Relaxant effect of a novel calcium-activated potassium channel modulator on human myometrial spontaneous contractility in vitro

Aim: To investigate the effect of 4,5‐dichloro‐1,3‐diethyl‐1,3‐dihydro‐benzoimidazol‐2‐one (NS4591), a novel SK/IK channels positive modulator, on human myometrial activity. Methods: Organ bath studies were performed on myometrial preparations obtained from women undergoing elective caesarean section at term (N = 11) or hysterectomy (N = 11). NS4591 was added cumulatively in the concentration range of 0.3–30 μm . In separate experiments, the effects of pre‐incubation of muscle preparation with the SK or IK channel blockers apamin (1 μm ) and TRAM34 (10 μm ) on the outcomes of NS4591 were evaluated. Simultaneous vehicle controls were performed for all experiments. The effects of drugs were studied on spontaneous contractions. Results: NS4591 exerted an inhibitory effect on myometrial contractions in muscle strips from non‐pregnant and pregnant women. The contractility in non‐pregnant and pregnant myometrium was reduced to the following values respectively: amplitude 20.65 ± 7.38% (P < 0.001) and 42.85 ± 11.04% (P < 0.05) and area under the curve 11.72 ± 7.39% (P < 0.001) and 34.84 ± 10.50% (P < 0.001) and are reflective of 30 μm NS4591 compared to vehicle control. In non‐pregnant tissue, apamin partially reduced the inhibitory effects of NS4591, but we observed relaxation mediated by NS4591 despite pre‐incubation with TRAM34. In contrast, in pregnant tissue, neither apamin nor TRAM34 could reverse the relaxatory effects of NS4591. Conclusion: Our findings imply that SK/IK channels are present and functional in myometrium from pregnant and non‐pregnant women. The SK/IK channel‐positive modulator NS4591 exerts relaxation of human myometrium in vitro, and this may have implications for the clinical management of preterm labour.     

Modeling hippocampal neurogenesis across the lifespan in seven species

The aim of this study was to estimate the number of new cells and neurons added to the dentate gyrus across the lifespan, and to compare the rate of age-associated decline in neurogenesis across species. Data from mice (Mus musculus), rats (Rattus norvegicus), lesser hedgehog tenrecs (Echinops telfairi), macaques (Macaca mulatta), marmosets (Callithrix jacchus), tree shrews (Tupaia belangeri), and humans (Homo sapiens) were extracted from 21 data sets published in 14 different reports. Analysis of variance (ANOVA), exponential, Weibull, and power models were fit to the data to determine which best described the relationship between age and neurogenesis. Exponential models provided a suitable fit and were used to estimate the relevant parameters. The rate of decrease of neurogenesis correlated with species longevity (r = 0.769, p = 0.043), but not body mass or basal metabolic rate. Of all the cells added postnatally to the mouse dentate gyrus, only 8.5% (95% confidence interval [CI], 1.0% to 14.7%) of these will be added after middle age. In addition, only 5.7% (95% CI 0.7% to 9.9%) of the existing cell population turns over from middle age and onward. Thus, relatively few new cells are added for much of an animal's life, and only a proportion of these will mature into functional neurons.     

Functional interaction of Junctophilin 2 with small‐ conductance Ca2+‐activated potassium channel subtype 2(SK2) in mouse cardiac myocytes

Aim

Junctophilins (JPs), a protein family of the junctional membrane complex, maintain the close conjunction between cell surface and intracellular membranes in striate muscle cells mediating the crosstalk between extracellular Ca²⁺ entry and intracellular Ca²⁺ release. The small‐conductance Ca²⁺‐activated K⁺ channels are activated by the intracellular calcium and play an essential role in the cardiac action potential profile. Molecular mechanisms of regulation of the SK channels are still uncertain. Here, we sought to determine whether there is a functional interaction of junctophilin type 2 (JP2) with the SK channels and whether JP2 gene silencing might modulate the function of SK channels in cardiac myocytes.

