Strychnine Binding Research Articles

Glycine receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

The inhibitory glycine receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on Glycine Receptors) is a member of the Cys-loop superfamily of transmitter-gated ion channels that includes the zinc activated channels, GABAA, nicotinic acetylcholine and 5-HT3 receptors [63]. The receptor is expressed either as a homo-pentamer of α subunits, or a complex now thought to harbour 2α and 3β subunits [30, 7], that contain an intrinsic anion channel. Four differentially expressed isoforms of the α-subunit (α1-α4) and one variant of the β-subunit (β1, GLRB, P48167) have been identified by genomic and cDNA cloning. Further diversity originates from alternative splicing of the primary gene transcripts for α1 (α1INS and α1del), α2 (α2A and α2B), α3 (α3S and α3L) and β (βΔ7) subunits and by mRNA editing of the α2 and α3 subunit [80, 91, 18]. Both α2 splicing and α3 mRNA editing can produce subunits (i.e., α2B and α3P185L) with enhanced agonist sensitivity. Predominantly, the mature form of the receptor contains α1 (or α3) and β subunits while the immature form is mostly composed of only α2 subunits. RNA transcripts encoding the α4-subunit have not been detected in adult humans. The N-terminal domain of the α-subunit contains both the agonist and strychnine binding sites that consist of several discontinuous regions of amino acids. Inclusion of the β-subunit in the pentameric glycine receptor contributes to agonist binding, reduces single channel conductance and alters pharmacology. The β-subunit also anchors the receptor, via an amphipathic sequence within the large intracellular loop region, to gephyrin. The latter is a cytoskeletal attachment protein that binds to a number of subsynaptic proteins involved in cytoskeletal structure and thus clusters and anchors hetero-oligomeric receptors to the synapse [86, 51, 53]. G-protein βγ subunits enhance the open state probability of native and recombinant glycine receptors by association with domains within the large intracellular loop [122, 121]. Intracellular chloride concentration modulates the kinetics of native and recombinant glycine receptors [94]. Intracellular Ca2+ appears to increase native and recombinant glycine receptor affinity, prolonging channel open events, by a mechanism that does not involve phosphorylation [24].

Kavalactones from Kava (Piper methysticum) root extract as modulators of recombinant human glycine receptors.

Roots of kava (Piper methysticum) plant are used in almost all Pacific Ocean cultures to prepare a drink with sedative, anesthetic and euphoric properties. One of the main active ingredients of the extract are kava lactones. Here, kava root CO2 extract and three kavalactones, DL-kavain, dihydrokavain and yangonin (isolated from whole extract by column chromatography) were tested for their inhibitory action on recombinant homomeric human α1 glycine receptors expressed in HEK293 cells. Kava CO2 root extract, as well as the individual components DL-kavain, dihydrokavain and yangonin inhibited glycine receptor activity in a dose-dependent manner. DL-kavain was the most potent inhibitor (IC50 = 0.077 ± 0.002 mm), followed by yangonin (IC50 = 0.31 ± 0.04 mm) and dihydrokavain (IC50 = 3.23 ± 0.10 mm) which were 4- and 40-fold less active than DL-kavain, respectively. Application of kava root extract did not reduce maximum currents, but increased EC50 of glycine. Simultaneous application of kava extract and strychnine showed additive inhibition, suggesting that binding of kavalactones and strychnine on the receptor is mutually exclusive. Overall, kavalactones exert a moderate inhibitory effect on the human α1 glycine receptor with DL-kavain being the most potent constituent.

Characterization of the binding of strychnine with bovine β-lactoglobulin and human lysozyme using spectroscopic, kinetic and molecular docking analysis

The binding interaction of a well known alkaloid strychnine (STN) with the mammalian milk protein β-lactoglobulin and human lysozyme has been explored by using several spectroscopic techniques along with computational studies.

Oxime Ethers of (E)-11-Isonitrosostrychnine as Highly Potent Glycine Receptor Antagonists.

