of explicit demands on attentional performance, but involving similar basic operant components as the sustained attention task. The effects of intrabasalis infusions of NMDA (1, 3 and 6nmol) and APV (3, 10 and 20nmol) were tested in separate groups of animals. Infusion of neither drug affected the animals' response accuracy in the cued visual discrimination task, indicating that performance in this task remains insufficient to activate basal forebrain NMDA receptors. Infusions of APV in sustained attention task-performing animals selectively decreased the animals' ability to detect visual signals, but spared their ability to reject non-signal events. Conversely, infusions of NMDA into the basal forebrain did not affect the animals' hit rate but increased their number of false alarms, i.e. "claims" for signals in non-signal trials. The concentrations of NMDA infused into the basal forebrain did not result in neurotoxic effects as demonstrated by a separate experiment, which indicated neurodegeneration following the infusion of 30 nmol NMDA as visualized by the Fluoro-Jade method.The effects of APV correspond with the attentional consequences of other manipulations known to impair the functions of cortical cholinergic input. Conversely, the effects of NMDA infusions agree with the hypothesis that overactivity of cortical cholinergic inputs mediates an abnormal overprocessing of the stimulus situation. Basal forebrain NMDA receptor manipulations assist in determining the role of this neuronal system in cognitive processes.     

N-methyl-D-aspartate receptor-dependent long-term potentiation in CA1 region affects synaptic expression of glutamate receptor subunits and associated proteins in the whole hippocampus

Long term potentiation in hippocampus, evoked by high-frequency stimulation, is mediated by two major glutamate receptor subtypes, alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptors and N-methyl-D-aspartate receptors. Receptor subunit composition and its interaction with cytoplasmic proteins constitute different pathways regulating synaptic plasticity. Here, we provide further evidence that N-methyl-D-aspartate receptor-mediated long term potentiation evoked at hippocampal CA1 region of rats induced by high-frequency stimulation of the Schaffer collateral-commissural pathway in vivo is not dependent on N-methyl-D-aspartate receptor subunit NR2B. Applying semi-quantitative immunoblotting, we found that in the whole tetanized hippocampus, synaptic expression of the N-methyl-D-aspartate and alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate receptor subunits (NR1, NR2A, glutamate receptor 1) and their associated partners, e.g. synaptic associated protein 97, postsynaptic density protein 95, alpha subunit of Ca2+/calmodulin-dependent protein kinase II, neuronal nitricoxide synthase, increased 180 min post-high-frequency stimulation. Moreover, phosphorylation of Ca2+/calmodulin-dependent protein kinase II at thr286 and glutamate receptor 1 at ser831 was increased 30 min post-high-frequency stimulation and blocked by N-methyl-D-aspartate receptor antagonists (AP-5 and MK-801). In sham group and controls, these changes were not observed. The expression of several other synaptic proteins (NR2B, glutamate receptors 2/3, N-ethylmaleimide sensitive factor) was not affected by long term potentiation induction. In hippocampal homogenates, the level of these proteins remained unchanged. These data indicate that N-methyl-D-aspartate receptor-dependent long term potentiation in CA1 region in vivo mainly affects the synaptic expression of glutamate receptor subunits and associated proteins in the whole hippocampus. The alteration of molecular aspects can play a role in regulating the long-lasting synaptic modification in hippocampal long term potentiation in vivo.     

Single channel properties of the N-methyl-D-aspartate receptor channel using NMDA and NMDA agonists: on-cell recordings

Summary The cell-attached patch-clamp mode has been applied in cultured rat hippocampal neurons to record single channel currents through the ion channel which is coupled to activation of the N-methyl-D-aspartate (NMDA) receptor. A channel, with a conductance of 37 pS, was studied with either NMDA or D-cis-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD) in the patch pipette. The mean open time of the channels with NMDA in the pipette was near 5 ms at -80 mV and was diminished about 0.7 ms for each 20 mV of hyperpolarization. The mean open times with ACPD in the pipette were longer at all voltages studied and for both agonists the mean open times showed an exponential dependence on patch potential. Distributions for the channel open times were generally well-fit with single exponentials, whereas distributions for closed times required two-component fits. In some instances, the open distributions also showed a second, rapid time component. When Mg² was included in the pipette (40 or 100 M), the mean open times were significantly diminished with the effect increasing with the magnitude of the hyperpolarization. Both the amplitudes and mean open times of the NMDA channel were strongly modulated by temperature with Q₁₀ values in excess of 2.5.     

