Disease-Associated Variants in GRIN1, GRIN2A and GRIN2B genes: Insights into NMDA Receptor Structure, Function, and Pathophysiology.

N-methyl-D-aspartate receptors (NMDARs) are a subtype of ionotropic glutamate receptors critical for synaptic transmission and plasticity, and for the development of neural circuits. Rare or de-novo variants in GRIN genes encoding NMDAR subunits have been associated with neurodevelopmental disorders characterized by intellectual disability, developmental delay, autism, schizophrenia, or epilepsy. In recent years, some disease-associated variants in GRIN genes have been characterized using recombinant receptors expressed in non-neuronal cells, and a few variants have also been studied in neuronal preparations or animal models. Here we review the current literature on the functional evaluation of human disease-associated variants in GRIN1, GRIN2A and GRIN2B genes at all levels of analysis. Focusing on the impact of different patient variants at the level of receptor function, we discuss effects on receptor agonist and co-agonist affinity, channel open probability, and receptor cell surface expression. We consider how such receptor-level functional information may be used to classify variants as gain-of-function or loss-of-function, and discuss the limitations of this classification at the synaptic, cellular, or system level. Together this work by many laboratories worldwide yields valuable insights into NMDAR structure and function, and represents significant progress in the effort to understand and treat GRIN disorders. Keywords: NMDA receptor , GRIN genes, Genetic variants, Electrophysiology, Synapse, Animal models.

Application of spike sorting algorithm to neuronal signals originated from boron doped diamond micro-electrode arrays.

In this work we report on the implementation of methods for data processing signals from microelectrode arrays (MEA) and the application of these methods for signals originated from two types of MEAs to detect putative neurons and sort them into subpopulations. We recorded electrical signals from firing neurons using titanium nitride (TiN) and boron doped diamond (BDD) MEAs. In previous research, we have shown that these methods have the capacity to detect neurons using commercially-available TiN-MEAs. We have managed to cultivate and record hippocampal neurons for the first time using a newly developed custom-made multichannel BDD-MEA with 20 recording sites. We have analysed the signals with the algorithms developed and employed them to inspect firing bursts and enable spike sorting. We did not observe any significant difference between BDD- and TiN-MEAs over the parameters, which estimated spike shape variability per each detected neuron. This result supports the hypothesis that we have detected real neurons, rather than noise, in the BDD-MEA signal. BDD materials with suitable mechanical, electrical and biocompatibility properties have a large potential in novel therapies for treatments of neural pathologies, such as deep brain stimulation in Parkinson's disease.

Molecular basis of TRPA1 regulation in nociceptive neurons. A review.

Transient receptor potential A1 (TRPA1) is an excitatory ion channel that functions as a cellular sensor, detecting a wide range of proalgesic agents such as environmental irritants and endogenous products of inflammation and oxidative stress. Topical application of TRPA1 agonists produces an acute nociceptive response through peripheral release of neuropeptides, purines and other transmitters from activated sensory nerve endings. This, in turn, further regulates TRPA1 activity downstream of G-protein and phospholipase C-coupled signaling cascades. Despite the important physiological relevance of such regulation leading to nociceptor sensitization and consequent pain hypersensitivity, the specific domains through which TRPA1 undergoes post-translational modifications that affect its activation properties are yet to be determined at a molecular level. This review aims at providing an account of our current knowledge on molecular basis of regulation by neuronal inflammatory signaling pathways that converge on the TRPA1 channel protein and through modification of its specific residues influence the extent to which this channel may contribute to pain.

Electrode-less measurement of cell layers impedance.

This paper is devoted to yet unpublished electrode-less methods (ELM) of cell layers impedance measurement based on transformer principle. The main advantage of ELM is elimination uncertainties caused by interface between electrodes and measured electrolyte. The method of avoiding distortion caused by non-ideal transformer transfer function ("deconvolution") and errors caused by residual voltage is described. The modification of original transformer based method allowing to measure an impedance of inserted object is proposed. Results of several calibration measurements confirming the proper function of ELM including example of transepithelial resistance of cells layer are presented. Crucial parts of measuring system and recommendation for their realization are included.

The interaction of quaternary reversible acetylcholinesterase inhibitors with the nicotinic receptor.

