SEARCH · PubMed Health

Results for “coupled patch model”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

PubMed1997

A simple hardware model for the direct observation of voltage-clamp performance under realistic conditions.

A new hardware cell model for electrophysiological recording has been constructed which allows for the assessment of voltage clamp accuracy in different recording situations. Each compartment consists of a capacitor in parallel with a variable resistor and can be connected to other compartments by a variable axial resistance. The simulated membrane resistance can be changed extrinsically by a command voltage input which is optically coupled to the cell without any direct galvanic contact. Each compartment possesses a buffer amplifier which reads out the potential at the simulated membrane element, (e.g. 'somatic' or 'dendritic' potential). The model allows for the direct observation of typical situations and problems arising in electrophysiological experiments. We used the model to monitor deviations between the 'intracellular' and the command voltage, e.g. due to series resistance errors. We also used the model to simulate synaptic currents which were generated by triangular membrane conductance changes. The results demonstrate the strong influence of synaptic location and series resistance on voltage clamp fidelity. The cell model is a new and easy-to-handle tool for the observation of voltage control under realistic experimental conditions.

Animals↗

PubMed2001

Evidence for the action potential mediating the changes to contraction observed in cardiac hypertrophy in the rabbit.

BACKGROUND: We investigated the effects of cardiac hypertrophy on intracellular calcium (Ca(2+)) homeostasis, the amounts of proteins involved in calcium regulation and the influence of the action potential on such changes. METHODS: Cardiac hypertrophy was induced in rabbits by constriction of the ascending aorta. They were kept for 6 weeks then the heart was removed and left ventricular myocytes isolated. A portion of these myocytes was immediately frozen and stored for subsequent protein analyses using Western blotting. RESULTS: After aortic banding, cardiac myocyte two-dimensional area and membrane capacitance were increased by 53% and 23% respectively. Hypertrophy prolonged cell contraction and relaxation and the corresponding Indo-1 Ca(2+) transients. Hypertrophied cells displayed longer action potentials but Ca(2+) current densities were unchanged compared with myocytes from sham hearts. If Ca(2+) was released from the sarcoplasmic reticulum using rapid cooling, so bypassing the normal mechanisms involved in excitation-contraction coupling, then no functional differences between hypertrophied and control cells could be observed. Western blot analysis showed that the amounts of sarcoplasmic reticulum Ca(2+) ATPase, its regulatory protein phospholamban and the sodium/calcium exchanger were unchanged whereas the amount of calsequestrin was increased by 65% and the alpha(1) subunit of the sodium/potassium ATPase was reduced by 72%. These changes do not appear to evoke functional consequences under these conditions. CONCLUSION: In this model of cardiac hypertrophy, the increase in action potential duration is responsible for changes in contraction and relaxation.

Action Potentials↗

PubMed2004

Endothelial potassium channels, endothelium-dependent hyperpolarization and the regulation of vascular tone in health and disease.

1. The elusive nature of endothelium-derived hyperpolarizing factor (EDHF) has hampered detailed study of the ionic mechanisms that underlie the EDHF hyperpolarization and relaxation. Most studies have relied on a pharmacological approach in which interpretations of results can be confounded by limited specificity of action of the drugs used. Nevertheless, small-, intermediate- and large-conductance Ca2+-activated K+ channels (SKCa, IKCa and BKCa, respectively) have been implicated, with inward rectifier K+ channels (KIR) and Na+/K+-ATPase also suggested by some studies. 2. Endothelium-dependent membrane currents recorded using single-electrode voltage-clamp from electrically short lengths of arterioles in which the smooth muscle and endothelial cells remained in their normal functional relationship have provided useful insights into the mechanisms mediating EDHF. Charybdotoxin (ChTx) or apamin reduced, whereas apamin plus ChTx abolished, the EDHF current. The ChTx- and apamin-sensitive currents both reversed near the expected K+ equilibrium potential, were weakly outwardly rectifying and displayed little, if any, time- or voltage-dependent gating, thus having the biophysical and pharmacological characteristics of IKCa and SKCa channels, respectively. 3. The IKCa and SKCa channels occur in abundance in endothelial cells and their activation results in EDHF-like hyperpolarization of these cells. There is little evidence for a significant number of these channels in healthy, contractile vascular smooth muscle cells. 4. In a number of blood vessels in which EDHF occurs, the endothelial and smooth muscle cells are coupled electrically via myoendothelial gap junctions. In contrast, in the adult rat femoral artery, in which the smooth muscle and endothelial layers are not coupled electrically, EDHF does not occur, even though acetylcholine evokes hyperpolarization in the endothelial cells. 5. In vivo studies indicate that EDHF contributes little to basal conductance of the vasculature, but it contributes appreciably to evoked increases in conductance. 6. Endothelium-derived hyperpolarizing factor responses are diminished in some diseases, including hypertension, pre-eclampsia and some models of diabetes. 7. The most economical explanation for EDHF in vitro and in vivo in small vessels is that it arises from the activation of IKCa and SKCa channels in endothelial cells. The resulting endothelial hyperpolarization spreads via myoendothelial gap junctions to result in the EDHF-attributed hyperpolarization and relaxation of the smooth muscle.