N-methyl-D-aspartate receptor subunit changes are associated with lead-induced deficits of long-term potentiation and spatial learning

The present study demonstrates that impairments of spatial learning and hippocampal long-term potentiation in rats chronically exposed to lead are associated with changes in gene and protein expression of N-methyl-D-aspartate receptor subunits. Rats exposed to 750 and 1500 ppm lead acetate were found to exhibit deficits in acquisition of a water maze spatial learning task. Furthermore, lead-exposed rats show dose-dependent reductions in the maintenance of in vivo hippocampal long-term potentiation induced in entorhinal cortex-dentate gyrus synapses. We found an unexpected, but significant (P<0.05), correlation between spatial learning and long-term potentiation when control and lead-exposed rats were analysed as a single, combined population. Dentate gyrus NR1 subunit messenger RNA was reduced 18% and 28% by exposure to 750 and 1500 ppm lead acetate, respectively. NR2A subunit messenger RNA was reduced 18% but only in the dentate gyrus of rats exposed to 1500 ppm lead acetate. No significant changes in dentate NR2B messenger RNA expression were measured in either of the lead-exposed groups. NR1 subunit protein was reduced 24% and 58% in hippocampal homogenates from rats exposed to 750 and 1500 ppm lead acetate. In contrast, no changes in NR2A or NR2B subunit protein were observed in the same hippocampal homogenates.These data show that reductions of specific N-methyl-D-aspartate receptor subunits are associated with deficits of both hippocampal long-term potentiation and spatial learning, induced in rats by chronic exposure to environmentally relevant levels of lead. These findings strongly suggest that the effects of lead on N-methyl-D-aspartate receptors may be the mechanistic basis for lead-induced deficits in cognitive function.     

N-methyl-D-aspartate receptor blockade affects polysialylated neural cell adhesion molecule expression and synaptic density during striatal development.

Glutamatergic neurons innervate the striatum and form asymmetric synapses with the dendritic spines of striatal efferent neurons. The role of glutamate in striatal development, however, remains largely unknown. Previous studies have shown a dramatic increase in the density of asymmetric synapses in the rat striatum during the third postnatal week, followed by a decrease to adult levels by postnatal day 25. At the same time, the highly polysialylated form of the neural cell adhesion molecule becomes progressively restricted to synaptic regions and then disappears. We have now examined the effects of antagonists of the N-methyl-D-aspartate subtype of glutamatergic receptors on the expression of the polysialylated form of the neural cell adhesion molecule and on synaptic density during this late period of striatal development. Peripheral administration of the N-methyl-D-aspartate receptor antagonist dizocilpine maleate markedly decreased immunoreactivity for the highly polysialylated form of the neural cell adhesion molecule in the dorsolateral striatum and cerebral cortex when drug treatment included postnatal day 20, but not earlier in development. This effect was regionally specific and loss of the polysialylated neural cell adhesion molecule in the striatum was reproduced by the local administration of dizocilpine maleate, DL-2-amino-5-phosphonovalerate or ketamine on postnatal day 20. Quantitative ultrastructural studies of synaptic density with the physical disector method performed after one of the regimens inducing loss of the polysialylated neural cell adhesion molecule (postnatal days 18-20) revealed a 30% decrease in asymmetric synapses in the dorsolateral striatum of treated rats. Symmetric synapses, which presumably do not use glutamate, were not affected. The data indicate that N-methyl-D-aspartate receptors play a role in the late stages of synaptogenesis in the striatum and suggest that a subset of synapses expressing immunoreactivity for the highly polysialylated form of the neural cell adhesion molecule may be dependent on N-methyl-D-aspartate receptor stimulation during a critical period of striatal development.     

Neurochemical aspects of the N-methyl-D-aspartate receptor complex.

