Paired Turbulence and Light do not Produce a Supralinear Calcium Increase in Hermissenda

The sea slug Hermissenda learns to associate light and hair cell stimulation, but not when the stimuli are temporally uncorrelated. Memory storage, which requires an elevation in calcium, occurs in the photoreceptors, which receive monosynaptic input from hair cells that sense acceleration stimuli such as turbulence. Both light and hair cell activity increase calcium concentration in the photoreceptor, but it is unknown whether paired calcium signals combine supralinearly to initiate memory storage. A correlate of memory storage is an enhancement of the long lasting depolarization (LLD) after light offset, which is attributed to a reduction in voltage dependent potassium currents; however, it is unclear what causes the LLD in the untrained animal.These issues were addressed using a multi-compartmental computer model of phototransduction, calcium dynamics, and ionic currents of the Hermissenda photoreceptor. Simulations of the interaction between light and hair cell activity show that paired stimuli do not produce a greater calcium increase than unpaired stimuli. This suggests that hair cell activity is acting via some other pathway to initiate memory storage. In addition, simulations show that a potassium leak channel, which closes with an increase in calcium, is required to produce both the untrained LLD and the enhanced LLD due to the decrease in voltage dependent potassium currents. Thus, the expression of this correlate of classical conditioning may depend on a leak potassium current.     

D-Tubocurarine-Sensitive Component of Calcium-Dependent Potassium Current in Guinea Pig <Emphasis Type="Italic">Taenia Coli</Emphasis> Myocytes

Using a patch-clamp technique in the whole-cell configuration, we studied transmembrane ion currents in isolated single smooth muscle cells of the guinea pig taenia coli. A depolarizing step shift of the membrane potential from −50 mV was accompanied by the appearance of an outward current. Application of d-tubocurarine (d-TK) or a nonselective blocker of voltage-dependent potassium channels, tetraethylammonium (TEA), led to a decrease in the outward current. Application of d-TK against the background of the action of TEA additionally decreased the outward current. Analysis of the current-voltage (I–V) relationships of the d-TK-sensitive current showed that this current is practically voltage-independent. At the same time, an inflection of the I–V curve of the potassium current within the segment of maximum activation of the voltage-dependent potassium current is indicative of the sensitivity of this current to the intracellular Ca²⁺ concentration. Therefore, the calcium-activated potassium current through small-conductance calcium-dependent potassium channels includes a d-TK-sensitive voltage-independent component. Using depolarizing shifts of the membrane potential, we observed high- and low-amplitude spontaneous outward currents (SOCs) in many studied cells, i.e., the effect of an increase in the conductance of calcium-dependent potassium channels as a result of periodic release of Ca²⁺ from the intracellular stores. Application of d-TK led to a decrease in the frequency of low-amplitude SOCs and exerted nearly no influence on the high-amplitude SOCs under study. Neirofiziologiya/Neurophysiology, Vol. 37, No. 3, pp. 271–277, May–June, 2005.     

Normal and mutant rhodopsin activation measured with the early receptor current in a unicellular expression system.

The early receptor current (ERC) represents molecular charge movement during rhodopsin conformational dynamics. To determine whether this time-resolved assay can probe various aspects of structure-function relationships in rhodopsin, we first measured properties of expressed normal human rhodopsin with ERC recordings. These studies were conducted in single fused giant cells containing on the order of a picogram of regenerated pigment. The action spectrum of the ERC of normal human opsin regenerated with 11-cis-retinal was fit by the human rhodopsin absorbance spectrum. Successive flashes extinguished ERC signals consistent with bleaching of a rhodopsin photopigment with a normal range of photosensitivity. ERC signals followed the univariance principle since millisecond-order relaxation kinetics were independent of the wavelength of the flash stimulus. After signal extinction, dark adaptation without added 11-cis-retinal resulted in spontaneous pigment regeneration from an intracellular store of chromophore remaining from earlier loading. After the ERC was extinguished, 350-nm flashes overlapping metarhodopsin-II absorption promoted immediate recovery of ERC charge motions identified by subsequent 500-nm flashes. Small inverted R(2) signals were seen in response to some 350-nm flashes. These results indicate that the ERC can be photoregenerated from the metarhodopsin-II state. Regeneration with 9-cis-retinal permits recording of ERC signals consistent with flash activation of isorhodopsin. We initiated structure-function studies by measuring ERC signals in cells expressing the D83N and E134Q mutant human rhodopsin pigments. D83N ERCs were simplified in comparison with normal rhodopsin, while E134Q ERCs had only the early phase of charge motion. This study demonstrates that properties of normal rhodopsin can be accurately measured with the ERC assay and that a structure-function investigation of rapid activation processes in analogue and mutant visual pigments is feasible in a live unicellular environment.     