Acetylcholinesterase inhibitors (AChEIs) are used in the treatment of myasthenia gravis (MG). We investigated the effects of AChEIs on peripheral nicotinic receptors (nAChR), which play a crucial role in the treatment of MG symptoms. The positive modulation of those receptors by AChE inhibitors could have an added value to the anti-AChE activity and might be useful in the therapy of MG. Furthermore, to estimate the potential drawbacks of the compounds, cytotoxicity has been assessed on various cell lines. The whole-cell mode of the patch-clamp method was employed. The experiments were performed on medulloblastoma/rhabdomyosarcoma cell line TE671 expressing human embryonic muscle-like receptor with subunits alpha2betagammadelta. The effect of the compounds on cell viability was measured by standard MTT assay (Sigma Aldrich) on ACHN (renal cell adenocarcinoma), HeLa (immortal cell line derived from a cervical carcinoma), HEPG2 (hepatocellular carcinoma) and BJ (skin fibroblasts) cell lines. No positive modulation by the tested AChE inhibitors was observed. Moreover, the compounds exhibited antagonistic activity on the peripheral nAChR. Standard drugs used in MG treatment were shown to be less potent inhibitors of muscle-type nAChR than the newly synthesized compounds. The new compounds showed very little effect on cell viability, and toxicities were comparable to standards. Newly synthesized AChEIs inhibited peripheral nAChR. Furthermore, the inhibition was higher than that of standards used for the treatment of MG. They could be used for the study of nAChR function, thanks to their high antagonizing potency and fast recovery of receptor activity after their removal. However, since no positive modulation was observed, the new compounds do not seem to be promising candidates for MG treatment, even though their cytotoxic effect was relatively low.

Structure, function, and pharmacology of NMDA receptor channels.

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.

Oxime reactivators and their in vivo and in vitro effects on nicotinic receptors.

Current treatment of organophosphorus poisoning, resulting in overstimulation and desensitization of muscarinic and nicotinic receptors by acetylcholine (ACh), consists of the administration of atropine and oxime reactivators. However, no versatile oxime reactivator has been developed yet and some mortality still remains after application of standard atropine treatment, probably due to its lack of antinicotinic action. In our study, we focused on the interesting non-acetylcholinesterase property of oximes, i.e. antinicotinic effect of reactivators. Two standard reactivators (HI-6, obidoxime) and two new compounds (K027 and K203) were chosen for in vitro (patch clamp) and in vivo (nerve-evoked muscle contraction) testings. Both examinations showed antinicotinic effects of the reactivators. In vitro inhibition of acetylcholine-evoked currents by obidoxime, HI-6 and K203 was equivalent while K027 was less potent. Similar order of potency was observed by the in vivo examinations. We thus confirm previous in vitro results, which describe antinicotinic effects of oxime reactivators, and furthermore, we show in vivo antagonism of oxime reactivators exerted by the inhibition of ACh effect on the nicotinic receptor in the neuromuscular junction. Taking together, the effects of tested oxime reactivators indicate an antagonism on both embryonic and adult form of the muscle nicotinic receptors.

Allostery and cooperativity in the interaction of drugs with ionic channel receptors.

Allostery is an essential property of many physiological mechanisms. Cooperativity together with allostery is observed in the behavior of multisubunit receptors. Here we summarize and compare several approaches to the description and analysis of allosteric phenomena with emphasis on the receptors connected to ionic channels as a model. Several simplified methods are discussed in comparison with the microscopic kinetic scheme, affinity-efficacy separation and a thermodynamic approach.

Activation and modulation of ligand-gated ion channels.

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

Chloride cotransport in the membrane of earthworm body wall muscles.

The resting membrane potential (V(m)) of isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris was studied by glass microelectrodes. The inhibition of chloride permeability by low pH did not affect V(m) of the muscle fibers in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris which was -48.7 mV (inside negative) at pH 7.3 and -49.1 at pH 5.6. On the other hand, bathing the muscles in Cl(-) and Na(+)-free solutions, or application of the chloride transporter inhibitor furosemide and Na(+)-K(+)-ATPase inhibitor ouabain depolarized the V(m) by 3-5 mV. The effects of a Cl(-) -free solution and ouabain were not additive. This demonstrates relatively small contribution of equilibrium potential for Cl(-) to the resting membrane potential and electrogenic effect of Na(+)K(+)-ATPase which is dependent on the supply of Na(+)(i) ions by furosemide-sensitive and Cl(-)(e)- and Na(+)(e)-dependent electroneutral transport (most probably Na(+)K(+)Cl(-) cotransport).

Different degree of cooperativity in adult, embryonic and mutated mouse muscle nicotinic receptors.