The N-methyl-D-aspartic acid (NMDA)-sensitive subclass of brain excitatory amino acid receptors is supposed to be a receptor-ionophore complex consisting of at least 3 different major domains including an NMDA recognition site, glycine (Gly) recognition site and ion channel site. Biochemical labeling of the NMDA domain using [3H]L-glutamic acid (Glu) as a radioactive ligand often meets with several critical methodological pitfalls and artifacts that cause a serious misinterpretation of the results. Treatment of brain synaptic membranes with a low concentration of Triton X-100 induces a marked disclosure of [3H]Glu binding sensitive to displacement by NMDA with a concomitant removal of other several membranous constituents with relatively high affinity for the neuroactive amino acid. The NMDA site is also radiolabeled by the competitive antagonist (+/-)-3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid that reveals possible heterogeneity of the site. The Gly domain is sensitive to D-serine and D-alanine but insensitive to strychnine, and this domain seems to be absolutely required for an opening of the NMDA channels by agonists. The ionophore domain is radiolabeled by a non-competitive type of NMDA antagonist that is only able to bind to the open but not closed channels. The binding of these allosteric antagonists is markedly potentiated by NMDA agonists in a manner sensitive to antagonism by isosteric antagonists in brain synaptic membranes and additionally enhanced by further inclusion of Gly agonists through the Gly domain. Furthermore, physiological and biochemical responses mediated by the NMDA receptor complex are invariably potentiated by several endogenous polyamines, suggesting a novel polyamine site within the complex. At any rate, activation of the NMDA receptor complex results in a marked influx of Ca2+ as well as Na+ ions, which subsequently induces numerous intracellular metabolic alterations that could be associated with neuronal plasticity or excitotoxicity. Therefore, any isosteric and allosteric antagonists would be of great benefit for the therapy and treatment of neurodegenerative disorders with a risk of impairing the acquisition and formation process of memories.     

The bundle crossing region is responsible for the inwardly rectifying internal spermine block of the Kir2.1 channel

Inward rectifier potassium channels conduct K⁺ across the cell membrane more efficiently in the inward than outward direction in physiological conditions. Voltage-dependent and flow-dependent blocks of outward K⁺ currents by intracellular polyamines (e.g., spermine (SPM)) have been proposed as the major mechanisms underlying inward rectification. In this study, we show that the SPM blocking affinity curve is shifted according to the shift in K⁺ reversal potential. Moreover, the kinetics of SPM entry to and exit from the binding site are correlatively slowed by specific E224 and E299 mutations, which always also disrupt the flux coupling feature of SPM block. The entry rates carry little voltage dependence, whereas the exit rates are e-fold decelerated per ～15 mV depolarization. Interestingly, the voltage dependence remains rather constant among WT and quite a few different mutant channels. This voltage dependence offers an unprecedented chance of mapping the location (electrical distance) of the SPM site in the pore because these kinetic data were obtained along the preponderant direction of K⁺ current flow (outward currents for the entry rate and inward currents for the exit rate) and thus contamination from flow dependence should be negligible. Moreover, double mutations involving E224 and A178 or M183 seem to alter the height of the same asymmetrical barrier between the SPM binding site and the intracellular milieu. We conclude that the SPM site responsible for the inward rectifying block is located at an electrical distance of ～0.5 from the inside and is involved in a flux coupling segment in the bundle crossing region of the pore. With preponderant outward K⁺ flow, SPM is “pushed” to the outmost site of this segment (～D172). On the other hand, the blocking SPM would be pushed to the inner end of this segment (～M183–A184) with preponderant inward K⁺ flow. Moreover, E224 and E299 very likely electrostatically interact with the other residues (e.g., R228, R260) in the cytoplasmic domain and then allosterically keep the bundle crossing region in an open conformation appropriate for the flux coupling block of SPM.     

Mutation analysis of the N-methyl-D-aspartate receptor NR1 subunit gene (GRIN1) in schizophrenia

Dysfunction of N-methyl-D-aspartate (NMDA) type ionotropic glutamate receptors has been implicated in the etiology of schizophrenia based on psychotomimetic properties of the antagonist phencyclidine (PCP) and observation that mice expressing low levels of the N-methyl-D-aspartate receptor NR1 subunit exhibit behavioral alterations that may be ameliorated by neuroleptic drugs. Based on the hypothesis that some schizophrenic patients have functionally deficient mutation(s) of the gene encoding N-methyl-D-aspartate receptor NR1 subunit (GRIN1), we screened 48 Japanese patients with schizophrenia for mutations in the coding region of the GRIN1 gene. Four variants, IVS2-22T>C, IVS2-12G>A, IVS4-34C>T, and 1719G/A (Pro516Pro), were identified. No non-synonymous mutation was detected. No significant association was suggested by case-control comparisons. Results indicate that genomic variations of the GRIN1 gene are not likely to be involved substantially in the etiology of schizophrenia.     