A series of (E)-11-isonitrosostrychnine oxime ethers, 2-aminostrychnine, (strychnine-2-yl)propionamide, 18-oxostrychnine, and N-propylstrychnine bromide were synthesized and evaluated pharmacologically at human α1 and α1β glycine receptors in a functional fluorescence-based and a whole-cell patch-clamp assay and in [3H]strychnine binding studies. 2-Aminostrychnine and the methyl, allyl, and propargyl oxime ethers were the most potent α1 and α1β antagonists in the series, displaying IC50 values similar to those of strychnine at the two receptors. Docking experiments to the strychnine binding site of the crystal structure of the α3 glycine receptor indicated the same orientation of the strychnine core for all analogues. For the most potent oxime ethers, the ether substituent was accommodated in a lipophilic receptor binding pocket. The findings identify the oxime hydroxy group as a suitable attachment point for linking two strychnine pharmacophores by a polymethylene spacer and are, therefore, important for the design of bivalent ligands targeting glycine receptors.

Agonist and antagonist binding in human glycine receptors.

The human glycine receptor (hGlyR) is an anion-permeable ligand-gated channel that is part of a larger superfamily of receptors called the Cys-loop family. hGlyRs are particularly amenable to single-channel recordings, thus making them a model experimental system for understanding the Cys-loop receptor family in general. Understanding the relationship between agonist binding and efficacy in Cys-loop receptors should improve our future prospects for making specific agonists or antagonists. However, at present, there is no high-resolution structure for the complete hGlyR, and thus, modeling is needed to provide a physical framework on which to interpret single-channel data. The structure of the glutamate-gated chloride channel from Caenorhabditis elegans shows a much higher level of sequence identity to human hGlyR than previous templates such as AChBP or the bacterial channels, GLIC and ELIC. Thus, we constructed a model of the hGlyR and validated it against previously reported mutagenesis data. We used molecular dynamics to refine the model and to explore binding of both an agonist (glycine) and an antagonist (strychnine). The model shows excellent agreement with previous data but also suggests some unique features: (i) a water molecule that forms part of the binding site and allows us to account for some previous results that were difficult to reconcile, (ii) an interaction of the glycine agonist with S129, and (iii) an effect from E211, both of which we confirmed with new site-directed mutagenesis and patch clamp recordings. Finally, examination of the simulations suggests that strychnine binding induces movement to a conformational state distinct from the glycine-bound or apo state, not only within the ligand-binding domain but also in the transmembrane domain.

Thermodynamic studies of ligand binding to the human homopentameric glycine receptor using isothermal titration calorimetry

In this work, we describe a process for production of a Pichia pastoris strain which overproduces large quantities of the human glycine receptor. Subsequent purification yielded functional, uniform protein with expression yields of up to 5 mg per liter cell culture. As the wild-type protein is prone to proteolytic degradation, the labile sites were removed by mutagenesis resulting in an intracellular loop 2 deletion mutant with N-terminal modifications. This variant of the receptor is both stable during purification and storage on ice for up to a week as a complex with an antagonist. The quality of the protein is suitable for biophysical characterization and structural studies. The interaction of the agonist glycine and the antagonist strychnine with purified protein was analyzed by isothermal titration calorimetry. Strychnine binding is driven enthalpically with a K(D) of 138 ± 55 nM, a ΔH of -9708 ± 1195 cal/mol and a ΔS of -1.0 ± 4.1 cal/mol/K, whereas glycine binding is driven by entropy with a K(D) of 3.2 ± 0.8 μM, a ΔH of -2228 ± 1012 cal/mol and ΔS of 17.7 ± 2.8 cal/mol/K. Strychnine and glycine binding is competitive with a stoichiometry of one ligand molecule to one pentameric glycine receptor.

Characterization of Two Mutations, M287L and Q266I, in the α1 Glycine Receptor Subunit That Modify Sensitivity to Alcohols

Glycine receptors (GlyRs) are inhibitory ligand-gated ion channels. Ethanol potentiates glycine activation of the GlyR, and putative binding sites for alcohol are located in the transmembrane (TM) domains between and within subunits. To alter alcohol sensitivity of GlyR, we introduced two mutations in the GlyR α1 subunit, M287L (TM3) and Q266I (TM2). After expression in Xenopus laevis oocytes, both mutants showed a reduction in glycine sensitivity and glycine-induced maximal currents. Activation by taurine, another endogenous agonist, was almost abolished in the M287L GlyR. The ethanol potentiation of glycine currents was reduced in the M287L GlyR and eliminated in Q266I. Physiological levels of zinc (100 nM) potentiate glycine responses in wild-type GlyR and also enhance the ethanol potentiation of glycine responses. Although zinc potentiation of glycine responses was unchanged in both mutants, zinc enhancement of ethanol potentiation of glycine responses was absent in M287L GlyRs. The Q266I mutation decreased conductance but increased mean open time (effects not seen in M287L). Two lines of knockin mice bearing these mutations were developed. Survival of homozygous knockin mice was impaired, probably as a consequence of impaired glycinergic transmission. Glycine showed a decreased capacity for displacing strychnine binding in heterozygous knockin mice. Electrophysiology in isolated neurons of brain stem showed decreased glycine-mediated currents and decreased ethanol potentiation in homozygous knockin mice. Molecular models of the wild-type and mutant GlyRs show a smaller water-filled cavity within the TM domains of the Q266I α1 subunit. The behavioral characterization of these knockin mice is presented in a companion article (J Pharmacol Exp Ther 340:317-329, 2012).

