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PubMed1998

Three residues predicted by molecular modeling to interact with the purine moiety alter ligand binding and channel gating in cyclic nucleotide-gated channels.

Cytoplasmic cAMP and cGMP are soluble cellular messengers that directly activate cyclic nucleotide-gated (CNG) channels. These channels mediate sensory transduction in photoreceptors and olfactory neurons. The closely related CNG channels in these cell types have different nucleotide activation profiles, and we have investigated the molecular basis of their nucleotide selectivity properties. Previously, we predicted that the purine moiety of the nucleotide interacts with residues F533, K596, and D604 (bovine rod alpha CNG channel subunit sequences) of the nucleotide binding domain. In this study, we replaced these three residues with the corresponding residues of the bovine olfactory CNG channel. Mutations at each position altered the nucleotide activation of the rod CNG channels. In a mutant where K596 was replaced with arginine, cAMP-activated currents were enhanced 8-12-fold, suggesting that residue 596 influences channel gating. Thermodynamic cycle analysis of the data showed that (1) the residues are energetically coupled and (2) energetic coupling exists between the potentiating effects of Ni2+ and the replacement of F533 with tyrosine. These data suggest that changes in one of the residues alter the purine contacts with the other residues and that F533 communicates with the C-linker region of the channel involved in Ni2+ potentiation.

Amino Acid Substitution↗

PubMed2003

Inhibition of Caenorhabditis elegans social feeding by FMRFamide-related peptide activation of NPR-1.

Social and solitary feeding in natural Caenorhabditis elegans isolates are associated with two alleles of the orphan G-protein-coupled receptor (GPCR) NPR-1: social feeders contain NPR-1 215F, whereas solitary feeders contain NPR-1 215V. Here we identify FMRFamide-related neuropeptides (FaRPs) encoded by the flp-18 and flp-21 genes as NPR-1 ligands and show that these peptides can differentially activate the NPR-1 215F and NPR-1 215V receptors. Multicopy overexpression of flp-21 transformed wild social animals into solitary feeders. Conversely, a flp-21 deletion partially phenocopied the npr-1(null) phenotype, which is consistent with NPR-1 activation by FLP-21 in vivo but also implicates other ligands for NPR-1. Phylogenetic studies indicate that the dominant npr-1 215V allele likely arose from an ancestral npr-1 215F gene in C. elegans. Our data suggest a model in which solitary feeding evolved in an ancestral social strain of C. elegans by a gain-of-function mutation that modified the response of NPR-1 to FLP-18 and FLP-21 ligands.

Action Potentials↗

PubMed1997

Long-term potentiation of glial synaptic currents in cerebellar culture.

Glial cells in the brain express neurotransmitter receptors and can respond appropriately to application of exogenous neurotransmitters such as glutamate. However, activation of receptors by endogenous, synaptically released transmitter has been difficult to demonstrate directly. Using cell-pair recording in cerebellar cultures from embryonic mouse, it is shown that activation of a cerebellar granule neuron can give rise to a rapid inward current in an adjacent glial cell. This current is mediated by activation of Ca2+-permeable AMPA/kainate receptors and is largely independent of glutamate reuptake or gap junctional coupling. Furthermore, prolonged stimulation of the granule neuron at 4 Hz can give rise to long-term potentiation (LTP) of the glial synaptic current that has similar properties to LTP of granule neuron-Purkinje neuron synaptic transmission--its induction is independent of postsynaptic depolarization, postsynaptic Ca2+ influx, or glutamate receptor activation but requires presynaptic Ca2+ influx. These findings suggest a model in which cerebellar LTP is both induced and expressed presynaptically and therefore may be detected by either neuronal or glial postsynaptic cells.

Animals↗

PubMed1995

A molecular model of low-voltage-activated calcium conductance.