Animals↗

PubMed1981

Inhibition of the mobility of mouse lymphocyte surface immunoglobulins by locally bound concanavalin A.

Fluorescence photobleaching recovery was used to study directly and quantitatively the inhibition of the lateral mobility of surface immunoglobulins (sIg) on mouse lymphocytes by localized binding of concanavalin A (Con A) coupled to platelets. Up to a threshold occupancy of about 10% of the upper cell surface by Con A-platelets, the diffusion coefficient and mobile fraction of sIg remained as in untreated cells (5.3 X 10(-10) cm2/sec and 0.65, respectively). At higher surface occupancy, these values decreased to 8 X 10(-11) cm2/sec and 0.11. The magnitude of the effect was independent of the percentage occupancy above the threshold and of the distance from the bound Con A-platelets, indicating a cooperative and propagated phenomenon. Treatment with colchicine or cytochalasin B separately induced only partial reversal of the Con A-induced modulation. Treatment with both reversal of the Con A-induced modulation. Treatment with both drugs together was synergistic and fully reversed the mobility inhibition. The modulation was unaffected by NaN3 and 2-deoxyglucose, suggesting no dependence on metabolic energy. Con A-platelets did not affect the mobility of a lipid probe. Models for the Con A-induced modulation and the relationship between the effects of Con A on sIg mobility and patch formation are discussed.

Animals↗

PubMed1992

Single-membrane and cell-to-cell permeability properties of dissociated embryonic chick lens cells.

Ion channels are believed to play an important role in the maintenance of lens transparency. In order to ascribe junctional and nonjunctional permeability properties to specific lens cell types, embryonic chick lenses were enzymatically dissociated into cell clusters, cell pairs and single cells, and both cell-to-cell and single-membrane permeability properties were characterized with the patch-clamp technique. Double patch-clamp experiments and single patch-clamp experiments with Lucifer yellow in the pipette demonstrated that the cells in the dissociated preparation were well coupled, the average conductance between pairs being 42 +/- 27 nS. Double patch-clamp experiments also revealed single cell-to-cell channel events with a predominant unitary conductance of 286 +/- 38 pS. Whole-cell measurements of surface membrane conductance indicate heterogeneity within the population of dissociated embryonic chick lens cells: 63% of the cells have a voltage-independent leak current, 14% of the cells have a potassium-selective inward-rectifier current, and 23% of the cells have a current which turns off with positive voltage on a time scale on the order of seconds. The time constant for this turnoff is voltage dependent.

Animals↗

PubMed1997

Oscillation of gap junction electrical coupling in the mouse pancreatic islets of Langerhans.