Methods

Association of JP2 with SK2 channel in mouse heart tissue as well as HEK293 cells was studied using in vivo and in vitro approaches. siRNA knockdown of JP2 gene was assessed by real‐time PCR. The expression of proteins was analysed by Western blotting. Ca²⁺‐activated K⁺ current (I_K,Ca) in infected adult mouse cardiac myocytes was recorded using whole‐cell voltage‐clamp technique. The intracellular Ca²⁺ transient was measured using an IonOptix photometry system.

Results

We showed for the first time that JP2 associates with the SK2 channel in native cardiac tissue. JP2, via the membrane occupation and recognition nexus (MORN motifs) in its N‐terminus, directly interacted with SK2 channels. A colocalization of the SK2 channel with its interaction protein of JP2 was found in the cardiac myocytes. Moreover, we demonstrated that JP2 is necessary for the proper cell surface expression of the SK2 channel in HEK293. Functional experiments indicated that knockdown of JP2 caused a significant decrease in the density of I_K,Ca and reduced the amplitude of the Ca²⁺ transient in infected cardiomyocytes.

Conclusion

The present data provide evidence that the functional interaction between JP2 and SK2 channels is present in the native mouse heart tissue. Junctophilin 2, as junctional membrane complex (JMC) protein, is an important regulator of the cardiac SK channels.     

Gating of the shaker potassium channel is modulated differentially by N-glycosylation and sialic acids

N-linked glycans, including sialic acids, are integral components of ion channel complexes. To determine if N-linked sugars can modulate a rapidly inactivating K⁺ channel, the glycosylated Drosophila melanogaster Shaker K⁺ channel (ShB) and the N-glycosylation-deficient mutant (ShNQ), were studied under conditions of full and reduced sialylation. Through an apparent electrostatic mechanism, full sialylation induced uniform and significant hyperpolarizing shifts in all measured voltage-dependent ShB gating parameters compared to those measured under conditions of reduced sialylation. Steady-state gating of ShNQ was unaffected by changes in sialylation and was nearly identical to that observed for ShB under conditions of reduced sialylation, indicating that N-linked sialic acids were wholly responsible for the observed effects of sialic acid on ShB gating. Interestingly, the rates of transition among channel states and the voltage-independent rates of activation and inactivation were significantly slower for ShNQ compared to ShB. Both effects were independent of sialylation, indicating that N-linked sugars other than sialic acids alter ShB gating kinetics but have little to no effect on the steady-state distribution of channels among states. The effect of sialic acids on channel gating, particularly inactivation gating, and the impact of other N-linked sugars on channel gating kinetics are unique to the ShB isoform. Thus, ShB gating is modulated by two complementary but distinct sugar-dependent mechanisms, (1) an N-linked sialic acid-dependent surface charge effect and (2) a sialic acid-independent effect that is consistent with N-linked sugars affecting the stability of ShB among its functional states.     

Sodium and potassium channels in epithelial cells from thymus glands and thymomas of myasthenia gravis patients

In both normal and neoplastic epithelial cells from human thymus glands and thymomas, respectively, we found voltage-gated sodium and potassium channels that resemble the adult-type Na channel and the delayed outward rectifier K channel, respectively, of human skeletal muscle and mammalian nervous system. These voltage-gated ion channels might be part of a communication system between epithelial cells and other components of the microenvironment of the thymus.     

Visualization of sequential conversion of human intermediately reprogrammed stem cells into iPS cells

Analysis of gene expression in single cells is required to understand somatic cell reprogramming into human induced pluripotent stem cells (iPSCs). To facilitate this, we established intermediately reprogrammed stem cells (iRSCs), pre‐iPSC lines. The iRSC‐iPSC conversion system enables the reproducible monitoring of reprogramming events and the analysis of progressive gene expression profiles using single‐cell microarray analysis and genome editing. Here, single‐cell microarray analysis showed the stage‐specific sequential gene activation during the conversion of iRSCs into iPSCs, using OCT4, TDGF1 and E‐CADHERIN as marker genes. Out of 75 OCT4‐related genes, which were significantly up‐regulated after the activation of OCT4, and entry into the mesenchymal‐to‐epithelial transition (MET), LIN28 (LIN28A) and FOXO1 were selected for applying to gene expression visualization. Multicolored visualization was achieved by the genome editing of LIN28 or FOXO1 with mCherry into OCT4‐GFP iRSCs. Fluorescent analysis of gene activity in individual cells showed that OCT4 was dispensable for maintenance, but required for activation, of the LIN28 and FOXO1 expression in reprogramming.     