Glycine receptor internalization by protein kinases activation

Although glycine-induced currents in the central nervous system have been proven to be modulated by protein kinases A (PKA) and C (PKC), the mechanism is not well understood. In order to better comprehend the mechanism involved in this phenomenon, we tested the PKA and PKC activation effect on the specific [(3) H]glycine and [(3) H]strychnine binding to postsynaptic glycine receptor (GlyR) in intact rat retina. The specific binding constituted about 20% of the total radioligand binding. Kinetic analysis of the specific binding exhibited a sigmoidal behavior with three glycine and two strychnine binding sites and affinities of 212 nM for [(3) H]glycine and 50 nM for [(3) H]strychnine. Specific radioligand binding was decreased (60-85%) by PKA and PKC activation, an effect that was blocked by specific kinases inhibitors, as well as by cytochalasin D. GlyR expressed in the plasma membrane decreased about 50% in response to kinases activation, which was consistent with an increase of the receptor in the microsomal fraction when PKA was activated. Moreover, immunoprecipitation studies indicated that these kinases lead to a time-dependent receptor phosphorylation. Our results suggest that in retina, GlyR is cross-regulated by G protein-coupled receptors, activating PKA and PKC.

Benzodiazepine activity: is interaction with the glycine receptor, as evidenced by displacement of strychnine binding, a useful criterion?

Benzodiazepine activity: is interaction with the glycine receptor, as evidenced by displacement of strychnine binding, a useful criterion?

Mapping of Disulfide Bonds within the Amino-terminal Extracellular Domain of the Inhibitory Glycine Receptor

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel and a member of the superfamily of cysteine loop (Cys-loop) neurotransmitter receptors, which also comprises the nicotinic acetylcholine receptor (nAChR). Within the extracellular domain (ECD), the eponymous Cys-loop harbors two conserved cysteines, assumed to be linked by a superfamily-specific disulfide bond. The GlyR ECD carries three additional cysteine residues, two are predicted to form a second, GlyR-specific bond. The configuration of none of the cysteines of GlyR, however, had been determined directly. Based on a crystal structure of the nAChRalpha1 ECD, we generated a model of the human GlyRalpha1 where close proximity of the respective cysteines was consistent with the formation of both the Cys-loop and the GlyR-specific disulfide bonds. To identify native disulfide bonds, the GlyRalpha1 ECD was heterologously expressed and refolded under oxidative conditions. By matrix-assisted laser desorption ionization time-of-flight mass spectrometry, we detected tryptic fragments of the ECD indicative of disulfide bond formation for both pairs of cysteines, as proposed by modeling. The identity of tryptic fragments was confirmed using chemical modification of cysteine and lysine residues. As evident from circular dichroism spectroscopy, mutagenesis of single cysteines did not impair refolding of the ECD in vitro, whereas it led to partial or complete intracellular retention and consequently to a loss of function of full-length GlyR subunits in human embryonic kidney 293 cells. Our results indicate that the GlyR ECD forms both a Cys-loop and a GlyR-specific disulfide bond. In addition, cysteine residues appear to be important for protein maturation in vivo.

Novel Regulatory Site within the TM3–4 Loop of Human Recombinant α3 Glycine Receptors Determines Channel Gating and Domain Structure

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants alpha3L and alpha3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3-4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3-4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3-4 loops of alpha3L and alpha3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of alpha3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3-4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3-4 loop of alpha3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.

