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Results for “coupled patch model”

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At least 145 records · Page 8Linked to original sources

PubMed2003

Effects of extracellular sodium on mu-opioid receptors coupled to potassium channels coexpressed in Xenopus oocytes.

Wild-type or mutant H297N or H297Q of the mu-opioid receptor were co-expressed with the inwardly rectifying potassium channel GIRK1 in oocytes from Xenopus laevis. Under voltage clamp, pairs of concentration response curves were generated using the agonist normorphine in a bathing medium containing 38.5 mM sodium or an identical medium in which the sodium was replaced by an equimolar concentration of choline. The maximum currents were greater in the presence of sodium by about 30% at wild-type receptors and by about 100% at the mutant receptors. The EC(50) values tended to increase somewhat as well, though these differences reached statistical significance only for the mutant H297Q. Flame photometry detected no change in the intracellular sodium or potassium concentrations of oocytes, suggesting that the effect of sodium was solely extracellular. Thus sodium, long known for its effects on in vitro ligand binding at mu-opioid receptors, also affects overall transduction as revealed in the Xenopus oocyte model of a complete, living cell system.

Animals↗

PubMed1996

cAMP promotes gap junctional coupling in T84 cells.

The effect of intracellular adenosine 3',5'-cyclic monophosphate (cAMP) on dye and electrical coupling was studied in T84 cells, a cell line often used as a model for epithelial cell fluid secretion. Injections of lucifer yellow (LY) into single cells within a cluster of control cells resulted in LY localization to 1.3 +/- 0.2 (mean +/- SE) cells within a cluster. Twenty-six percent of control T84 cell pairs were electrically coupled as assayed by the dual patch-clamp technique. Treatment of cells with agents that either increase intracellular cAMP and/or activate protein kinase A (PKA) increased dye localization to 3.8 +/- 0.6 cells and the proportion of electrically coupled cell pairs to 65%. No electrical coupling was observed in the presence of the Rp diastereomer of adenosine-3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA antagonist. Excess of Rp-cAMPS prevented cell coupling elicited by 20 microM of the Sp diastereomer of adenosine-3',5'-cyclic monophosphothioate. Expression of connexin 32 mRNA, but not of connexins 26, 43, or 45, was detected by reverse transcription-polymerase chain reaction. These results suggest that communication between T84 cells is modulated by PKA, providing a mechanism for regulating multicellular activity, such as fluid secretion.

Cell Line↗

PubMed2001

Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R asymmetry by the mouse node.

Genetic analyses in Drosophila have demonstrated that the multipass membrane protein Smoothened (Smo) is essential for all Hedgehog signaling. We show that Smo acts epistatic to Ptc1 to mediate Shh and Ihh signaling in the early mouse embryo. Smo and Shh/Ihh compound mutants have identical phenotypes: embryos fail to turn, arresting at somite stages with a small, linear heart tube, an open gut and cyclopia. The absence of visible left/right (L/R) asymmetry led us to examine the pathways controlling L/R situs. We present evidence consistent with a model in which Hedgehog signaling within the node is required for activation of Gdf1, and induction of left-side determinants. Further, we demonstrate an absolute requirement for Hedgehog signaling in sclerotomal development and a role in cardiac morphogenesis.[Dedicated to Rosa Beddington, a pioneer in mammalian embryology].

Animals↗

PubMed2001

Smoothened mutants reveal redundant roles for Shh and Ihh signaling including regulation of L/R symmetry by the mouse node.

Animals↗

PubMedDate not supplied

Advances in membrane receptor screening and analysis.