PolyADP-ribosylation is required for long-term memory formation in mammals

Recoverin is suggested to inhibit rhodopsin kinase (GRK1) at high [Ca²⁺] in the dark state of the photoreceptor cell. Decreasing [Ca²⁺] terminates inhibition and facilitates phosphorylation of illuminated rhodopsin (Rh*). When recoverin formed a complex with GRK1, it did not interfere with the phosphorylation of a C-terminal peptide of rhodopsin (S338-A348) by GRK1. Furthermore, while GRK1 competed with transducin on interaction with rhodopsin and thereby suppressed GTPase activity of transducin, recoverin in the complex with GRK1 did not influence this competition. Constructs of GRK1 that encompass its N-terminal, catalytic or C-terminal domains were used in pull-down assays and surface plasmon resonance analysis to monitor interaction. Ca²⁺-recoverin bound to the N-terminus of GRK1, but did not bind to the other constructs. GRK1 interacted with rhodopsin also by its N-terminus in a light-dependent manner. No interaction was observed with the C-terminus. We conclude that inhibition of GRK1 by recoverin is not the result of their direct competition for the same docking site on Rh*, although the interaction sites of GRK1/Rh* and GRK1/recoverin partially overlap. The N-terminus of GRK1 is recognized by Rh* leading to a conformational change which moves the C-terminus of Rh* into the catalytic kinase groove. Ca²⁺-recoverin interacting with the N-terminus of GRK1 prevents this conformational change and thus blocks Rh* phosphorylation by GRK1.     

Modulation of Burst Frequency by Calcium-Dependent Potassium Channels in the Lamprey Locomotor System: Dependence of the Activity Level

It is crucial to determine the effects on the network level of a modulation of intrinsic membrane properties. The role calcium-dependent potassium channels, K_Ca, in the lamprey locomotor system has been investigated extensively. Earlier experimental studies have shown that apamin, which affects one type of K_Ca, increases the cycle duration of the locomotor network, due to effects on the burst termination. The effects of apamin were here larger when the network had a low level of activity (burst frequency 0.5 to 1 Hz) as compared to a higher rate (>2 Hz). By using a previously developed simulation model based on the lamprey locomotor network, we show that the model could account for the frequency dependence of the apamin modulation, if only the K_Ca conductance activated by Ca²⁺ entering during the action potential was altered and not the K_Ca conductance activated by Ca²⁺ entering through NMDA channels. The present simulation model of the spinal network in the lamprey can thus account for earlier experimental results with apamin on the network and cellular level that previously appeared enigmatic.     

Role of Multiple Calcium and Calcium-Dependent Conductances in Regulation of Hippocampal Dentate Granule Cell Excitability

We have constructed a detailed model of a hippocampal dentate granule (DG) cell that includes nine different channel types. Channel densities and distributions were chosen to reproduce reported physiological responses observed in normal solution and when blockers were applied. The model was used to explore the contribution of each channel type to spiking behavior with particular emphasis on the mechanisms underlying postspike events. T-type calcium current in more distal dendrites contributed prominently to the appearance of the depolarizing after-potential, and its effect was controlled by activation of BK-type calcium-dependent potassium channels. Co-activation and interaction of N-, and/or L-type calcium and AHP currents present in somatic and proximal dendritic regions contributed to the adaptive properties of the model DG cell in response to long-lasting current injection. The model was used to predict changes in channel densities that could lead to epileptogenic burst discharges and to predict the effect of altered buffering capacity on firing behavior. We conclude that the clustered spatial distributions of calcium related channels, the presence of slow delayed rectifier potassium currents in dendrites, and calcium buffering properties, together, might explain the resistance of DG cells to the development of epileptogenic burst discharges.     