In vitro chondrogenesis and in vivo repair of osteochondral defect with human induced pluripotent stem cells

The purpose of this study was to investigate the chondrogenic features of human induced pluripotent stem cells (hiPSCs) and examine the differences in the chondrogenesis between hiPSCs and human bone marrow-derived MSCs (hBMMSCs). Embryoid bodies (EBs) were formed from undifferentiated hiPSCs. After EBs were dissociated into single cells, chondrogenic culture was performed in pellets and alginate hydrogel. Chondro-induced hiPSCs were implanted in osteochondral defects created on the patellar groove of immunosuppressed rats and evaluated after 12 weeks. The ESC markers NANOG, SSEA4 and OCT3/4 disappeared while the mesodermal marker BMP-4 appeared in chondro-induced hiPSCs. After 21 days of culture, greater glycosaminoglycan contents and better chondrocytic features including lacuna and abundant matrix formation were observed from chondro-induced hiPSCs compared to chondro-induced hBMMSCs. The expression of chondrogenic markers including SOX-9, type II collagen, and aggrecan in chondro-induced hiPSCs was comparable to or greater than chondro-induced hBMMSCs. A remarkably low level of hypertrophic and osteogenic markers including type X collagen, type I collagen and Runx-2 was noted in chondro-induced hiPSCs compared to chondro-induced hBMMSCs. hiPSCs had significantly greater methylation of several CpG sites in COL10A1 promoter than hBMMSCs in either undifferentiated or chondro-induced state, suggesting an epigenetic cause of the difference in hypertrophy. The defects implanted with chondro-induced hiPSCs showed a significantly better quality of cartilage repair than the control defects, and the majority of cells in the regenerated cartilage consisted of implanted hiPSCs.     

Transient expression of FOXP3 in human activated nonregulatory CD4+ T cells

Foxp3 plays a key role in CD4+ CD25+ T(reg) cell function in mice and represents a specific marker for these cells. Despite the strong association between FOXP3 expression and regulatory function in fresh human T cells, little is known about the dynamics of endogenous FOXP3 expression and its relation to the suppressive function in activated human T cells. Here, we addressed the dynamics of FOXP3 expression during human CD4+ T cell activation by plate-bound anti-CD3 Ab as well as the relationship between its expression and regulatory function at the single-cell level. Our data show that FOXP3 is expressed in a high percentage of activated T cells after in vitro stimulation of human CD4+ CD25- cells. FOXP3 expression is strongly associated with hyporesponsiveness of activated T cells, but is not directly correlated with their suppressive capabilities, as we demonstrate that it is also expressed in activated nonsuppressive T cells. However, in this nonsuppressive T cell population, FOXP3 expression is transient, while it is stably expressed in activated T cells that do display suppressive function, and in natural CD4+ CD25++ T(reg) cells. These data indicate that expression of endogenous FOXP3, in humans, is not sufficient to induce regulatory T cell activity or to identify T(reg) cells.     

Endothelial progenitor cells from human dental pulp-derived iPS cells as a therapeutic target for ischemic vascular diseases

Human dental pulp cells (hDPCs) are a valuable source for the generation of patient-specific human induced pluripotent stem cells (hiPSCs). An advanced strategy for the safe and efficient reprogramming of hDPCs and subsequent lineage-specific differentiation is a critical step toward clinical application. In present research, we successfully generated hDPC-iPSCs using only two non-oncogenic factors: Oct4 and Sox2 (2F hDPC-hiPSCs) and evaluated the feasibility of hDPC-iPSCs as substrates for endothelial progenitor cells (EPCs), contributing to EPC-based therapies. Under conventional differentiation conditions, 2F hDPC-hiPSCs showed higher differentiation efficiency, compared to hiPSCs from other cell types, into multipotent CD34⁺ EPCs (2F-hEPCs) capable to differentiate into functional endothelial and smooth muscle cells. The angiogenic and neovasculogenic activities of 2F-hEPCs were confirmed using a Matrigel plug assay in mice. In addition, the therapeutic effects of 2F-hEPC transplantation were confirmed in mouse models of hind-limb ischemia and myocardial infarction. Importantly, 2F-EPCs effectively integrated into newly formed vascular structures and enhanced neovascularization via likely both direct and indirect paracrine mechanisms. 2F hDPC-hiPSCs have a robust capability for the generation of angiogenic and vasculogenic EPCs, representing a strategy for patient-specific EPC therapies and disease modeling, particularly for ischemic vascular diseases.     

