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At least 127 records · Page 7Linked to original sources
PubMed2006

New roles for a key glycine and its neighboring residue in potassium channel gating.

Potassium channel activation regulates cellular excitability in cells such as neurons and heart. Ion channel activity relies on a switching mechanism between two conformations, the open and closed states, known as gating. It has been suggested that potassium channels are gated via a pivoted mechanism of the pore-lining helix. Our analysis suggests that hinging occurs at the residue immediately preceding the central glycine of the inner helix. Furthermore, we show that the highly conserved central glycine is necessary to prevent constraining interactions with critical residues in its vicinity, including those located in the selectivity filter. We show that such interactions can impair channel function, and that upon their removal channel activity can be restored.

Amino Acid Sequence↗
PubMed1994

Mechanosensitive fibroblasts in the sino-atrial node region of rat heart: interaction with cardiomyocytes and possible role.

The positive chronotropic response of the heart to stretch of the right atrium is one of the major mechanisms adjusting the heart rate to variations in venous return on a beat-by-beat basis. The precise pathway of this mechano-electric feedback and its cellular basis are uncertain. In this study, a possible contribution of mechanosensitive fibroblasts, abundant in the sino-atrial node region, was investigated using a mathematical model of the electrical interaction of a mechanosensitive fibroblast and a sino-atrial pacemaker cell. Electrophysiological evidence for a bio-electrical interaction of mechanosensitive fibroblasts with surrounding cardiomyocytes has been studied in (i) the isolated spontaneously beating atrium of rat hearts, and (ii) cell cultures of the neonatal rat heart. These investigations were performed using (i) double-barrelled floating microelectrodes for intracellular potential registrations, and (ii) the double whole cell patch-clamp technique. It was shown that cardiac fibroblasts and surrounding cardiomyocytes can be either electrically well isolated from each other, or coupled both capacitively and electrotonically. The electrophysiological data obtained were incorporated into the OXSOFT HEART program. Assuming that equivalent coupling may occur between mechanosensitive fibroblasts and sino-atrial pacemaker cells, a heterologous cell pair consisting of one fibroblast and one sino-atrial node myocyte connected by ten to thirty single gap junctional channels with a conductance of 30 pS was modelled. The model of the electrotonic interaction of these cells showed that stretch of the fibroblast during atrial diastole, simulating increased atrial wall tension during atrial filling, can raise the spontaneous depolarization rate of the pacemaker cell in a stretch-dependent manner by up to 24%. These results show that cardiac mechanosensitive fibroblasts could form a cellular basis for the positive chronotropic response of the heart to stretch of the right atrium.

Animals↗
PubMed1999

Switched single-electrode voltage-clamp amplifiers allow precise measurement of gap junction conductance.

Measurement of gap junction conductance (gj) with patch-clamp amplifiers can, due to series resistance problems, be subject to considerable errors when large currents are measured. Formulas developed to correct for these errors unfortunately depend on exact estimates of series resistance, which are not always easy to obtain. Discontinuous single-electrode voltage-clamp amplifiers (DSEVCs) were shown to overcome series resistance problems in single whole cell recording. With the use of two synchronized DSEVCs, the simulated gj in a model circuit can be measured with a maximum error of <5% in all recording situations investigated (series resistance, 5-47 MOmega; membrane resistance, 20-1,000 MOmega; gj, 1-100 nS). At a very low gj of 100 pS, the error sometimes exceeded 5% (maximum of 15%), but the error was always <5% when membrane resistance was >100 MOmega. The precision of the measurements is independent of series resistance, membrane resistance, and gj. Consequently, it is possible to calculate gj directly from Ohm's law, i.e., without using correction formulas. Our results suggest that DSEVCs should be used to measure gj if large currents must be recorded, i.e., if cells are well coupled or if membrane resistance is low.

Animals↗
PubMed2000

Assessment of the percutaneous absorption of trichloroethylene in rats and humans using MS/MS real-time breath analysis and physiologically based pharmacokinetic modeling.