1. T-type or low-voltage-activated Ca2+ channels (IT) expressed in the mammalian thalamus play an important physiological role in the induction of membrane potential oscillations and in the regulation of the timing of synaptic signaling. On the basis of recent molecular studies on Ca2+, Na+, and K+ channels, a structure-kinetic model has been proposed for IT. 2. The model considers that the pore-opening process of IT is governed by three of the four protein domains that are arranged asymmetrically across the membrane plane. The three cytoplasmic linkers connecting individual channel domains serve as inactivation gates. On a membrane voltage change, each domain may engage independently into a sequential, thermodynamically coupled conformational change, thereby causing channel activation and inactivation. 3. A linear Marcov chain reaction with coupled channel activation and inactivation was adopted to test the kinetic feasibility of this trimeric model in a single-compartmental thalamic cell. The differential equations for voltage-dependent rate constants and transitional rates were solved to produce macroscopic T-type Ca2+ current. 4. Our simulation results indicate that this novel structure-kinetic model produces excellent predictions of the macroscopic behaviors of IT, and, more importantly, it provides some new insights into the microscopic mechanism underlying channel recovery.

Animals↗

PubMed2001

Functional properties of Cav1.3 (alpha1D) L-type Ca2+ channel splice variants expressed by rat brain and neuroendocrine GH3 cells.

Ca(2+) enters pituitary and pancreatic neuroendocrine cells through dihydropyridine-sensitive channels triggering hormone release. Inhibitory metabotropic receptors reduce Ca(2+) entry through activation of pertussis toxin-sensitive G proteins leading to activation of K(+) channels and voltage-sensitive inhibition of L-type channel activity. Despite the cloning and functional expression of several Ca(2+) channels, those involved in regulating hormone release remain unknown. Using reverse transcription-polymerase chain reaction we identified mRNAs encoding three alpha(1) (alpha(1A), alpha(1C), and alpha(1D)), four beta, and one alpha(2)-delta subunit in rat pituitary GH(3) cells; alpha(1B) and alpha(1S) transcripts were absent. GH(3) cells express multiple alternatively spliced alpha(1D) mRNAs. Many of the alpha(1D) transcript variants encode "short" alpha(1D) (alpha(1D-S)) subunits, which have a QXXER amino acid sequence at their C termini, a motif found in all other alpha(1) subunits that couple to opioid receptors. The other splice variants identified terminate with a longer C terminus that lacks the QXXER motif (alpha(1D-L)). We cloned and expressed the predominant alpha(1D-S) transcript variants in rat brain and GH(3) cells and their alpha(lD-L) counterpart in GH(3) cells. Unlike alpha(1A) channels, alpha(1D) channels exhibited current-voltage relationships similar to those of native GH(3) cell Ca(2+) channels, but lacked voltage-dependent G protein coupling. Our data demonstrate that alternatively spliced alpha(1D) transcripts form functional Ca(2+) channels that exhibit voltage-dependent, G protein-independent facilitation. Furthermore, the QXXER motif, located on the C terminus of alpha(1D-S) subunit, is not sufficient to confer sensitivity to inhibitory G proteins.

Alternative Splicing↗

PubMed2002

Modulation of acetylcholine receptor channel kinetics by hydrocortisone.

The kinetics of the nicotinic acetylcholine receptor (AChR) channel were analysed in the presence of hydrocortisone (HC, 100-400 microM), an electrically neutral steroid. The channel open time decreased, and in contrast to control conditions did not show any voltage dependency. However, HC induced a new (blocked) component in the closed time distribution, with a time constant that decreased with membrane hyperpolarization. HC decreased also, in a concentration-dependent way, the open time per burst. After coupling HC to bovine serum albumin, to restrict the place of steroid action at the external surface of the membrane, a voltage dependency of steroid action persisted. The effects of HC on the open and blocked time constants did not depend on agonist concentration, but was dependent on the type of agonist used (acetylcholine or nicotine). These results support the hypothesis that HC molecules bind near the agonist binding site.

Acetylcholine↗

PubMed1991

Induction of K-channel expression in a neuroblastoma cell line.