1. Pancreatic beta-cells oscillate synchronously when grouped in islets. Coupling seems essential to maintain this oscillatory behaviour, as isolated cells are unable to oscillate. This allows the islet to be used as a model system for studying the role of coupling in the generation of oscillatory patterns. 2. Pairs of beta-cells were intracellularly recorded in islets. beta-Cells oscillated synchronously. Propagated voltage deflections were observed as a function of glucose concentration and of the distance between the recording electrodes. Space constants were smaller in the silent than in the active phases, suggesting a higher intercellular connection in the active phases. 3. Coupling coefficients and estimated coupling conductances were larger in the active than in the silent phases. 4. Coupling coefficients and coupling conductances changed dynamically and in phase with the membrane potential oscillations, pointing to an active modulation of the gap junctions. 5. We hypothesize a role for coupling in the generation of the oscillatory events, providing different levels of permeability dependent on the state of conductance during the oscillatory phases.

Animals↗

PubMed2004

Phosphodiesterase 4D forms a cAMP diffusion barrier at the apical membrane of the airway epithelium.

We demonstrated previously that Calu-3 airway epithelial cells sense adenosine on their luminal surface through adenosine A2B receptors coupled to adenylyl cyclase. Occupancy of these receptors leads to activation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel through protein kinase A (PKA) anchored at the apical membrane. Because luminal A2B receptor activation does not raise total cellular cAMP levels, we hypothesized that activation of phosphodiesterases (PDEs) confines cAMP generated by apical A2B receptors to a microdomain that includes the CFTR channel. Using reverse transcription-PCR, Western blotting, and activity measurements, PDE4D was identified as the major PDE species in airway epithelia. Consistent with these results, inhibitors of PDE4, but not PDE3, selectively abolished the lateral confinement of cAMP signaling in apical membrane patches during cell-attached recordings. Furthermore, stimulation of the CFTR in excised apical patches by rolipram and RS25344 indicated that PDE4 is localized in close proximity to the CFTR channel. Indeed, immunohistochemistry of human airway sections revealed that PDE4D is localized in the apical domain of the cell. PDE4 was activated after luminal adenosine exposure in a PKA-dependent manner. Because PDE4 activity is positively regulated by PKA, our results support a model whereby the PDE diffusion barrier is proportional to the degree of receptor stimulation. These findings underscore the concept that subcellular localization of individual PDE isozymes is an important mechanism for confining cAMP signaling to functional domains within cells.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2002

Membrane stretch accelerates activation and slow inactivation in Shaker channels with S3-S4 linker deletions.

At low P(open)(V) Shaker exhibits pronounced stretch-activation. Possible explanations for Shaker's sensitivity to tension include 1) Shaker channels are sufficiently distensible that stretch produces novel channel states and 2) Shaker channels expand in the plane of the membrane during voltage gating. For channels expressed in oocytes, we compared effects of patch stretch on Shaker and mutants that retain their voltage-gating ability but activate sluggishly because all or most of the S3-S4 linker has been deleted. Deletants had 10, 5, or 0 amino acid (aa) linkers, whereas wild-type is 31 aa. In deletants, though activation is exceptionally slow, slow inactivation is exceptionally quick; the resulting kinetic match was a bonus that allowed effects of stretch to be followed simultaneously in both processes. With the intact linker, an approximately 3 orders of magnitude mismatch in the two processes makes this impracticable. Standard stretch stimuli increased the rates and extent of activation by about the same degree in wild type and deletants, with effects especially pronounced near the foot of G(V). In deletants (where slow inactivation is strongly coupled to activation) stretch also accelerated slow inactivation. Maximum conductances were unaffected by stretch in all variants. In ramp clamp dose experiments, near-lytic patch stretch acted, for all variants, like a approximately 10 mV hyperpolarizing shift. These results suggested that, whether basal rates were high (wild type) or low (deletants), stretch acted by facilitating voltage-dependent activation. Channel activity was therefore simulated with/without "tension," tension being simulated via rate changes at voltage-dependent closed-closed transitions that might involve in-plane expansion (explanation 2). Simulated Delta P(open) arising from approximately 2 kT of "mechanical gating energy" mimicked experimental effects seen with comfortably sub-lytic stretch.

Animals↗

PubMed1996

Mechanisms of the human intestinal H+-coupled oligopeptide transporter hPEPT1.