Evidence for direct roles of calcium in photosynthesis

Calcium may function directly in several aspects of photosynthesis. It appears to modulate activity of the phosphatase enzymes in the carbon reduction cycle and also to regulate chloroplast NAD⁺ kinase activity through a calmodulin-like protein. Some evidence supports a calcium function in the water-splitting complex, and other evidence indicates a reaction center function in photosystem II. Calcium in reaction center II may be tightly bound in chloroplasts and weakly bound in blue-green algal thylakoids. Free calcium concentration in stroma is probably <10^–6 M, although the absolute concentration is not yet known. Intrathylakoid calcium content is likely very high. Stromal calcium may regulate several enzyme activities, while intrathylakoid calcium may promote photosystem II constitutively. Results to date demonstrate the need for more attention to cation composition in studies of both light and dark reactions of photosynthesis, and the need to identify free calcium levels in chloroplasts.     

Evidence for a Multi-ion Pore Behavior in the Plant Potassium Channel KAT1

KAT1 is a cloned voltage-gated K⁺ channel from the plant Arabidopsis thaliana L., which displays an inward rectification reminiscent of `anomalous' rectification of the i <sub>f</sub> pacemaker current recorded in animal cells. Macroscopic conductance of KAT1 expressed in Xenopus oocytes was 5-fold less in pure Rb<sup>+</sup> solution than in pure K<sup>+</sup> solution, and negligible in pure Na<sup>+</sup> solution. Experiments in different K<sup>+</sup>/Na<sup>+</sup> or K<sup>+</sup>/Rb<sup>+</sup> mixtures revealed deviations from the principle of independence and notably two anomalous effects of the K<sup>+</sup>/Rb<sup>+</sup> mole fraction (i.e., the ratio [K<sup>+</sup>]/([K<sup>+</sup>]+[Rb<sup>+</sup>])). First, the KAT1 deactivation time constant was both voltage- and mole fraction-dependent (a so-called `foot in the door' effect was thus observed in KAT1 channel). Second, when plotted against the K⁺/Rb⁺ mole fraction, KAT1 conductance values passed through a minimum. This minimum is more important for two pore mutants of KAT1 (T259S and T260S) that displayed an increase in P_Rb/P_K. These results are consistent with the idea that KAT1 conduction requires several ions to be present simultaneously within the pore. Therefore, this atypical `green' member of the Shaker superfamily of K⁺ channels further shows itself to be an interesting model as well for permeation as for gating mechanism studies. Received: 9 February 1998/Revised: 28 July 1998     

Evidence for proton shuffling in a thioredoxin-like protein during catalysis

Proteins of the thioredoxin (Trx) superfamily catalyze disulfide-bond formation, reduction and isomerization in substrate proteins both in prokaryotic and in eukaryotic cells. All members of the Trx family with thiol-disulfide oxidoreductase activity contain the characteristic Cys-X-X-Cys motif in their active site. Here, using Poisson-Boltzmann-based protonation-state calculations based on 100-ns molecular dynamics simulations, we investigate the catalytic mechanism of DsbL, the most oxidizing Trx-like protein known to date. We observed several correlated transitions in the protonation states of the buried active-site cysteine and a neighboring lysine coupled to the exposure of the active-site thiolate. These results support the view of an internal proton shuffling mechanism during oxidation crucial for the uptake of two electrons from the substrate protein. Intramolecular disulfide-bond formation is probably steered by the conformational switch facilitating interaction with the active-site thiolate. A consistent catalytic mechanism for DsbL, probably conferrable to other proteins of the same class, is presented. Our results suggest a functional role of hydration entropy of active-site groups.     