Whole-cell currents were examined in mouse neuroblastoma cells of the N2AB-1 line. In standard culture medium, N2AB-1 cells exhibited large voltage-dependent Na currents but no discernible K currents. Treatment of N2AB-1 cells with either dimethylsulfoxide (DMSO) in low-serum medium or with retinoic acid (RA) caused the expression of delayed rectifier K currents. Currents from two types of K channel with single channel slope conductances of 15.0 pS and 6.4 pS were observed in outside-out patches from cells of both treatment groups. Thus, while N2AB-1 cells did not exhibit K currents under standard culture conditions, they did possess the gene(s) encoding K channels. The treatments caused other changes that were not directly linked to K-channel expression. RA treatment caused neurite extension in most, but not all, N2AB-1 cells; however, all RA-treated cells, including those without neurites, expressed K currents. RA treatment did not suppress cell division or cause hypertrophy. In contrast, treatment with DMSO/low serum suppressed cell division and caused cellular hypertrophy, but did not cause long neurites to form. Thus, the regulation of K channels was not coupled in a simple fashion to properties that have been associated with a differentiated neuronal phenotype: neurite elaboration, changes in cell size, and inhibition of cell division. These results suggest that N2AB-1 cells may be a good model system for investigating the processes regulating K-channel expression.

4-Aminopyridine↗

PubMed1999

Functional characterization of the Betaine/gamma-aminobutyric acid transporter BGT-1 expressed in Xenopus oocytes.

Betaine is an osmolyte accumulated in cells during osmotic cell shrinkage. The canine transporter mediating cellular accumulation of the osmolyte betaine and the neurotransmitter gamma-aminobutyric acid (BGT-1) was expressed in Xenopus oocytes and analyzed by two-electrode voltage clamp and tracer flux studies. Exposure of oocytes expressing BGT-1 to betaine or gamma-aminobutyric acid (GABA) depolarized the cell membrane in the current clamp mode and induced an inward current under voltage clamp conditions. At 1 mM substrate the induced currents decreased in the following order: betaine = GABA > diaminobutyric acid = beta-alanine > proline = quinidine > dimethylglycine > glycine > sarcosine. Both the Vmax and Km of GABA- and betaine-induced currents were voltage-dependent, and GABA- and betaine-induced currents and radioactive tracer uptake were strictly Na+-dependent but only partially dependent on the presence of Cl-. The apparent affinity of GABA decreased with decreasing Na+ concentrations. The Km of Na+ also depended on the GABA and Cl- concentration. A decrease of the Cl- concentration reduced the apparent affinity for Na+ and GABA, and a decrease of the Na+ concentration reduced the apparent affinity for Cl- and GABA. A comparison of 22Na+-, 36Cl--, and 14C-labeled GABA and 14C-labeled betaine fluxes and GABA- and betaine-induced currents yielded a coupling ratio of Na+/Cl-/organic substrate of 3:1:1 or 3:2:1. Based on the data, a transport model of ordered binding is proposed in which GABA binds first, Na+ second, and Cl- third. In conclusion, BGT-1 displays significant functional differences from the other members of the GABA transporter family.

Animals↗

PubMed2006

Spike-timing-dependent plasticity of inhibitory synapses in the entorhinal cortex.

Actions of inhibitory interneurons organize and modulate many neuronal processes, yet the mechanisms and consequences of plasticity of inhibitory synapses remain poorly understood. We report on spike-timing-dependent plasticity of inhibitory synapses in the entorhinal cortex. After pairing presynaptic stimulations at time t(pre) with evoked postsynaptic spikes at time t(post) under pharmacological blockade of excitation we found, via whole cell recordings, an asymmetrical timing rule for plasticity of the remaining inhibitory responses. Strength of response varied as a function of the time interval Deltat = t(post) - t(pre): for Deltat > 0 inhibitory responses potentiated, peaking at a delay of 10 ms. For Deltat < 0, the synaptic coupling depressed, again with a maximal effect near 10 ms of delay. We also show that changes in synaptic strength depend on changes in intracellular calcium concentrations and demonstrate that the calcium enters the postsynaptic cell through voltage-gated channels. Using network models, we demonstrate how this novel form of plasticity can sculpt network behavior efficiently and with remarkable flexibility.

Animals↗

PubMed2001

The kinetics of inhibition of nicotinic acetylcholine receptors by (+)-tubocurarine and pancuronium.