Plasticity at glycinergic synapses in dorsal cochlear nucleus of rats with behavioral evidence of tinnitus

Fifteen percent to 35% of the United States population experiences tinnitus, a subjective “ringing in the ears”. Up to 10% of those afflicted report severe and disabling symptoms. Tinnitus was induced in rats using unilateral, 1 h, 17 kHz-centered octave-band noise (116 dB SPL) and assessed using a gap-startle method. The dorsal cochlear nucleus (DCN) is thought to undergo plastic changes suggestive of altered inhibitory function during tinnitus development. Exposed rats showed near pre-exposure auditory brainstem response (ABR) thresholds for clicks and all tested frequencies 16 weeks post-exposure. Sound-exposed rats showed significantly worse gap detection at 24 and 32 kHz 16 weeks following sound exposure, suggesting the development of chronic, high frequency tinnitus. Message and protein levels of α 1–3, and β glycine receptor subunits (GlyRs), and the anchoring protein, gephyrin, were measured in DCN fusiform cells 4 months following sound exposure. Rats with evidence of tinnitus showed significant GlyR α 1 protein decreases in the middle and high frequency regions of the DCN while α 1 message levels were paradoxically increased. Gephyrin levels showed significant tinnitus-related increases in sound-exposed rats suggesting intracellular receptor trafficking changes following sound exposure. Consistent with decreased α 1 subunit protein levels, strychnine binding studies showed significant tinnitus-related decreases in the number of GlyR binding sites, supporting tinnitus-related changes in the number and/or composition of GlyRs. Collectively, these findings suggest the development of tinnitus is likely associated with functional GlyR changes in DCN fusiform cells consistent with previously described behavioral and neurophysiologic changes. Tinnitus related GlyR changes could provide a unique receptor target for tinnitus pharmacotherapy or blockade of tinnitus initiation.

Synthesis of heteroaromatic tropeines and heterogeneous binding to glycine receptors

Heteroaromatic carboxylic esters of (nor)tropine were synthesized. Tropine esters displaced [ 3H]strychnine binding to glycine receptors of rat spinal cord with low Hill slopes. Two-site displacement resulted in nanomolar IC 50,1 and micromolar IC 50,2 values, and IC 50,2/IC 50,1 ratios up to 615 depending on the heteroaromatic rings and N-methyl substitution. Nortropeines displayed high affinity and low heterogeneity. IC 50,1 and IC 50,2 values of tropeines did not correlate suggesting different binding modes/sites. Glycine potentiated only the nanomolar displacement reflecting positive allosteric interactions and potentiation of ionophore function. Affinities of three (nor)tropeines were different for glycine receptors but identical for 5-HT 3 receptors.

Age-related changes in glycine receptor subunit composition and binding in dorsal cochlear nucleus

Age-related hearing loss, presbycusis, can be thought of, in part, as a slow progressive peripheral deafferentation. Previous studies suggest that certain deficits seen in presbycusis may partially result from functional loss of the inhibitory neurotransmitter glycine in dorsal cochlear nucleus (DCN). The present study assessed age-related behavioral gap detection changes and neurochemical changes of postsynaptic glycine receptor (GlyRs) subunits and their anchoring protein gephyrin in fusiform cells of young (7–11 months) and aged (28–33 months) Fischer brown Norway (FBN) rats. Aged rats showed significantly (20–30 dB) elevated auditory brainstem-evoked response thresholds across all tested frequencies and worse gap detection ability compared to young FBN rats. In situ hybridization and quantitative immunocytochemistry were used to measure GlyR subunit message and protein levels. There were significant age-related increases in the α 1 subunit message with significant age-related decreases in α 1 subunit protein. Gephyrin message and protein showed significant increases in aged DCN fusiform cells. The pharmacologic consequences of these age-related subunit changes were assessed using [ 3H] strychnine binding. In support of the age-related decrease of α 1 subunit protein levels in DCN, there was a significant age-related decrease in the total number of GlyR binding sites with no significant change in affinity. These age-related changes may reflect an effort to reestablish a homeostatic balance between excitation and inhibition impacting on DCN fusiform cells by downregulation of inhibitory function in the face of an age-related loss of peripheral input. Age-related decrease in presynaptic glycine release results in altered subunit composition and this may correlate with loss of temporal coding of the aged fusiform cell in DCN. The previously reported role for gephyrin in retrograde intracellular receptor subunit trafficking could contribute to the α 1 decrease in the face of increased message.