Subunit-specific mechanisms and proton sensitivity of NMDA receptor channel block

We have compared the potencies of structurally distinct channel blockers at recombinant NR1/NR2A, NR1/NR2B, NR1/NR2C and NR1/NR2D receptors. The IC₅₀ values varied with stereochemistry and subunit composition, suggesting that it may be possible to design subunit-selective channel blockers. For dizocilpine (MK-801), the differential potency of MK-801 stereoisomers determined at recombinant NMDA receptors was confirmed at native receptors in vitro and in vivo . Since the proton sensor is tightly linked both structurally and functionally to channel gating, we examined whether blocking molecules that interact in the channel pore with the gating machinery can differentially sense protonation of the receptor. Blockers capable of remaining trapped in the pore during agonist unbinding showed the strongest dependence on extracellular pH, appearing more potent at acidic pH values that promote channel closure. Determination of p K _a values for channel blockers suggests that the ionization of ketamine but not of other blockers can influence its pH-dependent potency. Kinetic modelling and single channel studies suggest that the pH-dependent block of NR1/NR2A by (−)MK-801 but not (+)MK-801 reflects an increase in the MK-801 association rate even though protons reduce channel open probability and thus MK-801 access to its binding site. Allosteric modulators that alter pH sensitivity alter the potency of MK-801, supporting the interpretation that the pH sensitivity of MK-801 binding reflects the changes at the proton sensor rather than a secondary effect of pH. These data suggest a tight coupling between the proton sensor and the ion channel gate as well as unique subunit-specific mechanisms of channel block.     

In vivo modulation of N-methyl-D-aspartate receptor-dependent long-term potentiation by the glycine modulatory site.

The role of the glycine modulatory site in N-methyl-D-aspartate receptor function was examined by determining the effect of the glycine site antagonist, 7-chlorokynurenic acid, on the induction of long-term potentiation at the commissural-CA1 synapse in anesthetized rats. Robust long-term potentiation of population excitatory postsynaptic potentials and population spike responses recorded extracellularly in the stratum pyramidale and in stratum radiatum of CA1 developed after high frequency stimulation (100 Hz for 1 s) of commissural fibers during continuous intrahippocampal administration of vehicle solution (0.15 M NaCl). In contrast, infusion of either 7-chlorokynurenic acid (400 microM) or of the N-methyl-D-aspartate receptor antagonist, D-2-amino-5-phosphonovaleric acid (100 microM), significantly attenuated or completely blocked the development of long-term potentiation. When 7-chlorokynurenic acid was infused together with the glycine analog, D-serine (1 mM), long-term potentiation developed that was comparable to that observed in control animals. Intrahippocampal administration of D-serine alone was associated with slightly greater magnitude of long-term potentiation than observed in control animals. Collectively, these findings establish that in intact hippocampus, activity at the glycine modulatory site is necessary for activation of the N-methyl-D-aspartate receptor complex. Furthermore, these results suggest that the glycine modulatory site may not be fully saturated in vivo, and thus can serve to regulate N-methyl-D-aspartate receptor function.     

Opposite effects of spermine and arcaine on responses of N-methyl-D-aspartate receptors expressed in Xenopus oocytes.

The ability of polyamines to modulate N-methyl-D-aspartate (NMDA) receptor function was investigated in Xenopus oocytes injected with rat brain mRNA. Whereas spermine and spermidine augmented NMDA/glycine-induced inwards currents, arcaine (1,4-diguanidinobutane) and 1,10-diaminodecane inhibited the response. The potency of arcaine to inhibit NMDA/glycine-induced currents was unaffected by spermine; similarly, arcaine did not influence the potency of spermine, but did reduce the maximal response to spermine. These findings demonstrate that polyamines exert both positive and negative modulatory control of the NMDA receptor expressed in Xenopus oocytes, and suggest that spermine and arcaine act at distinct sites in the NMDA receptor complex.     