Equilibrium conditions of neurotransmitter concentration and receptor binding are never achieved during synaptic transmission at the neuromuscular junction. Thus, it is important to determine the binding kinetics of drugs that act this synapse. Previous determinations of the dissociation rate of (+)-tubocurarine have produced inconsistent results ranging from 0.1 to 4000/s. Here, we used a direct approach to measure association (l(on)) and dissociation (l(on)) rates for two competitive antagonists (clinically used as nondepolarizing muscle relaxants), pancuronium and (+)-tubocurarine, at nicotinic acetylcholine receptors (nAChR). We made macroscopic current recordings from outside-out patches of BC3H-1 cells expressing embryonic mouse muscle nAChR. We used a three-tube rapid perfusion system to make timed applications of antagonists and acetylcholine to the patch. We made independent measurements of the equilibrium inhibition (IC(50)) and the kinetics of onset and recovery of antagonist inhibition at 20 to 23 degrees C. Rate constants were calculated from the predictions of a single (high-affinity) site model of competitive inhibition. For pancuronium: IC(50) = 5.5 +/- 0.5 nM (mean +/- S.D.), l(on) = 2.7 +/- 0.9 x 10(8) M(-1) s(-1), l(off) = 2.1 +/- 0.7/s [corrected] x 10(8)/s. For (+)-tubocurarine: IC(50) = 41 +/- 2 nM, l(on) = 1.2 +/- 0.2 x 10(8) M(-1) s(-1), l(off) = 5.9 +/- 1.3/s. The kinetic results are consistent with the equilibrium results in that l(off)/l(on) is in good agreement with the IC(50) values. All differences between the antagonists are significant at the p < 0.001 level. The higher affinity of pancuronium is caused by a faster association rate (2.2-fold) coupled with a slower dissociation rate (2.8-fold). The association rates of both antagonists are comparable with or greater than the association rate for acetylcholine binding to nAChR.

Animals↗

PubMed1998

Characterization of the growth of 2D protein crystals on a lipid monolayer by ellipsometry and rigidity measurements coupled to electron microscopy.

We present here some sensitive optical and mechanical experiments for monitoring the process of formation and growth of two-dimensional (2D) crystals of proteins on a lipid monolayer at an air-water interface. The adsorption of proteins on the lipid monolayer was monitored by ellipsometry measurements. An instrument was developed to measure the shear elastic constant (in plane rigidity) of the monolayer. These experiments have been done using cholera toxin B subunit (CTB) and annexin V as model proteins interacting with a monosialoganglioside (GM1) and dioleoylphosphatidylserine (DOPS), respectively. Electron microscopy observations of the protein-lipid layer transferred to grids were systematically used as a control. We found a good correlation between the measured in-plane rigidity of the monolayer and the presence of large crystalline domains observed by electron microscopy grids. Our interpretation of these data is that the crystallization process of proteins on a lipid monolayer passes through at least three successive stages: 1) molecular recognition between protein and lipid-ligand, i.e., adsorption of the protein on the lipid layer; 2) nucleation and growth of crystalline patches whose percolation is detected by the appearance of a non-zero in-plane rigidity; and 3) annealing of the layer producing a slower increase of the lateral or in-plane rigidity.

Annexin A5↗

PubMed2006

A dynamic model of excitation-contraction coupling during acidosis in cardiac ventricular myocytes.

Acidosis in cardiac myocytes is a major factor in the reduced inotropy that occurs in the ischemic heart. During acidosis, diastolic calcium concentration and the amplitude of the calcium transient increase, while the strength of contraction decreases. This has been attributed to the inhibition by protons of calcium uptake and release by the sarcoplasmic reticulum, to a rise of intracellular sodium caused by activation of sodium-hydrogen exchange, decreased calcium binding affinity to Troponin-C, and direct effects on the contractile machinery. The relative contributions and concerted action of these effects are, however, difficult to establish experimentally. We have developed a mathematical model to examine altered calcium-handling mechanisms during acidosis. Each of the alterations was incorporated into a dynamical model of pH regulation and excitation-contraction coupling to predict the time courses of key ionic species during acidosis, in particular intracellular pH, sodium and the calcium transient, and contraction. This modeling study suggests that the most significant effects are elevated sodium, inhibition of sodium-calcium exchange, and the direct interaction of protons with the contractile machinery; and shows how the experimental data on these contributions can be reconciled to understand the overall effects of acidosis in the beating heart.