The development and validation of noninvasive techniques for estimating the dermal bioavailability of solvents in contaminated soil and water can facilitate the overall understanding of human health risk. To assess the dermal bioavailability of trichloroethylene (TCE), exhaled breath was monitored in real time using an ion trap mass spectrometer (MS/MS) to track the uptake and elimination of TCE from dermal exposures in rats and humans. A physiologically based pharmacokinetic (PBPK) model was used to estimate total bioavailability. Male F344 rats were exposed to TCE in water or soil under occluded or nonoccluded conditions by applying a patch to a clipper-shaved area of the back. Rats were placed in off-gassing chambers and chamber air TCE concentration was quantified for 3-5 h postdosing using the MS/MS. Human volunteers were exposed either by whole-hand immersion or by attaching patches containing TCE in soil or water on each forearm. Volunteers were provided breathing air via a face mask to eliminate inhalation exposure, and exhaled breath was analyzed using the MS/MS. The total TCE absorbed and the dermal permeability coefficient (K(P)) were estimated for each individual by optimization of the PBPK model to the exhaled breath data and the changing media and/or dermal patch concentrations. Rat skin was significantly more permeable than human skin. Estimates for K(P) in a water matrix were 0.31 +/- 0.01 cm/h and 0.015 +/- 0.003 cm/h in rats and humans, respectively. K(P) estimates were more than three times higher from water than soil matrices in both species. K(P) values calculated using the standard Fick's Law equation were strongly affected by exposure length and volatilization of TCE. In comparison, K(P) values estimated using noninvasive real-time breath analysis coupled with the PBPK model were consistent, regardless of volatilization, exposure concentration, or duration.

Administration, Cutaneous↗
PubMed2005

Kinetically critical sites of femoral head roughening for wear rate acceleration in total hip arthroplasty.

Polyethylene wear acceleration from (scratching) damage to the femoral head is a recognized hazard from constructs prone to generate third-body debris, but the phenomenon is nebulous and therefore often is subordinated to more direct and immediate considerations. To help delineate tangible quantitative relationships between counterface roughening and accelerated polyethylene wear, an experimentally validated sliding-distance-coupled finite element model of total hip replacement wear was adapted to incorporate regions of localized femoral head roughening. This computational formulation was used systematically to identify the sites on the femoral head for which a given severity of local roughening (parameterized in terms of roughening patch size and tribologic wear coefficient) was most consequential in terms of elevated polyethylene wear. Two such sites, of nominally comparable kinetic importance, were consistently evident throughout a wide range of roughening severities. These critical sites were located quasi-superiorly near the sagittal midline of the head, one slightly anterior and one slightly posterior of the coronal midline.

Arthroplasty, Replacement, Hip↗
PubMed2000

Coupled oscillator model of the dopaminergic neuron of the substantia nigra.

Calcium imaging using fura-2 and whole cell recording revealed the effective location of the oscillator mechanism on dopaminergic neurons of the substantia nigra, pars compacta, in slices from rats aged 15-20 days. As previously reported, dopaminergic neurons fired in a slow rhythmic single spiking pattern. The underlying membrane potential oscillation survived blockade of sodium currents with TTX and was enhanced by blockade of voltage-sensitive potassium currents with TEA. Calcium levels increased during the subthreshold depolarizing phase of the membrane potential oscillation and peaked at the onset of the hyperpolarizing phase as expected if the pacemaker potential were due to a low-threshold calcium current and the hyperpolarizing phase to calcium-dependent potassium current. Calcium oscillations were synchronous in the dendrites and soma and were greater in the dendrites than in the soma. Average calcium levels in the dendrites overshot steady-state levels and decayed over the course of seconds after the oscillation was resumed after having been halted by hyperpolarizing currents. Average calcium levels in the soma increased slowly, taking many cycles to achieve steady state. Voltage clamp with calcium imaging revealed the voltage dependence of the somatic calcium current without the artifacts of incomplete spatial voltage control. This showed that the calcium current had little or no inactivation and was half-maximal at -40 to -30 mV. The time constant of calcium removal was measured by the return of calcium to resting levels and depended on diameter. The calcium sensitivity of the calcium-dependent potassium current was estimated by plotting the slow tail current against calcium concentration during the decay of calcium to resting levels at -60 mV. A single compartment model of the dopaminergic neuron consisting of a noninactivating low-threshold calcium current, a calcium-dependent potassium current, and a small leak current reproduced most features of the membrane potential oscillations. The same currents much more accurately reproduced the calcium transients when distributed uniformly along a tapering cable in a multicompartment model. This model represented the dopaminergic neuron as a set of electrically coupled oscillators with different natural frequencies. Each frequency was determined by the surface area to volume ratio of the compartment. This model could account for additional features of the dopaminergic neurons seen in slices, such as slow adaptation of oscillation frequency and may produce irregular firing under different coupling conditions.