Potassium transport in Neurospora. Evidence for a multisite carrier at high pH

At low extracellular pH (4–6), net uptake of potassium by Neurospora is a simple exponential process which obeys Michaelis kinetics as a function of [K]_o. At high pH, however, potassium uptake becomes considerably more complex, and can be resolved into two distinct exponential components. The fast component (time constant = 1.2 min) is matched quantitatively by a rapid loss of sodium; it is attributed to ion exchange within the cell wall, since it is comparatively insensitive to low temperature and metabolic inhibitors. By contrast, the slower component (time constant = 10.9 min) is inhibited markedly at 0°C and by CN and deoxycorticosterone, and is thought to represent carrier-mediated transport of potassium across the cell membrane. This transport process exhibits sigmoid kinetics as a function of [K]_o; the data can be fitted satisfactorily by two different two-site models (one involving a carrier site and a modifier site, the other an allosteric model). Either of these models could also accommodate the simple Michaelis kinetics at low pH.     

Zebrafish heart as a model for human cardiac electrophysiology

The zebrafish (Danio rerio) has become a popular model for human cardiac diseases and pharmacology including cardiac arrhythmias and its electrophysiological basis. Notably, the phenotype of zebrafish cardiac action potential is similar to the human cardiac action potential in that both have a long plateau phase. Also the major inward and outward current systems are qualitatively similar in zebrafish and human hearts. However, there are also significant differences in ionic current composition between human and zebrafish hearts, and the molecular basis and pharmacological properties of human and zebrafish cardiac ionic currents differ in several ways. Cardiac ionic currents may be produced by non-orthologous genes in zebrafish and humans, and paralogous gene products of some ion channels are expressed in the zebrafish heart. More research on molecular basis of cardiac ion channels, and regulation and drug sensitivity of the cardiac ionic currents are needed to enable rational use of the zebrafish heart as an electrophysiological model for the human heart.     

Evidence for Two Distinct Forms of Fatty Acid Cyclooxygenase in Brain

Abstract: The enzymatic metabolism of [¹⁴C]arachidonic acid (AA) was studied with microsomes prepared from rabbit medulla. Prostaglandin E₂ (PGE₂) levels, measured either by radiochemistry or radioimmunoassay, rose rapidly and abruptly plateaued within 5 min, while prostaglandin F_2a (PGF_2a) levels continued to rise for 30 min. The rapid termination of PGE2 biosynthesis was not the result of limited cofactor, substrate, or product feedback inhibition, nor was it due to PGE₂-9-ketoreductase activity. Inhibition of the PGH₂→ PGE₂ isomerase by arachidonic acid or its metabolites could not explain the abrupt halt in PGE₂ biosynthesis. Proof for two separate cyclooxygenases comes from our observation that a preincubation of the brain microsomes with unlabeled AA eliminated PGE₂ biosynthesis while PGF2o production continued. Further evidence to suggest two cyclooxygenases in brain is derived from the observation that indomethacin inhibited PGE₂ production at concentrations that did not affect PGF_2a biosynthesis. These results suggest that one fatty acid cyclooxygenase is closely associated with PGH₂→ PGE₂ isomerase and readily undergoes autodestruction and the second cyclooxygenase is associated with a PGH₂→ PGF_2a reductase and is somewhat resistant to arachidonate-induced destruction and to nonsteroidal antiinflammatory agents.     