Depressed respiratory response to hypoxia and increased central glycinergic inhibitions in a rat model of fetal alcohol syndrom

Introduction Environmental conditions during pregnancy may have profound effects on breathing. However, nothing is known about fetal alcohol exposure on respiratory adaptation to chemosensory challenges in the offspring while the deleterious effects of alcohol on CNS development are well described. We recently found that fetal alcohol exposure in rat slowed respiratory frequency in juvenile rats and that brainstem respiratory network became insensitive to GABA A agonist [1] . We now investigated both in vivo and in situ the respiratory response to low oxygen level and studied the role of glycinergic inhibitions. Methods Female rats had a 10% (v/v) ethanol solution as sole source of fluid for 4 weeks before mating, during gestation and lactation periods. We used 3 week-old offspring rats and measured breathing in vivo with plethysmography both in normal condition of oxygenation and during hypoxia (10% O 2 ). In an in situ perfused heart-brainstem preparation, we recorded phrenic nerve activity and its response to anoxia. Strychnine, a glycine receptor antagonist, was applied into the perfusate in a dose-dependent manner. Anoxia was tested again in presence of strychnine. [ 3 H] strychnine binding experiments were performed at the level of the brainstem. Heart and lungs were dissected and weights while blood samples were collected to assess for oxygen blood status. Results The response to hypoxia was abolished in vivo and phrenic nerve response to anoxia was reduced while gasping activity was unaffected in situ. In situ, strychnine applications demonstrated a high sensitivity of expiratory duration during normal oxygenation. Indeed, 0.2μM strychnine compensated the lower basal phrenic nerve frequency and also the response to anoxia. [ 3 H] strychnine binding experiments showed an increase in both Bmax and Kd. Heart and lungs developed normally after birth, and polycythemia was detected. These results demonstrate that ethanol exposure during brain growth spurt period in rat induced polycythemia, possibly to compensate the low respiratory frequency at rest, and increased glycinergic inhibitions within the central respiratory network that was responsible for the blunted response to low oxygen. Conclusion Exposure to ethanol during gestation and/or early life compromises respiratory chemoreflex in juvenile offspring and may possibly put the neonates at risk.

Blunted response to low oxygen of rat respiratory network after perinatal ethanol exposure: involvement of inhibitory control

Acute ethanol depresses respiration, but little is known about chronic ethanol exposure during gestation and breathing, while the deleterious effects of ethanol on CNS development have been clearly described. In a recent study we demonstrated that pre- and postnatal ethanol exposure induced low minute ventilation in juvenile rats. The present study analysed in juvenile rats the respiratory response to hypoxia in vivo by plethysmography and the phrenic (Phr) nerve response to ischaemia in situ. Glycinergic neurotransmission was assessed in situ with strychnine application and [(3)H]strychnine binding experiments performed in the medulla. After chronic ethanol exposure, hyperventilation during hypoxia was blunted in vivo. In situ Phr nerve response to ischaemia was also impaired, while gasping activity occurred earlier and recovery was delayed. Strychnine applications in situ (0.05-0.5 microM) demonstrated a higher sensitivity of expiratory duration in ethanol-exposed animals compared to control animals. Moreover, [(3)H]strychnine binding density was increased after ethanol and was associated with higher affinity. Furthermore, 0.2 microM strychnine in ethanol-exposed animals restored the low basal Phr nerve frequency, but also the Phr nerve response to ischaemia and the time to recovery, while gasping activity appeared even earlier with a higher frequency. Polycythaemia was present after ethanol exposure whereas lung and heart weights were not altered. We conclude that chronic ethanol exposure during rat brain development (i) induced polycythaemia to compensate for low minute ventilation at rest; (ii) impaired the respiratory network adaptive response to low oxygen because of an increase in central glycinergic tonic inhibitions, and (iii) did not affect gasping mechanisms. We suggest that ethanol exposure during early life can be a risk factor for the newborn respiratory adaptive mechanisms to a low oxygen environment.

Synthesis of (nor)tropeine (di)esters and allosteric modulation of glycine receptor binding

(Hetero)aromatic mono- and diesters of tropine and nortropine were prepared. Modulation of [ 3H]strychnine binding to glycine receptors of rat spinal cord was examined with a ternary allosteric model. The esters displaced [ 3H]strychnine binding with nano- or micromolar potencies and strong negative cooperativity. Coplanarity and distance of the ester moieties of diesters affected the binding affinity being nanomolar for isophthaloyl-bistropane and nortropeines. Nortropisetron had the highest affinity ( K A ∼ 10 nM). Two esters displayed negative cooperativity with glycine in displacement, while three esters of low-affinity and nortropisetron exerted positive cooperativity with glycine.