During the last decade there has been significant progress in the development of analytical techniques for the screening of ligand binding to membranes and membrane receptors. This review focuses on developments using label-free assays that facilitate ligand-membrane-receptor screening without the need for chemical-, biological- or radiological-labelled reagents. These assays include acoustic, optical surface plasmon resonance biosensing, sedimentation (analytical ultracentrifugation), chromatographic assays, isothermal titration calorimetry and differential scanning calorimetry. The merits and applications of cell-based screening systems and of different model membrane systems, including planar supported lipid layers, bead-supported membranes and lipid micro-arrays, are discussed. Recent advances involving more established techniques including intrinsic fluorescence, FRET spectroscopy, scintillation proximity assays and automated patch clamping are presented along with applications to peripheral membrane proteins, ion channels and G protein-coupled receptors. Novel high-throughput assays for determination of drug- and protein-partitioning in membranes are also highlighted. To aid the experimenter, a brief synopsis of the techniques commonly employed to purify and reconstitute membranes and membrane receptors is included.

Animals↗

PubMed2005

Estimating conductances of dual-recorded neurons within a network of coupled cells.

Simultaneous pre- and postsynaptic cell recordings are used to calculate gap junction conductance based on an equivalent electrical circuit of an electrically coupled pair of cells. This calculation is imprecise when recording from a cell pair that is coupled to neighboring cells providing indirect conductance paths between the recorded cells. Despite this imprecision, junctional conductance has been calculated for coupled cell networks during the past 40 years since a more accurate method was lacking. The present study simulated a three-dimensional network of electrically coupled heterogeneous neurons and used mathematical modeling to reduce the complexity to the simplest equations that could more accurately estimate the electrical properties of dual-recorded cells in the network. Analyses of the simulations showed that knowledge of the number of unrecorded cells directly linked to the recorded cells and of the voltage responses of these recorded cells were largely sufficient to accurately predict the direct junctional resistance linking the recorded cells as well as the input resistance of the recorded cells that would exist in the absence of junctional coupling. All model parameters could be obtained from real dual-intracellular penetrations which allow electrophysiological recordings and intracellular staining.

Animals↗

PubMedDate not supplied

Interactions between adjacent fibers in a cardiac muscle bundle.

A strand of cardiac muscle was modeled as a small bundle of individual fibers surrounded by a large volume conductor. The bundle is a uniform assembly of small identical cylindrical fibers, arranged as a series of concentric layers, and its behavior is examined in the presence (coupled bundle) or absence (uncoupled bundle) of transverse resistive coupling between adjacent fibers. Individual fibers are continuous cables of excitable membrane, with circumferential segmentation into 12 equal patches to make the membrane potential changes dependent upon the local interstitial potential. The minimum spacing (d) between adjacent fibers is used to modify the interstitial microstructural organization and the intracellular volume fraction (fi). When d is small enough (d < 0.01 micron), fi remains unchanged at its maximum of about 90%, the interstitial potential is large, the transverse interstitial resistance is high, and the proximity effect arising from the close juxtaposition of adjacent fibers is important. A surface fiber of the uncoupled bundle exhibits little sensitivity to changes in the interstitial microstructure, owing to the dominant influence of the external volume conductor, whereas the central fiber shows a large decrease in velocity, substantial waveshape modifications, and a large increase in interstitial potential as d is reduced. In the coupled bundle, all fibers adopt the same velocity during uniform propagation, owing to the strong transverse resistive coupling; when d is reduced in the range of d < 0.01 micron, the velocity and interstitial potential changes are less pronounced than in the uncoupled bundle. When d is large enough (d > 0.01 micron), the bundle behavior (coupled and uncoupled) approaches that obtained with a bidomain formulation.

Action Potentials↗

PubMed1999

Modeling gain and gradedness of Ca2+ release in the functional unit of the cardiac diadic space.