Comorbid Externalising Behaviour in AD/HD: Evidence for a Distinct Pathological Entity in Adolescence

While the profiling of subtypes of Attention Deficit Hyperactivity Disorder (AD/HD) have been the subject of considerable scrutiny, both psychometrically and psychophysiologically, little attention has been paid to the effect of diagnoses comorbid with AD/HD on such profiles. This is despite the greater than 80% prevalence of comorbidity under the DSM-IV-TR diagnostic definitions. Here we investigate the event related potential (ERP) and psychometric profiles of Controls, AD/HD, and comorbid AD/HD (particularly AD/HD+ODD/CD) groups on six neurocognitive tasks thought to probe the constructs of selective and sustained attention, response inhibition and executive function. Data from 29 parameters extracted from a child group (age range 6 to 12; 52 Controls and 64 AD/HD) and from an adolescent group (age range 13 to 17; 79 Controls and 88 AD/HD) were reduced via a Principal Components Analysis, the 6 significant eigenvectors then used as determinants of cluster membership via a Two-Step Cluster Analysis. Two clusters were found in the analysis of the adolescent age group - a cluster dominated by Control and AD/HD participants without comorbidity, while the second cluster was dominated by AD/HD participants with externalising comorbidity (largely oppositional defiant/conduct disorder ODD/CD). A similar segregation within the child age group was not found. Further analysis of these objectively determined clusters in terms of their clinical diagnoses indicates a significant effect of ODD/CD comorbidity on a concurrent AD/HD diagnosis. We conclude that comorbid externalising behaviour in AD/HD constitutes a distinct pathological entity in adolescence.     

Distinct signal transduction pathways for GABA-induced GABA(A) receptor down-regulation and uncoupling in neuronal culture: a role for voltage-gated calcium channels

Changes in GABA receptor (GABA(A)R) gene expression are detected in animal models of epilepsy, anxiety and in post-mortem schizophrenic brain, suggesting a role for GABA(A)R regulation in neurological disorders. Persistent (48 h) exposure of brain neurons in culture to GABA results in down-regulation of GABA(A)R number and uncoupling of GABA and benzodiazepine (BZD) binding sites. Given the central role of GABA(A)Rs in fast inhibitory synaptic transmission, GABA(A)R down-regulation and uncoupling are potentially important mechanisms of regulating neuronal excitability, yet the molecular mechanisms remain unknown. In this report we show that treatment of brain neurons in culture with tetrodotoxin, glutamate receptor antagonists, or depolarization with 25 mM K(+) fails to alter GABA(A)R number or coupling. Changes in neuronal activity or membrane potential are therefore not sufficient to induce either GABA(A)R down-regulation or uncoupling. Nifedipine, a voltage-gated Ca(2+) channel (VGCC) blocker, inhibits both GABA-induced increases in [Ca(2+)](i) and GABA(A)R down-regulation, suggesting that VGCC activation is required for GABA(A)R down-regulation. Depolarization with 25 mM K(+) produces a sustained increase in intracellular [Ca(2+)] without causing GABA(A)R down-regulation, suggesting that activation of VGCCs is not sufficient to produce GABA(A)R down-regulation. In contrast to GABA(A)R down-regulation, nifedipine and 25 mM K(+) fail to inhibit GABA-induced uncoupling, demonstrating that GABA-induced GABA(A)R down-regulation and uncoupling are mediated by independent molecular events. Therefore, GABA(A)R activation initiates at least two distinct signal transduction pathways, one of which involves elevation of intracellular [Ca(2+)] through VGCCs.     

Evidence that a Cerebellum-Enriched, Synaptic Junction Glycoprotein Is Related to Fodrin and Resists Extraction with Triton in a Calcium-Dependent Manner