Acidosis↗

PubMed2004

Spike timing of distinct types of GABAergic interneuron during hippocampal gamma oscillations in vitro.

Gamma frequency (30-100 Hz) network oscillations occur in the intact hippocampus during awake, attentive behavior. Here, we explored the underlying cellular mechanisms in an in vitro model of persistent gamma-frequency oscillations, induced by bath application of 20 microm carbachol in submerged hippocampal slices at 30 +/- 1 degrees C. Current-source density analysis of the field oscillation revealed a prominent alternating sink-source pair in the perisomatic and apical dendritic regions of CA3. To elucidate the active events generating these extracellular dipoles, we examined the firing properties of distinct neuron types. Visually guided unit recordings were obtained from individual CA3 neurons followed by intracellular labeling for anatomical identification. Pyramidal cells fired at 2.82 +/- 0.7 Hz, close to the negative peak of the oscillation (0.03 +/- 0.65 msec), and often in conjunction with a negative spike-like component of the field potential. In contrast, all phase-coupled interneurons fired after this negative peak. Perisomatic inhibitory interneurons fired at high frequency (18.1 +/- 2.7 Hz), shortly after the negative peak (1.97 +/- 0.95 msec) and were strongly phase-coupled. Dendritic inhibitory interneurons fired at lower frequency (8.4 +/- 2.4 Hz) and with less fidelity and a longer delay after the negative peak (4.3 +/- 1.1 msec), whereas interneurons with cell body in the stratum radiatum often showed no phase relationship with the field oscillation. The phase and spike time data of individual neurons, together with the current-source density analysis, support a synaptic feedback model of gamma oscillations primarily involving pyramidal cells and inhibitory cells targeting their perisomatic region.

Action Potentials↗

PubMed2003

The STC-1 cells express functional orexin-A receptors coupled to CCK release.

Orexins are newly discovered neuropeptides regulating feeding and vigilance and have been detected in neuroendocrine cells of the gut. Potential neuroendocrine functions of orexin are unknown. Therefore, the effects of orexin-A on the intestinal neuroendocrine cell line, STC-1, were investigated as a model system. RT-PCR demonstrated the presence of both OX(1) and OX(2) receptors. Stimulation with orexin-A produced a dose-dependent release of cholecystokinin (CCK), which was abolished by removal of extracellular Ca(2+) or the presence of the voltage-gated L-type Ca(2+)-channel blocker diltiazem (10 microM). Orexin-A (Ox-A) elevated intracellular Ca(2+), which was dependent on extracellular Ca(2+). Furthermore, orexin-A caused a membrane depolarization in the STC-1 cells. Ox-A neither elevated cAMP levels nor stimulated phosphoinositide turnover in these cells. These data demonstrate a functional orexin receptor in the STC-1 cell line. Ox-A produces CCK release in these cells, by a mechanism involving membrane depolarization and subsequently activation of L-type voltage-gated Ca(2+)-channels.

Animals↗

PubMed2003

Coupling gating with ion permeation in ClC channels.

In ClC chloride (Cl(-)) channels, unlike cation-selective ion channels, ion permeation is intimately coupled to fast gating. Recent research comparing the crystallographic structure of a bacterial ClC channel with functional studies of a Torpedo ClC channel suggests that gating depends on the negatively charged carboxyl group on a glutamate residue, which blocks the channel pore. In this model, the permeating Cl(-) competes with the carboxyl group for an anion-binding site in the channel pore. This model of Cl(-) competition with a glutamate gate helps explain the effect of intracellular Cl(-) on channel gating; the mechanism underlying the effects of extracellular Cl(-), however, remains to be determined, as does the nature of the Cl(-) channel slow gate.

Animals↗

PubMed1996

Quantal properties of spontaneous EPSCs in neurones of the guinea-pig dorsal lateral geniculate nucleus.