Adult and embryonic nicotinic receptors expressed in COS cells have similar affinities for acetylcholine but differ in their Hill coefficient. Parameters of wild-type receptors were compared with those of receptors with mutated delta and gamma subunits in selected negatively charged amino acids, which were expected to participate in agonist binding. A tentative scheme of affinities, allosteric interactions and channel gating efficacy was used for assessing the role of mutated amino acids in the channel function. In three models, the parameters of wild-type embryonic and adult receptors were compared with those of receptors with mutated delta and gamma subunits. The analysis of different models of channel activation indicates that negatively charged amino acids which were mutated in the delta subunit in embryonic receptors participate in channel gating and in allosteric interactions between subunits rather than directly in agonist binding. Changes in the gamma subunit in the embryonic receptors and delta subunit in the adult receptors could equally affect agonist binding, allosteric coupling between subunits or channel gating.

Citalopram inhibits L-type calcium channel current in rat cardiomyocytes in culture.

Selective serotonine reuptake inhibitors (SSRI) are believed to be less dangerous in the treatment of depressive disorder in comparison with tricyclic antidepressants (TCA) due to their relative lack of cardiotoxicity. Thus, we investigated the effect of citalopram (SSRI) on membrane electrophysiology in rat cardiomyocytes in tissue culture. The results were compared with those from amitriptyline (TCA). The whole-cell configuration patch-clamp technique was used. Both citalopram and amitriptyline exhibited the concentration-dependent inhibition of the L-type calcium channel current (ICa). Citalopram in concentrations of 3 microM and 10 microM inhibited peak calcium current by 2.7% and 8%, respectively. We demonstrated the same potency of citalopram and amitriptyline to inhibit ICa. These observations led us to conclude that citalopram and amitriptyline are drugs, which exhibit a similar potency for causing concentration-dependent inhibition of ICa.

Spatial distribution of spermine/spermidine content and K(+)-current rectification in frog retinal glial (Müller) cells.

Previous studies in retinal glial (Müller) cells have suggested that (1) the dominant membrane currents are mediated by K(+) inward-rectifier (Kir) channels (Newman and Reichenbach, Trends Neurosci 19:307-312, 1996), and (2) rectification of these Kir channels is due largely to a block of outward currents by endogenous polyamines such as spermine/spermidine (SPM/SPD) (Lopatin et al., Nature 372:366-369, 1994). In frog Müller cells, the degree of rectification of Kir-mediated currents is significantly higher in the endfoot than in the somatic membrane (Skatchkov et al., Glia 27:171-181, 1999). This article shows that in these cells there is a topographical correlation between the local cytoplasmic SPM/SPD immunoreactivity and the ratio of inward to outward K(+) currents through the surrounding membrane area. Throughout the retina, Müller cell endfeet display a high SPM/SPD immunolabel (assessed by densitometry) and a large inward rectification of K(+) currents, as measured by the ratio of inward to outward current produced by step changes in [K(+)](o). In the retinal periphery, Müller cell somata are characterized by roughly one-half of the SPM/SPD immunoreactivity and K(+)-current rectification as the corresponding endfeet. In the retinal center, Müller cell somata are virtually devoid of both SPM/SPD immunolabel and K(+)-current inward rectification. Comparing one region of the retina with another, we find an exponential correlation between the local K(+) rectification and the local SPM/SPD content. This finding suggests that the degree of inward rectification in a given membrane area is determined by the local cytoplasmic polyamine concentration.

Overexpression of the G protein G11alpha prevents desensitization of Ca2+ response to thyrotropin-releasing hormone.

Doubly transfected human embryonal kidney cells (clone E2M11 of the HEK 293 cell line) expressing both thyrotropin-releasing hormone (TRH) receptors and G11alpha protein in high amounts were used to analyze the desensitization phenomenon of the Ca2+-mobilizing pathway. Quite unexpectedly, we did not observe any significant desensitization of the [Ca2+]i response to TRH in these cells after repeated or prolonged incubation with the hormone (up to 5 h). Under the same conditions, the TRH-induced [Ca2+]i response was completely desensitized in the parent cell line (293-E2 cels) expressing TRH receptors alone. In both cell lines, inositol phosphate response was desensitized after TRH exposure, although basal levels of inositol phospates in TRH-pretreated cells were much higher than in "naive" TRH-unexposed cells. These data suggest a significant role of the G protein G11alpha in desensitization of the Ca2+-mobilizing pathway occuring after repeated or long-term exposure of target cells to TRH-receptor agonists.

Potassium buffering by Müller cells isolated from the center and periphery of the frog retina.