A model of the functional release unit (FRU) in rat cardiac muscle consisting of one dihydropyridine receptor (DHPR) and eight ryanodine receptor (RyR) channels, and the volume surrounding them, is formulated. It is assumed that no spatial [Ca2+] gradients exist in this volume, and that each FRU acts independently. The model is amenable to systematic parameter studies in which FRU dynamics are simulated at the channel level using Monte Carlo methods with Ca2+ concentrations simulated by numerical integration of a coupled system of differential equations. Using stochastic methods, Ca(2+)-induced Ca2+ release (CICR) shows both high gain and graded Ca2+ release that is robust when parameters are varied. For a single DHPR opening, the resulting RyR Ca2+ release flux is insensitive to the DHPR open duration, and is determined principally by local sarcoplasmic reticulum (SR) Ca2+ load, consistent with experimental data on Ca2+ sparks. In addition, single RyR openings are effective in triggering Ca2+ release from adjacent RyRs only when open duration is long and SR Ca2+ load is high. This indicates relatively low coupling between RyRs, and suggests a mechanism that limits the regenerative spread of RyR openings. The results also suggest that adaptation plays an important modulatory role in shaping Ca2+ release duration and magnitude, but is not solely responsible for terminating Ca2+ release. Results obtained with the stochastic model suggest that high gain and gradedness can occur by the recruitment of independent FRUs without requiring spatial [Ca2+] gradients within a functional unit or cross-coupling between adjacent functional units.

Animals↗

PubMed2005

K channel subconductance levels result from heteromeric pore conformations.

Voltage-gated K channels assemble from four identical subunits symmetrically arranged around a central permeation pathway. Each subunit harbors a voltage-sensing domain. The sigmoidal nature of the activation kinetics suggests that multiple sensors need to undergo a conformational change before the channel can open. Following activation, individual K channels alternate stochastically between two main permeation states, open and closed. This binary character of single channel behavior suggests the presence of a structure in the permeation pathway that can exist in only two conformations. However, single channel analysis of drk1 (K(v)2.1) K channels demonstrated the existence of four additional, intermediate conductance levels. These short-lived subconductance levels are visited when the channel gate moves between the closed and fully open state. We have proposed that these sublevels arise from transient heteromeric pore conformations, in which some, but not all, subunits are in the "open" state. A minimal model based on this hypothesis relates specific subconductance states with the number of activated subunits (Chapman et al., 1997). To stringently test this hypothesis, we constructed a tandem dimer that links two K channel subunits with different activation thresholds. Activation of this dimer by strong depolarizations resulted in the characteristic binary open-close behavior. However, depolarizations to membrane potentials in between the activation thresholds of the two parents elicited highly unusual single channel gating, displaying frequent visits to two subconductance levels. The voltage dependence and kinetics of the small and large sublevels associate them with the activation of one and two subunits, respectively. The data therefore support the hypothesis that subconductance levels result from heteromeric pore conformations. In this model, both sensor movement and channel opening have a subunit basis and these processes are allosterically coupled.

Allosteric Regulation↗

PubMed2006

The role of L- and T-type Ca2+ currents during the in vitro aging of murine myogenic (i28) cells in culture.

The age-related decline in skeletal muscle strength could, in part, result from alterations in the mechanism of excitation-contraction coupling, responsible for muscle contraction. In the present work, we used the in vitro aging of murine myogenic (i28) cells as a model, to investigate whether the inefficiency of aged satellite cells to generate functional skeletal muscle fibres could be partly due to defective voltage-dependent Ca2+ currents. The whole-cell patch clamp technique was employed to measure L- and T-type Ca2+ currents in myotubes derived from the differentiation and fusion of these cells reaching replicative senescence. Our data showed that the expression and the amplitude of these currents decreased significantly during in vitro aging. Moreover, the analysis of the L-type current evoked in young and old cells by positive voltage steps, revealed no differences in the kinetics of activation, but significant alterations in the rate of inactivation. These effects of in vitro aging on voltage-dependent Ca2+ currents could also be related to their inability to fuse into myotubes. Taken together, our data support the hypothesis that age-related effects on voltage-dependent L- and T-type currents could be one of the causes of the failure of satellite cells to efficiently counteract the impairment in muscle force.

Animals↗

PubMed1975

Simulation of action potential propagation in an inhomogeneous sheet of coupled excitable cells.