The hPEPT1 cDNA cloned from human intestine (Liang, R., Fei, Y.-J., Prasad, P. D., Ramamoorthy, S., Han, H., Yang-Feng, T. L., Hediger, M. A., Ganapathy, V., and Leibach, F. H. (1995) J. Biol. Chem. 270, 6456-6463) encodes a H+/oligopeptide cotransporter. Using two-microelectrode voltage-clamp in Xenopus oocytes expressing hPEPT1, we have investigated the transport mechanisms of hPEPT1 with regard to voltage dependence, steady-state kinetics, and transient charge movements. The currents evoked by 20 mM glycyl-sarcosine (Gly-Sar) at pH 5.0 were dependent upon membrane potential (Vm) between -150 mV and +50 mV. Gly-Sar-evoked currents increased hyperbolically with increasing extracellular [H+], with Hill coefficient approximately 1, and the apparent affinity constant (K0.5H) for H+ was in the range of 0.05 1 microM. K0.5 for Gly-Sar (K0.5GS) was dependent upon Vm and pH; at -50 mV, K0.5H was minimal (approximately 0.7 mM) at pH 6.0. Following step-changes in Vm, in the absence of Gly-Sar, hPEPT1 exhibited H+-dependent transient currents with characteristics similar to those of Na+-coupled transporters. These charge movements (which relaxed with time constants of 2-10 ms) were fitted to Boltzmann relations with maximal charge (Qmax) of up to 12 nC; the apparent valence was determined to be approximately 1. Qmax is an index of the level of transporter expression which for hPEPT1 was in the order of 1011/oocyte. In general our data are consistent with an ordered, simultaneous transport model for hPEPT1 in which H+ binds first.

Animals↗

PubMed2003

Voltage-dependent Ca2+ fluxes in skeletal myotubes determined using a removal model analysis.

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the "removal model fit" approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 microM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at -16 and -20 mV, respectively, compared with -2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 microM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

Animals↗

PubMed1984

Sequential events in calmodulin on binding with calcium and interaction with target enzymes.

khe conformational and functional events in calmodulin (CaM) are disproportionate to the mean saturation by Ca2+. The enhancement of intrinsic tyrosine fluorescence closely follows the appearance of species CaM X Can greater than or equal to 1; the exposure of the hydrophobic patch at the surface of CaM coincides with the appearance of CaM X Can greater than or equal to 2. For the activation of four different target enzymes, i.e., brain phosphodiesterase and adenylate cyclase, red blood cell Ca,Mg-ATPase, and skeletal muscle phosphorylase b kinase, CaM X Can greater than or equal to 3 is required. The different enzymes have the same affinity for the active species. The direct interaction of CaM with Ca2+ and phosphorylase b kinase has been analyzed according to the theory of energy coupling: whereas the first two stoichiometric calcium-binding constants in the complex are not significantly different from those of free CaM, the third Ca2+ binds with an affinity at least 10(6)-fold higher to enzyme-bound CaM than to free CaM, which corresponds to a free energy coupling of -7 kcal/mol CaM. The similarities in the activation mechanism of different enzymes suggest the existence of one unique CaM-binding domain. The characteristics of the interaction between CaM and melittin, a small amphiphatic cytotoxin, led us to propose melittin as a model for such a CaM-binding domain.

Allosteric Regulation↗

PubMed2006

Slo3 K+ channels: voltage and pH dependence of macroscopic currents.

The mouse Slo3 gene (KCNMA3) encodes a K(+) channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca(2+) and voltage-activated BK-type channel, the Slo3 alpha subunit contains a pore module with homology to voltage-gated K(+) channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K(+) channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich-type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (-300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (z(L)) of the Slo3 closed-open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.

Allosteric Regulation↗

PubMed2004

Regulation of KCNQ2/KCNQ3 current by G protein cycling: the kinetics of receptor-mediated signaling by Gq.