Action Potentials↗
PubMed2001

The sonic hedgehog receptor patched associates with caveolin-1 in cholesterol-rich microdomains of the plasma membrane.

The Hedgehog signaling pathway is involved in early embryonic patterning as well as in cancer; however, little is known about the subcellular localization of the Hedgehog receptor complex of Patched and Smoothened. Since Hh has been found in lipid rafts in Drosophila, we hypothesized that Patched and Smoothened might also be found in these cholesterol-rich microdomains. In this study, we demonstrate that both Smoothened and Patched are in caveolin-1-enriched/raft microdomains. Immunoprecipitation studies show that Patched specifically interacts with caveolin-1, whereas Smoothened does not. Fractionation studies show that Patched and caveolin-1 can be co-isolated from buoyant density fractions that represent caveolae/raft microdomains and that Patched and caveolin-1 co-localize by confocal microscopy. Glutathione S-transferase fusion protein experiments show that the interaction between Patched and caveolin-1 involves the caveolin-1 scaffolding domain and a Patched consensus binding site. Immunocytochemistry data and fractionation studies also show that Patched seems to be required for transport of Smoothened to the membrane. Depletion of plasmalemmal cholesterol influences the distribution of the Hh receptor complex in the caveolin-enriched/raft microdomains. These data suggest that caveolin-1 may be integral for sequestering the Hh receptor complex in these caveolin-enriched microdomains, which act as a scaffold for the interactions with the Hh protein.

Amino Acid Sequence↗
PubMed2003

Activation of the store-operated calcium current ICRAC can be dissociated from regulated exocytosis in rat basophilic leukaemia (RBL-1) cells.

In many cell types, the emptying of intracellular Ca2+ stores results in the opening of store-operated Ca2+ channels in the plasma membrane. However, the nature of the signal that couples store content to the opening of these Ca2+ channels is unclear. One model proposes that the Ca2+ channels are initially stored in cytoplasmic vesicles but inserted into the plasma membrane upon store depletion via a regulated exocytoytic mechanism (vesicular fusion model). Using the whole-cell patch-clamp technique to measure the store-operated Ca2+ current ICRAC and the capacitance method to monitor vesicular fusion, an indicator of exocytosis, we have investigated the effects of interfering with regulated exocytosis on the ability of ICRAC to activate. We find that the recombinant protein alpha-SNAP1-285, an inhibitor of exocytosis in many systems, suppresses such fusion but has no impact on the activation of ICRAC. A variety of other manoeuvres that interfere with vesicle trafficking and exocytosis were also without effect on ICRAC. Impairing constitutive exocytosis with brefeldin A reduced the extent of ICRAC, but this effect was less pronounced when current density was considered instead. Activation of ICRAC can therefore be clearly dissociated from an exocytotic mechanism, a finding that is not easily reconcilable with the vesicular fusion model.

Animals↗
PubMedDate not supplied

Membrane resonance and stochastic resonance modulate firing patterns of thalamocortical neurons.

We examined the interactions of subthreshold membrane resonance and stochastic resonance using whole-cell patch clamp recordings in thalamocortical neurons of rat brain slices, as well as with a Hodgkin-Huxley-type mathematical model of thalamocortical neurons. The neurons exhibited the subthreshold resonance when stimulated with small amplitude sine wave currents of varying frequency, and stochastic resonance when noise was added to sine wave inputs. Stochastic resonance was manifest as a maximum in signal-to-noise ratio of output response to subthreshold periodic input combined with noise. Stochastic resonance in conjunction with subthreshold resonance resulted in action potential patterns that showed frequency selectivity for periodic inputs. Stochastic resonance was maximal near subthreshold resonance frequency and a high noise level was required for detection of high frequency signals. We speculate that combined membrane and stochastic resonances have physiological utility in coupling synaptic activity to preferred firing frequency and in network synchronization under noise.