Cable theory and active equivalent circuits have been used to simulate the propagation of action potentials along a single nerve or muscle fiber by representing the cell as a unidimensional cable composed of isopotential segments. We extended this method to a two-dimensional sheet of cells which in many ways represents the atrium. Our method consisted of solving for the potential profile of a sheet composed of a large number of isopotential membrane patches, each of which was represented by an active equivalent circuit in which the ionic conductances were functions of voltage and time. The patches were arranged in a rectangular array with resistive interconnections that could be varied over the sheet. We used this model to study the effect of various inhomogeneities on conduction velocity and the resulting wave fronts in a sheet of excitable tissue. Some of these inhomogeneities included different effective internal resistances in the x and y directions, preferential pathways, and discrete regions of changing resistive connections. The results showed that very localized changes in membrane properties or cellular interconnections produce changes in the wave front over broad areas. This model provides a method for computing the wave fronts of action potential propagation in any two-dimensional inhomogeneous sheet of coupled excitable cells.

Action Potentials↗

PubMed2000

Coupling of the muscarinic m2 receptor to G protein-activated K(+) channels via Galpha(z) and a receptor-Galpha(z) fusion protein. Fusion between the receptor and Galpha(z) eliminates catalytic (collision) coupling.

G protein-activated K(+) channel (GIRK), which is activated by the G(betagamma) subunit of heterotrimeric G proteins, and muscarinic m2 receptor (m2R) were coexpressed in Xenopus oocytes. Acetylcholine evoked a K(+) current, I(ACh), via the endogenous pertussis toxin (PTX)-sensitive G(i/o) proteins. Activation of I(ACh) was accelerated by increasing the expression of m2R, suggesting a collision coupling mechanism in which one receptor catalytically activates several G proteins. Coexpression of the alpha subunit of the PTX-insensitive G protein G(z), Galpha(z), induced a slowly activating PTX-insensitive I(ACh), whose activation kinetics were also compatible with the collision coupling mechanism. When GIRK was coexpressed with an m2R x Galpha(z) fusion protein (tandem), in which the C terminus of m2R was tethered to the N terminus of Galpha(z), part of I(ACh) was still eliminated by PTX. Thus, the m2R of the tandem activates the tethered Galpha(z) but also the nontethered G(i/o) proteins. After PTX treatment, the speed of activation of the m2R x Galpha(z)-mediated response did not depend on the expression level of m2R x Galpha(z) and was faster than when m2R and Galpha(z) were coexpressed as separate proteins. These results demonstrate that fusing the receptor and the Galpha strengthens their coupling, support the collision-coupling mechanism between m2R and the G proteins, and suggest a noncatalytic (stoichiometric) coupling between the G protein and GIRK in this model system.

Animals↗

PubMed2004

Oscillations of membrane potential and cytosolic Ca(2+) concentration in SUR1(-/-) beta cells.

AIMS/HYPOTHESIS: SUR1(ABCC8)(-/-) mice lacking functional K(ATP) channels are an appropriate model to test the significance of K(ATP) channels in beta-cell function. We examined how this gene deletion interferes with stimulus-secretion coupling. We tested the influence of metabolic inhibition and galanin, whose mode of action is controversial. METHODS: Plasma membrane potential (Vm) and currents were measured with microelectrodes or the patch-clamp technique; cytosolic Ca(2+) concentrations ([Ca(2+)](c)) and mitochondrial membrane potential (DeltaPsi) were measured using fluorescent dyes. RESULTS: In contrast to the controls, SUR1(-/-) beta cells showed electrical activity even at a low glucose concentration. Continuous spike activity was measured with the patch-clamp technique, but with microelectrodes slow oscillations in Vm consisting of bursts of Ca(2+)-dependent action potentials were detected. [Ca(2+)](c) showed various patterns of oscillations or a sustained increase. Sodium azide did not hyperpolarize SUR1(-/-) beta cells. The depolarization of DeltaPsi evoked by sodium azide was significantly lower in SUR1(-/-) than SUR1(+/+) cells. Galanin transiently decreased action potential frequency and [Ca(2+)](c) in cells from both SUR1(-/-) and SUR1(+/+) mice. CONCLUSION/INTERPRETATION: The strong dependence of Vm and [Ca(2+)](c) on glucose concentration observed in SUR1(+/+) beta cells is disrupted in the knock-out cells. This demonstrates that both parameters oscillate in the absence of functional K(ATP) channels. The lack of effect of metabolic inhibition by sodium azide shows that in SUR1(-/-) beta cells changes in ATP/ADP no longer link glucose metabolism and Vm. The results with galanin suggest that this peptide affects beta cells independently of K(ATP) currents and thus could contribute to the regulation of beta-cell function in SUR1(-/-) animals.