Müller (radial glial) cells span the retina from the outer to the inner limiting membranes. They are the only glial cells found in the amphibian retina. The thickness of the frog (Rana pipiens) retina decreases by a factor of about four from the center to the periphery. Thus, Müller cells were isolated, by enzymatic dissociation, with stalk lengths from 20 to 140 microm. Their ability to transfer K(+) via the stalk between soma and endfoot was studied. Membrane currents were recorded using the whole-cell voltage-clamp technique with the pipette sealed to either the endfoot or the soma. Inward (I(KIN)) or outward (I(KO)) currents were elicited by rapid increases (3 to 10 mM) or decreases (3 to 1 mM) of the extracellular K(+) concentration ([K(+)](o)) either by local application (close or distant to the recording pipette) or around the entire cell (whole cell perfusion). For the long central cells, the ratio I(KIN)/I(KO) was 4.6 +/- 0.6 SE (n = 9) at the endfoot and 1.7 +/- 0.1 SE (n = 8) at the soma. In cells from the retinal periphery, the ratio I(KIN)/I(KO) was higher, 7.0 +/- 0.27 (n = 8) at the endfoot and 3.2 +/- 0.1 (n = 10) at the soma. The results suggest that there is less inward rectification in the somatic than in the endfoot membrane. As expected from previous studies, the sensitivity of the cells to K(+) was higher at the endfoot than at the soma. The amplitude of I(KIN) at the endfoot compared to the soma was about 8-fold for the long central cells but only about 1.5-fold for the short peripheral cells. Currents spread readily from endfoot to soma in the peripheral cells. In the long central Müller cells the soma and endfoot appeared electrotonically isolated. The "functional length constant", lambda, of cell stalk processes was about 70 microm. The relative decrement of large inward currents was stronger than that of smaller outward currents; this difference ("artificial rectification") is explained by a simple model, where larger currents (inward) are attenuated more than smaller (outward) currents. The data support the hypothesis that in the retinal periphery, Müller cells provide extensive spatial K(+) buffering from both plexiform layers into the vitreous body. In the central retina, however, such currents are limited within a short (interlaminar) range.

Specific binding to plasma membrane is the first step in the uptake of non-transferrin iron by cultured cells.

We studied transport of non-transferrin iron into HeLa cells adapted for growth in defined medium, containing either 5 micrograms/ml of iron-saturated transferrin (HeLa/Tf cells) or 5 microM ferric citrate (HeLa/Fe5 cells) as a source of iron. Employing 55Fe-ferric citrate, iron uptake by intact cells was compared with iron binding to isolated membranes. Uptake characteristics of both HeLa/Tf and HeLa/Fe5 cells seemed to be similar: Km = 14 microM and Vmax = 135 pmol Fe/min/10(5) cells for HeLa/Tf, Km = 22 microM and Vmax = 165 pmol Fe/min/10(5) cells for HeLa/Fe5. Increasing concentrations (0.3-1.2 microM) of 55Fe-ferric citrate, producing levels of free 55Fe which were independent of total Fe under the experimental conditions used, led to increased binding of 55Fe for both HeLa/Tf and HeLa/Fe5 cells (1.08-8.03 nmol Fe/h/10(5) cells). This corresponds with the suggestion that iron was bound in the form of ferric citrate rather than in the form of free iron. Dissociation constants of Fe binding, KD = 0.61 microM for HeLa/Tf and KD = 1.53 microM for HeLa/Fe5, were obtained from competition experiments. We conclude that specific binding sites for ferric citrate are constitutively expressed in plasma membrane and that their expression does not require the induction by the presence of ferric citrate. The uptake of non-transferrin iron is realized in at least two steps. The first step is iron binding to the specific binding sites in plasma membrane. The binding does not represent a limiting step of the uptake.

Analysis of kinetic properties of gamma-glutamyl transpeptidase from rat kidney.

The initial rate kinetics of rat kidney gamma-glutamyl transpeptidase were measured using L-gamma-glutamyl-p-nitroanilide and glycyl-glycine as the donor and the acceptor substrate, respectively. Experimental data were fitted with the initial rate equation, and the obtained results indicated that: (1) Michaelis constants for transpeptidation (Kb), autotranspeptidation (Ka), and hydrolysis (Kh) are 8.56 mmol/l, 2.02 mmol/l and 0.005 mmol/l, respectively. (2) The maximum rate of transpeptidation (Vb) exceeds that of hydrolysis (Vh) and autotranspeptidation (Va) 160 times and 5 times, respectively. (3) A comparison of the ratios of maximal rate: Michaelis constant of individual reactions shows that hydrolysis is approximately 10 times more efficient than the remaining two reactions. (4) Under routine conditions used for gamma-glutamyl transpeptidase estimation, transpeptidation is the prevalent reaction.