Animals↗
PubMed1988

Effect of agonist concentration on the lifetime of GABA-activated membrane channels in spinal cord neurons.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) hyperpolarizes spinal neurons by activating bicuculline sensitive GABAA receptors coupled to chloride permeable ionic channels in the cell membrane. Single channel measurements using outside-out membrane patches from cultured mouse spinal neurons showed that there is a large excess of brief openings of these channels, compared to the number predicted from a simple, two-state model of channel function. The cumulative open time distributions for channel openings were well fit by the sum of two exponential terms, a fast component with amplitude Nf and time constant TAUf, and a slow term with amplitude Ns and time constant TAUs. These kinetics could arise if monoliganded GABAA receptors triggered an open state less stable than that induced by the biliganded receptor. A prediction of this hypothesis is that the ratio Nf/Ns should decline in an approximately linear fashion with increasing doses of GABA. This was found to be the case, as Nf/Ns declined by a factor of 4.8 on increasing the GABA dose from 0.5 microM to 5 microM. Application of 1.25 microM GABA produced an intermediate value of Nf/Ns, as predicted. In contrast the parameters TAUf and TAUs were not significantly influenced by GABA concentration. Spontaneous, bicuculline sensitive currents were seen in some patches. These events resembled currents triggered by low doses of exogenous GABA, with regard to the values of Nf/Ns, TAUf and TAUs. These results suggest that the spontaneous currents are triggered by endogenous GABA molecules, present in the culture environment at a concentration of 0.5 microM or less.

Animals↗
PubMed2003

Heart failure -- a challenge to our current concepts of excitation-contraction coupling.

Development of novel therapeutic strategies for congestive heart failure (CHF) seems to be hampered by insufficient knowledge of the molecular machinery of excitation-contraction (EC) coupling in both normal and failing hearts. Cardiac hypertrophy and failure represent a multitude of cardiac phenotypes, and available invasive and non-invasive techniques, briefly reviewed here, allow proper quantification of myocardial function in experimental models even in rats and mice. Both reduced fractional shortening and reduced velocity of contraction characterize myocardial failure. Only when myocardial function is depressed in vivo can meaningful studies be done in vitro of contractility and EC coupling. Also, we point out potential limitations with the whole cell patch clamp technique. Two main factors stand out as explanations for myocardial failure. First, a basic feature of CHF seems to be a reduced Ca(2+) load of the sarcoplasmic reticulum (SR) mainly due to a low phosphorylation level of phospholamban. Second, there seems to be a defect of the trigger mechanism of Ca(2+) release from the SR. We argue that this defect only becomes manifest in the presence of reduced Ca(2+) reuptake capacity of the SR and that it may not be solely attributable to reduced gain of the Ca(2+)-induced Ca(2+) release (CICR). We list several possible explanations for this defect that represent important avenues for future research.

Animals↗
PubMed2002

Small-molecule modulators of Hedgehog signaling: identification and characterization of Smoothened agonists and antagonists.

BACKGROUND: The Hedgehog (Hh) signaling pathway is vital to animal development as it mediates the differentiation of multiple cell types during embryogenesis. In adults, Hh signaling can be activated to facilitate tissue maintenance and repair. Moreover, stimulation of the Hh pathway has shown therapeutic efficacy in models of neuropathy. The underlying mechanisms of Hh signal transduction remain obscure, however: little is known about the communication between the pathway suppressor Patched (Ptc), a multipass transmembrane protein that directly binds Hh, and the pathway activator Smoothened (Smo), a protein that is related to G-protein-coupled receptors and is capable of constitutive activation in the absence of Ptc. RESULTS: We have identified and characterized a synthetic non-peptidyl small molecule, Hh-Ag, that acts as an agonist of the Hh pathway. This Hh agonist promotes cell-type-specific proliferation and concentration-dependent differentiation in vitro, while in utero it rescues aspects of the Hh-signaling defect in Sonic hedgehog-null, but not Smo-null, mouse embryos. Biochemical studies with Hh-Ag, the Hh-signaling antagonist cyclopamine, and a novel Hh-signaling inhibitor Cur61414, reveal that the action of all these compounds is independent of Hh-protein ligand and of the Hh receptor Ptc, as each binds directly to Smo. CONCLUSIONS: Smo can have its activity modulated directly by synthetic small molecules. These studies raise the possibility that Hh signaling may be regulated by endogenous small molecules in vivo and provide potent compounds with which to test the therapeutic value of activating the Hh-signaling pathway in the treatment of traumatic and chronic degenerative conditions.

Animals↗
PubMed2005

Particle deposition onto micropatterned charge heterogeneous substrates: trajectory analysis.