活性氧对豚鼠心室肌细胞不同钾通道离子电流的影响

目的：揭示年头自由基导致跨膜电位变化的离子电流基础。方法：采用全细胞膜片钳技术,观察了Ｈ２Ｏ２（１ｍｍｏｌ／Ｌ）对豚鼠单个心室肌细胞不同钾通道影响。结果：Ｈ２Ｏ２使习心肌细胞静息电位（ＲＰ）降低,动作电位时程（ＡＰＤ）显著缩短的同时,明显抑制超极化时的内向整流钾电流（Ｉｋ１）,致（Ｉｋ１）正常呈Ｎ字型Ｉ－Ｖ曲 成一条斜线;增强延迟整流钾电流（ＩＫ）的快组分（ＩＫＲ）和慢组分（ＩＫＡ）,尤其ＩＫＲ２     

Hyperpolarization‐activated cation and T‐type calcium ion channel expression in porcine and human renal pacemaker tissues

Renal pacemaker activity triggers peristaltic upper urinary tract contractions that propel waste from the kidney to the bladder, a process prone to congenital defects that are the leading cause of pediatric kidney failure. Recently, studies have discovered that hyperpolarization‐activated cation (HCN) and T‐type calcium (TTC) channel conductances underlie murine renal pacemaker activity, setting the origin and frequency and coordinating upper urinary tract peristalsis. Here, we determined whether this ion channel expression is conserved in the porcine and human urinary tracts, which share a distinct multicalyceal anatomy with multiple pacemaker sites. Double chromagenic immunohistochemistry revealed that HCN isoform 3 is highly expressed at the porcine minor calyces, the renal pacemaker tissues, whereas the kidney and urinary tract smooth muscle lacked this HCN expression. Immunofluorescent staining demonstrated that HCN⁺ cells are integrated within the porcine calyx smooth muscle, and that they co‐express TTC channel isoform Cav3.2. In humans, the anatomic structure of the minor calyx pacemaker was assayed via hematoxylin and eosin analyses, and enabled the visualization of the calyx smooth muscle surrounding adjacent papillae. Strikingly, immunofluorescence revealed that HCN3⁺/Cav3.2⁺ cells are also localized to the human minor calyx smooth muscle. Collectively, these data have elucidated a conserved molecular signature of HCN and TTC channel expression in porcine and human calyx pacemaker tissues. These findings provide evidence for the mechanisms that can drive renal pacemaker activity in the multi‐calyceal urinary tract, and potential causes of obstructive uropathies.     

大鼠MSCs诱导分化为心肌样细胞时离子通道基因的表达

目的研究大鼠骨髓间充质干细胞(MSCs)体外向心肌样细胞分化过程中L-型钙通道(α1C)和瞬间外向钾通道(kv4.3)基因的表达。方法无菌条件下冲洗Wistar大鼠双侧股骨和胫骨的骨髓腔获得MSCs,体外培养传代纯化,5-氮杂胞苷(5-aza)诱导24 h,显微镜下观察诱导前后形态变化,RT-PCR鉴定α1C和kv4.3基因的表达。结果kv4.3基因诱导前有弱的表达,诱导后1、4、7和14 d表达明显逐渐增强(P<0.05);α1C基因诱导前有较强的表达,诱导后1、4、7和14 d表达却明显逐渐减弱(P<0.05)。结论α1C和kv4.3基因在维持MSCs的诱导分化过程中发挥重要作用。     