N-methyl-D-aspartate receptor activation is required for the induction of both early and late phases of long-term potentiation in rat hippocampal slices

The possible involvement of N-methyl-D-aspartate (NMDA) receptors in mechanisms enabling the maintenance of long-term potentiation (LTP) was investigated in rat hippocampal slices. The action of the specific NMDA receptor antagonists (-)-2-amino-7-phosphonoheptanoic acid (D-APH) and 2-amino-5-phosphonovaleric acid (DL-APV) as well as of the inactive isomer L-APH was tested on orthodromic population excitatory postsynaptic potential (EPSP) and population spike (PS) responses recorded extracellularly from CA1 pyramidal cells. If the active D-isomer of APH (10 microM) or DL-APV (50 microM), but not if L-APH was present during tetanization, both EPSP and spike potentiation were markedly reduced or even blocked for the whole recording period (8 h after tetanization). It is concluded that the NMDA receptor component expressed during tetanization is a necessary step not only for initiation but also for subsequent mechanisms enabling late phases of synaptic LTP. Some remaining potentiation of the population spike may be related to a second, NMDA-independent mechanism.     

1,10-Diaminodecane and 1,12-diaminododecane block NMDA receptor currents by an open channel mechanism.

In whole-cell recordings from cultured rat hippocampal neurons (VH = -60 mV), 1,10-diaminodecane (DA10) and 1,12-diaminododecane (DA12) produced a concentration-dependent block of NMDA-induced current (IC50 = 30 and 7 microM, resp.). In contrast, the diamines failed to affect AMPA and kainate currents. The inhibition of NMDA currents was highly voltage-dependent and was substantially relieved at positive holding potentials. In outside-out patches, DA10 and DA12 produced a voltage-dependent flickery block of NMDA-activated single-channel currents. These results indicate that DA10 and DA12 antagonize NMDA responses via an open channel mechanism. DA10 and DA12 have been proposed to be inverse agonists at the spermine facilitatory site on the NMDA receptor. However, the channel blocking effects of the diamines complicate the interpretation of their actions at this site.     

Interaction of Mg2+ and phencyclidine in use-dependent block of NMDA channels.

The interaction between Mg2+ and phencyclidine (PCP) in blocking open N-methyl-D-aspartate (NMDA) channels was investigated in Xenopus oocytes injected with rat brain mRNA. These receptors exhibit the pharmacological and physiological properties of the neuronal receptors, and the oocyte is readily amenable to electrical recording and application of well-controlled chemical stimuli. We found that Mg2+ at physiological concentrations greatly impeded the ability of PCP to block the NMDA channel. The interaction between Mg2+ and PCP was competitive; 0.5 mM Mg2+ caused a four-fold decrease in the potency of PCP in blocking open NMDA channels. Moreover, Mg2+ speeded the recovery from PCP block in the presence of agonist, suggesting that Mg2+ reduced reblock of NMDA channels by PCP that had escaped from open channels. Our observations suggest that the presence of Mg2+ in the channel tends to prevent PCP entry and block. Since depolarization is likely to reduce channel occupancy by Mg2+ more than that by PCP, neural activity may have an important influence on the actions of PCP and related drugs.     

Mutation of a glutamate receptor motif reveals its role in gating and delta2 receptor channel properties

Despite its importance in the cerebellum, the functions of the orphan glutamate receptor delta2 are unknown. We examined a mutant delta2 receptor channel in lurcher mice that was constitutively active in the absence of ligand. Because this mutation was within a highly conserved motif (YTANLAAF), we tested its effect on several glutamate receptors. Mutant delta2 receptors showed distinct channel properties, including double rectification of the current-voltage relationship, sensitivity to a polyamine antagonist and moderate Ca 2+ permeability, whereas other constitutively active mutant glutamate channels resembled wild-type channels in these respects. Moreover, the kinetics of ligand-activated currents were strikingly altered. We conclude that the delta2 receptor has a functional ion channel pore similar to that of glutamate receptors. The motif may have a role in the channel gating of glutamate receptors.     

Benzodiazepines block long-term potentiation in slices of hippocampus and piriform cortex.