ATP-Binding Cassette Transporters↗

PubMed2002

Heterotrimeric G-proteins activate Cl- channels through stimulation of a cyclooxygenase-dependent pathway in a model liver cell line.

Circulating hormones produce rapid changes in the Cl(-) permeability of liver cells through activation of plasma membrane receptors coupled to heterotrimeric G-proteins. The resulting effects on intracellular pH, membrane potential, and Cl(-) content are important contributors to the overall metabolic response. Consequently, the purpose of these studies was to evaluate the mechanisms responsible for G-protein-mediated changes in membrane Cl(-) permeability using HTC hepatoma cells as a model. Using patch clamp techniques, intracellular dialysis with 0.3 mm guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) increased membrane conductance from 10 to 260 picosiemens/picofarads due to activation of Ca(2+)-dependent Cl(-) currents that were outwardly rectifying and exhibited slow activation at depolarizing potentials. These effects were mimicked by intracellular AlF(4)(-) (0.03 mm) and inhibited by pertussis toxin (PTX), consistent with current activation through Galpha(i). Studies using defined agonists and inhibitors indicate that Cl(-) channel activation by GTPgammaS occurs through an indomethacin-sensitive pathway involving sequential activation of phospholipase C, mobilization of Ca(2+) from inositol 1,4,5-trisphosphate-sensitive stores, and stimulation of phospholipase A(2) and cyclooxygenase (COX). Accordingly, the conductance responses to GTPgammaS or to intracellular Ca(2+) were inhibited by COX inhibitors. These results indicate that PTX-sensitive G-proteins regulate the Cl(-) permeability of HTC cells through Ca(2+)-dependent stimulation of COX activity. Thus, receptor-mediated activation of Galpha(i) may be essential for hormonal regulation of liver transport and metabolism through COX-dependent opening of a distinct population of plasma membrane Cl(-) channels.

Adenosine Triphosphate↗

PubMed2004

Use of the covariance matrix in directly fitting kinetic parameters: application to GABAA receptors.

A new method of analysis is described that begins to explore the relationship between the phases of ion channel desensitization and the underlying states of the channel. The method, referred to as covariance fitting (CVF), couples Q-matrix calculations with a maximum likelihood algorithm to fit macroscopic desensitization data directly to kinetic models. Unlike conventional sum-of-squares minimization, CVF fits both the magnitude of the recorded current and the strength of the correlations between different time points. When applied to simulated data generated using various kinetic models with up to 11 free parameters, CVF leads to reasonable parameter estimates. Coupled with the likelihood ratio test, it accurately discriminates between models with different numbers of states, discriminates between most models with the same number but a different arrangement of states, and extracts meaningful information on the relationship between the desensitized states and the phases of macroscopic desensitization. When applied to GABA(A) receptor traces (outside out patches, alpha 1 beta 2 gamma 2S, 1 mM GABA, >2.5 s), a model with two open states and three desensitized states is favored. When applied to simulated data generated using a consensus model, CVF leads to reasonable parameter estimates and accurately discriminates between this and other models.

Algorithms↗

PubMed1999

Local regulation of the threshold for calcium sparks in rat ventricular myocytes: role of sodium-calcium exchange.