丹参素对猪冠脉平滑肌细胞钙激活钾通道的作用

目的观察丹参素(DS-182)对原代培养猪冠脉平滑肌细胞钙激活钾通道(KCa)的作用,从单个钾通道水平揭示其扩冠机制。方法采用膜片钳单通道电流记录技术。结果①猪冠脉平滑肌细胞KCa的单通道电导值高,为(291.74±4.89)pS;对膜电位变化及细胞内钙离子浓度变化敏感,能被5~20 mmol/L膜内侧四乙基铵(TEA)所阻断。②在细胞贴附式膜片方式下,细胞外应用10~20μmol/L的DS-182对通道具明显的激活效应;而在内面向外式膜片方式下,细胞内应用5~30μmol/L的DS-182对通道均无明显作用。结论DS-182对通道的激活作用是非直接的,需要一系列的胞内过程。     

A potassium ion channel is involved in cytokine production by activated human macrophages

Antibodies are powerful immunotherapeutic agents but their use for treating ocular disorders is limited by their poor penetration into the eye. We hypothesized that antibody fragments of relatively small size might penetrate the cornea more readily. Monovalent single chain variable region (scFv) antibody fragments and divalent miniantibodies were engineered from existing monoclonal antibodies, expressed in a bacterial expression system, and purified by metal ion affinity chromatography. Corneoscleral preparations from normal pig and cat eyes were mounted in a corneal perfusion chamber. Intact antibodies and antibody fragments were applied topically to the anterior corneal surface over 12-h periods, and samples were collected from the artificial anterior chamber. Similar experiments were performed with whole enucleated pig and human eyes. Penetration of antibodies and fragments was quantified by high-sensitivity flow cytometry on appropriate target cells. Both monovalent scFv and divalent miniantibody fragments (but not whole immunoglobulin molecules) passed through de-epithelialized and intact corneas after topical administration, and could be detected by antigen binding. Addition of 0.5% sodium caprate facilitated penetration through intact corneas. Topically-applied scFv was found to penetrate into the anterior chamber fluid of rabbit eyes in vivo. The engineered fragments were stable and resistant to ocular proteases. Monovalent and divalent antibody constructs of molecular weight 28 kD and 67 kD, respectively, can penetrate through intact corneas into the anterior chamber, with retention of appropriate antigen-binding activity. Such constructs may form novel therapeutic agents for topical ophthalmic use.     

s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP⁺ cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP⁺ cells can regenerate the mammary epithelial network. GFP⁺ mammary epithelial cells are p63⁺, CD24^mod, CD49f^high, and CD29^high; are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP⁺ basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells.     

Properties of a potassium-selective ion channel in human melanoma cells

Currents through ion channels were measured from cells of a human melanin-producing melanoma cell line (IRG 1) with the patch clamp technique. In these cells the most frequently observed channel is a potassium channel. The channel activates slowly at depolarizing voltage steps but does not inactivate. Single channel potassium currents can be measured in cell-attached patches at the resting potential of melanoma cells. The channel has a conductance of approximately 10 pS. As measured from the reversal potentials of single channel currents, the permeability ratio for sodium and potassium, P _Na/P _K, is between 0.03 and 0.04. Open probability is increased at positive potentials. Mean open times are prolonged at voltage steps to more positive potentials. Closed time histograms are fitted by two exponentials. The slow shut time is decreased at positive potentials. In whole cell measurements, cell conductance measured between –20 and +70 mV was reduced by 10 mM tetraethylammonium chloride from 6.4±1.2 nS (n=4) to 0.8±0.3 nS (n=3). Application of isoproterenol decreases the probability of the channel being open without any change in the single channel conductance. A possible role of the 10 pS potassium channel in the growth of melanoma cells is discussed.     