Receptor-mediated modulation of KCNQ channels regulates neuronal excitability. This study concerns the kinetics and mechanism of M1 muscarinic receptor-mediated regulation of the cloned neuronal M channel, KCNQ2/KCNQ3 (Kv7.2/Kv7.3). Receptors, channels, various mutated G-protein subunits, and an optical probe for phosphatidylinositol 4,5-bisphosphate (PIP2) were coexpressed by transfection in tsA-201 cells, and the cells were studied by whole-cell patch clamp and by confocal microscopy. Constitutively active forms of Galphaq and Galpha11, but not Galpha13, caused a loss of the plasma membrane PIP2 and a total tonic inhibition of the KCNQ current. There were no further changes upon addition of the muscarinic agonist oxotremorine-M (oxo-M). Expression of the regulator of G-protein signaling, RGS2, blocked PIP2 hydrolysis and current suppression by muscarinic stimulation, confirming that the Gq family of G-proteins is necessary. Dialysis with the competitive inhibitor GDPbetaS (1 mM) lengthened the time constant of inhibition sixfold, decreased the suppression of current, and decreased agonist sensitivity. Removal of intracellular Mg2+ slowed both the development and the recovery from muscarinic suppression. When combined with GDPbetaS, low intracellular Mg2+ nearly eliminated muscarinic inhibition. With nonhydrolyzable GTP analogs, current suppression developed spontaneously and muscarinic inhibition was enhanced. Such spontaneous suppression was antagonized by GDPbetaS or GTP or by expression of RGS2. These observations were successfully described by a kinetic model representing biochemical steps of the signaling cascade using published rate constants where available. The model supports the following sequence of events for this Gq-coupled signaling: A classical G-protein cycle, including competition for nucleotide-free G-protein by all nucleotide forms and an activation step requiring Mg2+, followed by G-protein-stimulated phospholipase C and hydrolysis of PIP2, and finally PIP2 dissociation from binding sites for inositol lipid on the channels so that KCNQ current was suppressed. Further experiments will be needed to refine some untested assumptions.

Cell Membrane↗

PubMed1998

Spatial changes in the transmembrane potential during extracellular electric stimulation.

The purpose of this study was to determine the spatial changes in the transmembrane potential caused by extracellular electric field stimulation. The transmembrane potential was recorded in 10 guinea pig papillary muscles in a tissue bath using a double-barrel microelectrode. After 20 S1 stimuli, a 10-ms square wave S2 shock field with a 30-ms S1-S2 coupling interval was given via patch shock electrodes 1 cm on either side of the tissue during the action potential plateau. Two shock strengths (2.1+/-0.2 and 6.5+/-0.6 V/cm) were tested with both shock polarities. The recording site was moved across the tissue along fibers with either 200 micrometer (macroscopic group [n=5], 12 consecutive recording sites over a 2. 2-mm tissue length in each muscle) or 20 micrometer (microscopic group [n=5], 21 consecutive recording sites over a 0.4-mm tissue length in each muscle) between adjacent recording sites. In the macroscopic group, the portion of the tissue toward the anode was hyperpolarized, whereas the portion toward the cathode was depolarized, with 1 zero-potential crossing from hyperpolarization to depolarization present near the center of the tissue. In the microscopic group, only 1 zero-potential crossing was observed in the center region of the tissue, whereas, away from the center, only hyperpolarization was observed toward the anode and depolarization toward the cathode. Although these results are consistent with predictions from field stimulation of continuous representations of myocardial structure, ie, the bidomain and cable equation models, they are not consistent with the prediction of depolarization-hyperpolarization oscillation from representations based on cellular-level resistive discontinuities associated with gap junctions, ie, the sawtooth model.

Animals↗

PubMed2006

Clustering of raft-associated proteins in the external membrane leaflet modulates internal leaflet H-ras diffusion and signaling.