Relationship between kinetic properties of gamma-glutamyl transpeptidase and the structure of its saccharide moiety.

Gamma-glutamyl transpeptidase (EC 2.3.2.2; GGT) is a plasma-membrane bound glycoenzyme, the saccharide moiety of which is rather heterogeneous and organ specific. It has been stated that GGT catalyses three types of reactions, i.e., hydrolysis, transpeptidation and autotranspeptidation. The initial velocity equation, involving all these reactions, is shown in the present report. Mathematical analysis of the equation resulting in a definition of the constant of half saturation (Khs). The value of Khs was used for characterization of kinetics of GGT from rat organs differing in the structure of GGT oligosaccharide chains. No significant organ differences were found, when the Khs values of GGT from the brain, kidney and pancreas equalled 0.61 mM, 0.68 mM and 0.68, respectively. On the contrary, when two different glycoforms of GGT from the pancreas were compared, distinct values of Khs were obtained (1.43 mM and 0.67 mM, respectively). It is therefore being suggested that the saccharide chains of GGT are involved in its kinetic properties. However, this effect is masked when the enzyme, non-fractionated into glycoforms, is analysed, even though the saccharide moiety is specific for the organ studied.

Electrophysiological characterization of GABAA receptors in anterior pituitary cells of newborn rats.

The gamma-aminobutyric acid (GABA)-ergic communication between the CNS and the anterior pituitary gland has been documented in numerous histochemical and biochemical studies but electrophysiological studies characterizing the GABAA receptor in the anterior pituitary are still lacking. In the present report we studied the GABA-induced current responses in cultured cells from the anterior pituitary gland of 6- to 10-day-old rats using the patch-clamp technique in the whole cell configuration. Fast application of GABA (100 microM) induced membrane currents in 90% of cells in 2-day-old cultures. The EC50 for GABA was 22.9 microM and the Hill coefficient was 1.8. The responses to GABA (10 microM) were inhibited by bicuculline (2 microM) to 14%, by picrotoxin (5 microM) to 21% and by zinc (10 microM) to 33%. Inhibition to 56% was observed with 6 microM strychnine. The GABA responses were sensitive to diazepam and pentobarbital. Half-maximal potentiation of responses to GABA (10 microM) was found with 1.0 microM diazepam and with 14.4 microM pentobarbital. The maximal potentiation of GABA responses was 222% for diazepam and 195% for pentobarbital. Pentobarbital (100 microM) did not induce any response in anterior pituitary cells in the absence of GABA. The application of GABA at concentrations 10 microM or higher, induced membrane currents that desensitized. Desensitization proceeded as a biexponential process with estimated fast and slow time constants which decreased with concentration. The responses to GABA (300 microM) desensitized to 93% with time constants of 1.4 and 5.3 s. Half-maximal desensitization was found with 13.4 microM GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of non-quantal release of acetylcholine in regulation of postsynaptic membrane electrogenesis.

In mammalian nerve-muscle preparations treated with an anticholinesterase, the acetylcholine (ACh) released non-quantally (NQR) reaches the postsynaptic receptors and causes a small depolarization of the membrane potential at the endplate region of the muscle fibres. Increase in quantal release potentiates the NQR and vice versa, the amplitude and the kinetic parameters of quantal miniature endplate currents (MEPCs) change during manipulation of NQR, indicating direct interaction between both types of release. Repetitive binding of ACh to postsynaptic receptors which prolongs the time course of MEPCs in anti-cholinesterase-treated endplates leads within 1-2 h to progressive desensitization in the presence of non-quantal release and to the subsequent shortening of the quantal responses. We have also investigated the effect of procedures known to modulate non-quantal acetylcholine release, on the small, but obvious, difference in the resting membrane potential between the endplate zone and other areas of the mouse muscle fibre. The resting membrane potential at the endplate zone with intact cholinesterase is more negative (by 2-4 mV) than in the endplate-free area. The experiments were performed to test the hypothesis that the hyperpolarization is caused by an electrogenic Na(+)-K+ pump operating during the action of ACh released in non-quantal form. Observations in favour of this idea are that both short-term denervation (which eliminates non-quantal but not quantal release) and ouabain abolish the local synaptic hyperpolarization and that subsequent application of low doses of ACh restores it. It follows, therefore, that the hyperpolarization is probably caused by a small but continuous ACh leakage from the nerve terminal.