1. Spontaneous non-NMDA glutamate receptor-mediated EPSCs were recorded with the whole-cell patch-clamp technique from twenty-six neurones in the dorsal lateral geniculate nucleus in thalamic slices from guinea-pig. 2. Amplitude distributions of the EPSCs were skewed towards larger values. The skewness could be accounted for by multiquantal properties. The multiquantal properties were most clearly demonstrated in four cells that had prominent peaks in the amplitude distribution, and peak separation approximately corresponding to the modal value. The amplitude distribution for all cells could be adequately fitted by a quantal model consisting of a sum of Gaussians with means equal to integer multiples of a quantal unit. The variance of each Gaussian was equal to the sum of the noise variance of the recordings and an additional non-negative variance which increased linearly with the number of the Gaussian in the series. The estimated mean quantal size was 152 +/- 37 pS. The estimated mean quantal coefficient of variation was 15%. Addition of tetrodotoxin did not significantly change any of the quantal parameters (n = 5). 3. The waveform of the EPSCs was similar for small and large events, and similar to that of events evoked by stimulation of retinal input fibres. There was a positive correlation between peak amplitude and rise time. This is the opposite of that expected if differences in electrotonic distances were to explain differences in amplitude. 4. The spontaneous EPSCs occurred randomly at an average frequency of 3.1 Hz. The events with amplitudes approximately equal to multiples of the quantal size were, in most cells, more numerous than could be accounted for by coincidence of randomly occurring events of quantal size. 5. The results indicate that spontaneous EPSCs can reflect more than a single quantum, and suggest that quantal events may be coupled due to action potential-independent near-synchronous multiquantal release of transmitter.

2-Amino-5-phosphonovalerate↗

PubMed1999

Action of internal pronase on the f-channel kinetics in the rabbit SA node.

1. The hyperpolarization-activated If current was recorded in inside-out macropatches from sino-atrial (SA) node myocytes during exposure of their intracellular side to pronase, in an attempt to verify if cytoplasmic f-channel domains are involved in both voltage- and cAMP-dependent gating. 2. Superfusion with pronase caused a quick, dramatic acceleration of channel opening upon hyperpolarization and slowing, rapidly progressing into full blockade, of channel closing upon depolarization; these changes persisted after wash off of pronase and were irreversible, indicating proteolytic cleavage of channel regions which contribute to gating. 3. If recorded from patches normally responding to cAMP became totally insensitive to cAMP following pronase treatment, indicating partial or total removal of channel regions involved in the cAMP-dependent activation. 4. The fully activated I-V relationship was not modified by pronase, indicating that internal proteolysis did not affect the f-channel conductance. 5. The changes in If kinetics induced by pronase were due to a large depolarizing shift of the f-channel open probability curve (56.5 +/- 1.1 mV, n = 7). 6. These results are consistent with the hypothesis that cytoplasmic f-channel regions are implicated in dual voltage- and cAMP-dependent gating; also, since pronase does not abolish hyperpolarization-activated opening, an intrinsic voltage-dependent gating mechanism must exist which is inaccessible to proteolytic cleavage. A model scheme able to account for these data thus includes an intrinsic gating mechanism operating at depolarized voltages, and a blocking mechanism coupled to cAMP binding to the channel.

Animals↗

PubMed1998

An evaluation of the signal and noise characteristics of four CCD-based film digitizers.

Film digitizers are common devices in radiology departments involved with picture archive and communication systems (PACS) and teleradiology. In this paper, we studied the performance of film digitizers based on charge-coupled device detectors (CCD digitizers), and compared this with the performance of a laser digitizer (the de facto standard). Our focus was on the assessment of signal, noise and useful optical density range performance. A function (L* delta D) derived from the Rose model was used to evaluate these parameters in absolute terms, based their predicted ability to detect objects of specific size and optical density difference with respect to background. We studied CCD digitizers from four different vendors and found that none was able to reliably operate up to the maximum density of 3.0 required to digitize plain radiographs, while the laser digitizer was capable of this task. Our analysis also indicated that two of the four CCD digitizers were adequate for digitizing laser-printed cross-sectional images in certain cases. Finally, our analysis indicated that digitization of SMPTE pattern films along with visual assessment of the 5% and 95% contrast patches was not sufficient for determining the utility of film digitizers for clinical tasks. Computation of the L* delta D function provides a useful means of assessing the performance of film digitizers (e.g., for acceptance testing and quality control), and this technique may be adaptable for evaluation of other digital imaging modalities.

Biophysical Phenomena↗