Abstract: Subcellular fractions from rat cerebellum and other tissues were examined for the presence of a 240K glycoprotein, designated GP-A. Previous results have shown that GP-A is enriched in cerebellum synaptic junction (SJ) fractions when compared to parent synaptic plasma membrane (SPM) fractions and is not detected in forebrain SPM or SJ fractions. In the present studies, GP-A was not detected in myelin, mitochondria, purified nuclei, or cytosolic fractions from cerebellum, but was present in microsomal fractions. GP-A is partially soluble in the non-ionic detergent Triton X-100 and is completely soluble when cerebellum SPMs are treated with the ionic detergent N-lauryl sarcosinate. The solubilization of GP-A from cerebellum membranes was shown to be a function of bound calcium ions, e.g., pretreating SPMs with 100 μM-1mM Ca²⁺ decreased the solubility of GP-A in Triton by approximately threefold. GP-A is a major concanavalin A (Con A)-binding glycoprotein in cerebellum SJ fractions and migrates on sodium dodecyl sulfate (SDS) gels with a slower relative mobility than the 235K/ 230K fodrin doublet. Comparisons between purified fodrin and the 235K/230K doublet in cerebellum and fore-brain synaptic fractions by two-dimensional peptide mapping indicated that they were identical. The Con A-binding property of GP-A was exploited to purify it by affinity chromatography with agarose-Con A. Peptide mapping comparisons between affinity-purified GP-A and GP-A in SPM and SJ fractions indicated that GP-A in synaptic fractions is apparently homogeneous. Peptide map comparisons between GP-A and 235K fodrin polypeptide indicated that these two synaptic components are highly related (50% of their respective peptides are shared). The 235K fodrin polypeptide in SJs reacted with anti-fodrin antisera on Western blots; however, GP-A failed to cross-react. These observations, together with results from previous studies, indicate that GP-A is highly enriched in cerebellum compared to other neuronal and nonneural tissues. Moreover, GP-A is enriched in SJs relative to SPM fractions, is related to fodrin, and is most likely a cell-surface glycoprotein at asymmetric synapses in cerebellum. GP-A may be involved in neuronal recognition or synaptic transmission in the cerebellum. The important role of calcium in synaptic transmission, together with the decreased solubility of GP-A in Triton that results from micromolar concentrations of calcium, suggest that GP-A may play a role in stabilizing cerebellar synaptic junctions.     

A method for determining the positions of polar hydrogens added to a protein structure that maximizes protein hydrogen bonding.

An automated method for the optimal placement of polar hydrogens in a protein structure is described. This method treats the polar, side chain hydrogens of lysine, serine, threonine, and tyrosine and the amino terminus of a protein. The program, called NETWORK, divides the potential hydrogen-bonding pairs of a protein into groups of interacting donors and acceptors. A search is conducted on each of the local groups to find an arrangement which forms the most hydrogen bonds. If two or more arrangements have the same number of hydrogen bonds, the arrangement with the shortest set of hydrogen bonds is selected. The polar hydrogens of the histidyl side chain are specifically treated, and the ionization state of this residue is allowed to change, if this change results in additional hydrogen bonds for the local group. The program will accept Protein Data Bank as well as Biosym-format coordinate files. Input and output routines can be easily modified to accept other coordinate file formats. The predictions from this method are compared to known hydrogen positions for bovine pancreatic trypsin inhibitor, insulin, RNase-A, and trypsin for which the neutron diffraction structures have been determined. The usefulness of this program is further demonstrated by a comparison of molecular dynamics simulations for the enzyme cytochrome P-450cam with and without using NETWORK.     

A mathematical model of ion movements in grey matter during a stroke

The development of cytotoxic oedema during a stroke consists in cell swelling and shrinking of the extracellular space. This phenomenon is triggered by ion movements through voltage-gated channels, exchangers and pumps. During ischaemia, sodium, calcium and chloride enter the neurons whereas potassium and glutamate are expelled out of the cells. A mathematical model is proposed to represent the long-term dynamics of membrane potentials, cell volumes and ionic concentrations in intracellular and extracellular spaces during a stroke and to study the influence of each ionic current on cell swelling. The model relies on electrophysiological mechanisms and takes into account the behaviour of two types of cells: neurons and also astrocytes known to play a key role in the excitotoxic process in grey matter. The results obtained when a severe or a moderate ischaemia is simulated are consistent with those observed in the in vitro and in vivo experiments. As this model appears to be robust, it is used to perform illustrative simulations aimed at studying the effect of some channel blockers on cell swelling. This approach may help to explore new therapeutic strategies in order to reduce stroke damage.