1. To determine whether Na+-Ca2+ exchange modulates Ca2+ sparks, we studied enzymatically isolated patch clamped rat ventricular myocytes loaded with the Ca2+-sensitive indicator fluo-3, using confocal microscopy at 20-22 C. Two-dimensional images of Ca2+ sparks were recorded at 240 Hz using a laser scanning confocal microscope, allowing observation of a large area of the cell (820 microm2) at one time. 2. At a holding potential of -75 mV, spontaneous sparks were infrequent. Removal of extracellular Na+ for 520 ms, which in the absence of pipette Na+ should block Na+-Ca2+ exchange bidirectionally, was associated with a fourfold increase in spark frequency, without a significant change in cytoplasmic [Ca2+], sarcoplasmic reticulum (SR) Ca2+ content, or spark intensity, size or time course. 3. These findings are consistent with a model of excitation-contraction coupling in which Na+-Ca2+ exchange locally regulates the resting Ca2+ concentration in the diadic cleft (T-tubule-SR junction), thereby modulating the threshold for triggering Ca2+ sparks.

Aniline Compounds↗

PubMed1998

Modalities of distortion of physiological voltage signals by patch-clamp amplifiers: a modeling study.

An extensive evaluation of the possible alterations affecting physiological voltage signals recorded with patch-clamp amplifiers (PCAs) working in the current-clamp (CC) mode was carried out by following a modeling approach. The PCA output voltage and current signals obtained during CC recordings performed under simplified experimental conditions were exploited to determine the equations describing the generation of error currents and voltage distortions by PCAs. The functions thus obtained were used to construct models of PCAs working in the CC mode, which were coupled to numerical simulations of neuronal bioelectrical behavior; this allowed us to evaluate the effects of the same PCAs on different physiological membrane-voltage events. The models revealed that rapid signals such as fast action potentials are preferentially affected, whereas slower events, such as low-threshold spikes, are less altered. Prominent effects of model PCAs on fast action potentials were alterations of their amplitude, duration, depolarization and repolarization speeds, and, most notably, the generation of spurious afterhyperpolarizations. Processes like regular firing and burst firing could also be altered, under particular conditions, by the model PCAs. When a cell consisting of more than one single intracellular compartment was considered, the model PCAs distorted fast equalization transients. Furthermore, the effects of different experimental and cellular parameters (series resistance, cell capacitance, temperature) on PCA-generated artifacts were analyzed. Finally, the simulations indicated that no off-line correction based on manipulations of the error-current signals returned by the PCAs can be successfully performed in the attempt to recover unperturbed voltage signals, because of alterations of the overall current flowing through the cell-PCA system.

Action Potentials↗

PubMed1996

Regulation of connexin-43, GFAP, and FGF-2 is not accompanied by changes in astroglial coupling in MPTP-lesioned, FGF-2-treated parkinsonian mice.

Basic fibroblast growth factor (bFGF; FGF-2) has potent trophic effects on developing and toxically impaired midbrain dopaminergic (DAergic) neurons which are crucially affected in Parkinson's disease. The trophic effects of FGF-2 are largely indirect, both in vitro and in vivo, and possibly involve intermediate actions of astrocytes and other glial cells. To further investigate the cellular and molecular mechanisms underlying the restorative actions of FGF-2, and to analyse in more detail the changes within astroglial cells in the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned striatum, we have studied striatal expression and regulation of connexin-43 (cx43), the principal gap junction protein of astroglial cells, along with the expression of glial fibrillary acidic protein (GFAP), FGF-2, and functional coupling. Our results show an immediate, yet transient increase in cx43 mRNA, and a sustained increase in FGF-2 mRNA, GFAP-positive cells, and cx43-immunoreactive punctata following the MPTP lesion, without any induction of functional coupling between astrocytes and other glial cells as revealed by dye coupling of patched cells. Unilateral administration of FGF-2 in a piece of gelfoam caused a further increase in cx43-positive punctata immediately adjacent to the implant, which was more pronounced than after application of a gelfoam containing the nontrophic control protein cytochrome C. These changes were parallelled by a small increase in cx43 protein determined by Western blot, but not by alterations in the coupling state of cells in the vicinity of the gelfoam implant. Although our data indicate that MPTP and exogenous FGF-2 may alter expression and protein levels of cx43, they do not support the notion that increases in cellular coupling may underly the trophic and widespread actions of FGF-2 in the MPTP-model of Parkinson's disease.

Animals↗