A trajectory analysis of particles near a micropatterned charged substrate under radial impinging jet flow conditions is presented to investigate the effect of surface charge heterogeneity on particle trajectory and deposition efficiency. The surface charge heterogeneity is modeled as concentric bands of specified width and pitch having positive and negative surface potentials. The flow distribution is obtained using finite element analysis of the governing Navier-Stokes equations. The particle trajectory analysis takes into consideration the hydrodynamic interactions, gravity, van der Waals and electrostatic double layer interactions. The presence of surface charge heterogeneity on the substrate gives rise to an oscillating particle trajectory near the collector surface due to repulsive and attractive forces. As a result of the coupled effects of hydrodynamic and colloidal forces, the particle trajectories and deposition efficiencies are increasingly affected by surface charge heterogeneity as one moves radially away from the stagnation point. The results indicate that it is possible to render collectors with up to 50% favorable surface fraction completely unfavorable by modifying the ratio of the radial to normal fluid velocity. Utilizing the real favorable area fraction of the collector, the patch model expression for calculating the deposition efficiency is modified for impinging jet flow geometry.

Journal Article↗
PubMed2004

Protection in a mouse peritonitis model mediated by iron-regulated outer-membrane protein of Salmonella typhi coupled to its Vi antigen.

Vi polysaccharide and iron-regulated outer-membrane proteins (IROMPs) were extracted and purified from the standard strain of Salmonella typhi, Ty2. These were then conjugated by chemical coupling using the carbodimide method. Vi-IROMP conjugate was tested for its ability to protect against colonization by S. typhi in different organs. Mice immunized with 2.5 microg Vi-IROMP conjugate showed the most protection, as the least bacterial colonization of spleen, liver and Peyer's patches was observed. Peritoneal macrophages obtained from conjugate-treated mice phagocytosed bacteria efficiently. Circulating antibodies and the delayed type hypersensitivity response elucidated by mouse foot-pad swelling was significantly higher in conjugate-treated animals. These results clearly demonstrate that an IROMP and polysaccharide conjugate of S. typhi prepared from the same strain has the potential to protect animals against challenge.

Animals↗
PubMed2006

Kinetics of calcium spikes in rat cardiac myocytes.

The local calcium release flux signals (calcium spikes) evoked by membrane depolarization were recorded at high temporal resolution (2000 lines s(-1)) in isolated ventricular myocytes of male rats, using combination of scanning confocal microscopy and the patch-clamp technique. The kinetic properties of calcium spikes were investigated. The time course of calcium spike activation could be described reliably by a model with higher-order (n = 3) kinetics, but not by a first-order exponential process. A model of calcium spike with calcium release termination coupled to its activation was preferential to a model with the release termination independent of its activation. Three fluorescent calcium dyes (OG-5N, fluo-3, and fluo-4) were compared for calcium spike measurements. Experimental measurements as well as simulations showed that the occurrence and latency of calcium spikes could be measured faithfully with all indicators, while the kinetics of calcium spikes was reliably traced only with OG-5N. Calcium spikes evoked by a step depolarization from -50 to 0 mV commenced with a mean latency of 4.1 +/- 0.2 ms and peaked 6.7 +/- 0.2 ms later. Their full amplitudes were normally distributed. The activation time constant of calcium spikes was 3.1 +/- 0.1 ms, and the time constant of termination was 5.5 +/- 0.2 ms. A negative correlation was observed between the observed amplitude of calcium spikes and their time constant of activation, but there was no correlation between their observed amplitude and time constant of termination, in agreement with the concept of steep calcium-dependent activation and fateful inactivation of calcium release flux.

Animals↗
PubMed2001

Modeling transdermal drug release.

The stratum corneum forms the outermost layer of the skin and is essentially a multilamellar lipid milieu punctuated by protein-filled corneocytes that augment membrane integrity and significantly increase membrane tortuosity. The lipophilic character of the stratum corneum, coupled with its intrinsic tortuosity, ensure that it almost always provides the principal barrier to the entry of drug molecules into the organism; the only exceptions being highly lipophilic species which might encounter problems at the stratum corneum-viable epidermis interface where they must partition into a predominantly aqueous environment. Drugs can be administered either as suspensions or as solutions and the formulation can range in complexity from a gel or an ointment to a multilayer transdermal patch. In this review we describe the theoretical principles used to describe transdermal release and we show that relatively simple membrane transport models based on the appropriate solution to Fick's second law of diffusion can be used to explain drug release kinetics into this complex biological membrane.

Administration, Cutaneous↗
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↗