Hyperekplexia mutation R271L of α1 glycine receptors potentiates allosteric interactions of nortropeines, propofol and glycine with [3H]strychnine binding

Human α1 and hyperekplexia mutant α1(R271L) glycine receptors (GlyRs) were transiently expressed in human embryonic kidney 293 cells for [3H]strychnine binding. Binding parameters were determined using a ternary allosteric model. The hyperekplexia mutation increased the positive cooperativity of 0.3mM propofol and glycine binding by about six times: the cooperativity factor β was 0.26 for α1 GlyRs and 0.04 for α1(R271L) GlyRs. Thus, propofol restored the potency of glycine impaired by the mutation. Five nortropeines, i.e. substituted benzoates of nortropine and a new compound, nortropisetron were prepared and also examined on [3H]strychnine binding. They showed nanomolar displacing potencies amplified by the hyperekplexia mutation. The affinity of nor-O-zatosetron (2.6nM) is one of the highest reported for GlyRs. This binding test offers an in vitro method to evaluate agents against neurological disorders associated with inherited mutations of GlyRs.

Protein kinases regulate glycine receptor binding in brain stem auditory nuclei after unilateral cochlear ablation

Glycinergic synaptic inhibition is part of acoustic information processing in brain stem auditory pathways and contributes to the regulation of neuronal excitation. We found previously that unilateral cochlear ablation (UCA) in young adult guinea pigs decreased [3H]strychnine binding activity in several brain stem auditory nuclei. This study determined if the UCA-induced deficit could be regulated by protein kinase C (PKC), protein kinase A (PKA) or Ca2+/calmodulin-dependent protein kinase II (CaMKII). The specific binding of [3H]strychnine was measured in slices of the dorsal (DCN), posteroventral (PVCN) and anteroventral (AVCN) cochlear nucleus (CN), the lateral (LSO) and medial (MSO) superior olive, and the inferior colliculus (IC) 145 days after UCA. Tissues from age-matched unlesioned animals served as controls. UCA induced deficits in specific binding in the AVCN, PVCN, and LSO on the ablated side and in the MSO bilaterally. These deficits were reversed by 3 μM phorbol 1,2-dibutyrate, a PKC activator, or 0.2 mM dibutyryl-cAMP, a PKA activator. However, 50 nM Ro31-8220, a PKC inhibitor, and 2 μM H-89, a PKA inhibitor, had no effect in unlesioned controls and after UCA. In contrast, 4 μM KN-93, a CaMKII inhibitor, relieved or reversed the UCA-induced binding deficits and elevated binding in the IC. These findings suggest that a UCA-induced down-regulation of glycine receptor synthesis may have occurred via reduced phosphorylation of proteins that control receptor synthesis; this effect was reversed by diminishing CaMKII activity or increasing PKC and PKA activity.

Study of the interaction of antiplasmodial strychnine derivatives with the glycine receptor

Strychnos icaja Baill. (Loganiaceae) is a liana found in Central Africa known to be an arrow and ordeal poison but also used by traditional medicine to treat malaria. Recently, many dimeric or trimeric indolomonoterpenic alkaloids with antiplasmodial properties have been isolated from its rootbark. Since these alkaloids are derivatives of strychnine, it was important, in view of their potential use as antimalarial drugs, to assess their possible convulsant strychnine-like properties. In that regard, their interaction with the strychnine-sensitive glycine receptor was investigated by whole-cell patch-clamp recordings on glycine-gated currents in mouse spinal cord neurons in culture and by [ 3H]strychnine competition assays on membranes from adult rat spinal cord. These experiments were carried out on sungucine (leading compound of the chemical class) and on the antiplasmodial strychnogucine B (dimeric) and strychnohexamine (trimeric). In comparison with strychnine, all compounds interact with a very poor efficacy and only at concentrations > 1 μM with both [ 3H]strychnine binding and glycine-gated currents. Furthermore, the effects of strychnine and protostrychnine, a monomeric alkaloid (without antiplasmodial activity) also isolated from S. icaja and differing from strychnine only by a cycle opening, were compared in the same way. The weak interaction of protostrychnine confirms the importance of the G cycle ring structure in strychnine for its binding to the glycine receptor and its antagonist properties.