Human CD4+ T cells stimulated by conserved adenovirus 5 hexon peptides recognize cells infected with different species of human adenovirus

The immune response against human adenovirus (HAdV) has gained interest because of the application of HAdV-based vectors in gene therapy and the high incidence of infections in pediatric recipients of allogeneic stem cell grafts. Because antiviral medication is frequently ineffective, the option of adoptive transfer of HAdV-specific donor-derived T cells in these immunocompromised patients is investigated. To generate good manufacturing practice-compatible reagents, a panel of 63 long, overlapping, peptides of the hexon protein was screened for recognition by T cells. Five conserved peptides of 30 amino acids were identified that were recognized by the majority of adult donors. CD4+ T cells from long-term cultures of PBMC, stimulated with this set of five peptides, recognized cells infected with HAdV serotypes belonging to different species. These data demonstrate that adult human T cells preferentially recognize conserved sequences of amino acid residues from a structural protein of HAdV. In the context of gene therapy, this observation may limit the beneficial effect of switching to HAdV-based vectors derived from less common serotypes of HAdV in an attempt to circumvent pre-existing immunity. However, this cross-reactivity benefits the application of HAdV-specific T cells for adoptive immunotherapy in immunocompromised transplant recipients.     

小鼠iPS细胞体外自发性分化及神经定向分化中基因表达谱的研究

目的:研究小鼠诱导多能干细胞(i PS细胞)在体外自发性分化和神经定向分化过程中的基因表达谱的特性。方法:在体外将i PS细胞分别进行形成类胚胎小体后自发性分化及成骨蛋白抑制剂Noggin诱导神经定向分化后,通过实时荧光定量PCR(q PCR)测定在分化过程中i PS细胞分化相关基因表达的变化。结果:i PS细胞形成类胚胎小体后胚胎外胚层标志基因(GFAP,Map2和Tu J1)及内胚层标记基因(Foxa2,GATA4和Sox17)的表达随分化而迅速增加,但中胚层标记基因(Bmp4,BRA,FGF5)表达水平变化不明显。在神经定向分化过程中,i PS细胞的神经标志物基因(Map2,Neu N,Tu J1和Sox1)及线粒体相关基因(12s,ND3,ND5,ND6、Cytb和Cox1)的表达都逐渐增加,且两者具有相同的增长趋势。结论:在自发性分化过程中小鼠i PS细胞具有自发向外胚层和内胚层分化的趋势;在神经定向分化过程中,i PS细胞的线粒体相关基因表达随着分化表达逐渐增加,并与神经细胞标记基因具有正相关性,表明线粒体的功能在i PS细胞神经定向分化中发挥重要作用。     

Calcium dependent gating of thel-glutamate activated,excitatory synaptic channel on crayfish muscle

Excitatory, glutamate-activated single channel currents were measured in outside-out patches of crayfish muscle. The open time of single channel openings, and the durations and rates of bursts were evaluated. These kinetic parameters were not appreciably affected by replacement of extracellular Na⁺ by Li⁺ or choline. Changes in extracellular Ca²⁺ concentration Ca_o also did not influence the duration of single openings. However the mean burst duration decreased for Ca_o<13.5 mM and the rate of bursts declined with a power of almost 2 in low Ca_o. At Ca_o<1 mM practically no channel openings were observed in presence of glutamate. In order to exclude more rapid desensitization of the glutamate receptors in low Ca_o as the cause of disappearance of channel openings, glutamate was applied in short pulses with a liquid-filament switch. In 0 Ca_o also a glutamate pulse did not trigger channel openings. In presence of 13.5 mM Ca_o, the inorganic Ca-channel blockers La³⁺ and Cd²⁺ diminished the duration and rate of bursts of channel openings in a similar manner as low Ca_o. The effects of low Ca_o and of Cd²⁺ were tested also on quantal postsynaptic currents, EPSCs, which were recorded through a perfused macro-patch-clamp electrode. At 1.4 mM Ca_o in the perfused electrode tip, spontaneous EPSCs were reduced at least by a factor of 4, and elicited EPSCs by a factor of 16. Application of Cd²⁺ had similarly strong effects on the EPSCs. Also the decay of EPSCs was shortened substantially in 1.4 mM Ca_o or 5 mM Cd²⁺.The inhibitory Cl^–-channel of crayfish muscle, activated by glutamate or GABA, also was studied in outside-out patches. The openings of this channel persisted in 0 Ca_o solutions; the block of channel openings in low Ca_o thus is a specific property of the excitatory channel. The action of Ca_o on the excitatory channel may be described as that of a cofactor to glutamate. A possible reaction scheme is proposed.Supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 220