The effects of two benzodiazepines, diazepam and triazolam, on long-term potentiation were tested in slices of hippocampus and piriform cortex. The drugs had little influence on baseline synaptic responses but both were very effective in blocking LTP elicited by theta pattern stimulation. The effects were fully reversible upon washout. Diazepam reduced the increase in burst responses that occurs during theta stimulation and thus appears to interfere with the initial triggering events for long-term potentiation. This may reflect the enhancing action of the drug on GABA-mediated inhibitory potentials. Triazolam did not detectably change the burst responses elicited by theta pattern stimulation. Experiments with slices of piriform cortex indicated that triazolam also failed to disrupt the development of long-term potentiation but instead caused the potentiation to decay back to baseline in 15-30 min. Triazolam thus seems to act on the mechanisms that stabilize long-term potentiation. These results provide a possible explanation for the amnestic effects of benzodiazepines in humans and animals and support the hypothesis that long-term potentiation contributes to memory encoding.     

Murine startle mutant Nmf11 affects the structural stability of the glycine receptor and increases deactivation

AbstractDysfunctional glycinergic inhibitory transmission underlies the debilitating neurological condition, hyperekplexia, which is characterised by exaggerated startle reflexes, muscle hypertonia and apnoea. Here we investigated the N46K missense mutation in the GlyR α1 subunit gene found in the ethylnitrosourea (ENU) murine mutant, Nmf11, which causes reduced body size, evoked tremor, seizures, muscle stiffness, and morbidity by postnatal day 21. Introducing the N46K mutation into recombinant GlyR α1 homomeric receptors, expressed in HEK cells, reduced the potencies of glycine, β‐alanine and taurine by 9‐, 6‐ and 3‐fold respectively, and that of the competitive antagonist strychnine by 15‐fold. Replacing N46 with hydrophobic, charged or polar residues revealed that the amide moiety of asparagine was crucial for GlyR activation. Co‐mutating N61, located on a neighbouring β loop to N46, rescued the wild‐type phenotype depending on the amino acid charge. Single‐channel recording identified that burst length for the N46K mutant was reduced and fast agonist application revealed faster glycine deactivation times for the N46K mutant compared with the WT receptor. Overall, these data are consistent with N46 ensuring correct alignment of the α1 subunit interface by interaction with juxtaposed residues to preserve the structural integrity of the glycine binding site. This represents a new mechanism by which GlyR dysfunction induces startle disease.

Abbreviations

DMEM

Dulbecco''s modified Eagle medium

DR

dose‐ratio

ENU

ethylnitrosourea

GFP

green fluorescent protein

GLRA1

GlyR α1 subunit gene

GLRB

GlyR β subunit gene

GluCl

glutamate‐activated Cl^˗ channel

GlyR

glycine receptor

HEK‐293

human embryonic kidney 293 cells

K_B

equilibrium dissociation constant

V_patch

trans‐patch potential

WT

wild type

     

Ca2+ and voltage dependence of cardiac ryanodine receptor channel block by sphingosylphosphorylcholine

The effect of sphingosylphosphorylcholine (SPC) on the cytoplasmic Ca(2+) and voltage dependence of channel gating by cardiac ryanodine receptors (RyR) was examined in lipid bilayer experiments. Micromolar concentrations of the lysosphingolipid SPC added to cis solutions rapidly and reversibly decreased the single-channel open probability (P(o)) of reconstituted RyR channels. The SPC-induced decrease in P(o) was marked by an increase in mean closed time and burst-like channel gating. Gating kinetics during intraburst periods were unchanged from those observed in the absence of the sphingolipid, although SPC induced a long-lived closed state that appeared to explain the observed decrease in channel P(o). SPC effects were observed over a broad range of cis [Ca(2+)] but were not competitive with Ca(2+). Interestingly, the sphingolipid-induced, long-lived closed state displayed voltage-dependent kinetics, even though other channel gating kinetics were not sensitive to voltage. Assuming SPC effects represent channel blockade, these results suggest that the blocking rate is independent of voltage whereas the unblocking rate is voltage dependent. Together, these results suggest that SPC binds directly to the cytoplasmic side of the RyR protein in a location in or near the membrane dielectric, but distinct from cytoplasmic Ca(2+) binding sites on the protein.