One of the least-explored aspects of cholesterol-enriched domains (rafts) in cells is the coupling between such domains in the external and internal monolayers and its potential to modulate transbilayer signal transduction. Here, we employed fluorescence recovery after photobleaching to study the effects of antibody-mediated patching of influenza hemagglutinin (HA) proteins [raft-resident wild-type HA and glycosylphosphatidylinositol-anchored HA, or the nonraft mutant HA(2A520)] on the lateral diffusion of internal-leaflet raft and nonraft Ras isoforms (H-Ras and K-Ras, respectively). Our studies demonstrate that the clustering of outer-leaflet or transmembrane raft-associated HA proteins (but not their nonraft mutants) retards the lateral diffusion of H-Ras (but not K-Ras), suggesting stabilized interactions of H-Ras with the clusters of raft-associated HA proteins. These modulations were paralleled by specific effects on the activity of H-Ras but not of the nonraft K-Ras. Thus, clustering raft-associated HA proteins facilitated the early step whereby H-Ras is converted to an activated, GTP-loaded state but inhibited the ensuing step of downstream signaling via the Mek/Erk pathway. We propose a model for the modulation of transbilayer signaling by clustering of raft proteins, where external clustering (antibody or ligand mediated) enhances the association of internal-leaflet proteins with the stabilized clusters, promoting either enhancement or inhibition of signaling.

Animals↗

PubMed1997

Comparison of the physiologically equivalent proteins cytochrome c6 and plastocyanin on the basis of their electrostatic potentials. Tryptophan 63 in cytochrome c6 may be isofunctional with tyrosine 83 in plastocyanin.

The blue copper protein plastocyanin and the heme protein cytochrome c6 differ in composition and in structure but perform the same function in the photosynthetic electron-transport chain. We compare these two proteins on the basis of their electrostatic potentials in order to understand the structural basis of their functional equivalence. In the first approach, we use a monopole-dipole approximation of the electrostatic potentials to superimpose the proteins. The resulting alignment suggests that Tyr51 in cytochrome c6 corresponds to Tyr83 in plastocyanin. But since Tyr51 is not conserved in all known cytochrome c6 sequences, a physiological role of this residue is questionable. In a more sophisticated approach, we applied the recently-developed Fame (flexible alignment of molecule ensembles) algorithm, in which molecules are superimposed by optimizing the similarity of their electrostatic potentials with respect to the relative orientation of the molecules. On the basis of the Fame alignments of plastocyanin and cytochrome c6, we analyze the docking and the electron-transfer reactions of these two proteins with its physiological reaction partner cytochrome f. We derive functional analogies for individual amino acids in possible electron-transfer paths in the interprotein redox reactions. We identify two surface patches in cytochrome c6 that may be involved in electron-transfer paths. The hydrophobic patch with the exposed heme edge in cytochrome c6 may be equivalent to the hydrophobic patch with His87 in plastocyanin, whereas Trp63 in cytochrome c6 may be equivalent to Tyr83 in plastocyanin. An aromatic amino acid is present at the position of Trp63 in all known cytochrome c6 sequences. The electronic coupling between the heme and the copper site on the one side and several potentially important amino acid residues on the other is analyzed by the Pathways method. We have proposed recently that Lys65 of cytochrome f and Tyr83 of plastocyanin form a cation-pi system, which may be involved in a two-step mechanism of the electron-transfer reaction between these two proteins from higher plants. Now we corroborate this proposal by analyzing available amino acid sequences.

Algorithms↗

PubMed1997

Kinetics of high-affinity K+ uptake in plants, derived from K(+)-induced changes in current-voltage relationships. A modelling approach to the analysis of carrier-mediated transport.

To investigate coupled, charge-translocating transport, it is imperative that the specific transporter current-voltage (IV) relationship of the transporter is separated from the overall membrane IV relationship. We report here a case study in which the currents mediated by the K(+)-H+ symporter, responsible for high-affinity K+ uptake in Arabidopsis thaliana (L.) Heynh. cv. Columbia roots, are analyzed with an enzyme kinetic reaction scheme. The model explicitly incorporates changes in membrane voltage and external substrate, and enables the derivation of the underlying symport IV relationships from the experimentally obtained difference IV data. Data obtained for high-affinity K+ transport in A. thaliana root protoplasts were best described by a 1:1 coupled K(+)-H+ symport-mediated current with a parallel, outward non-linear K+ pathway. Furthermore, the large predictive value of the model was used to describe symport behaviour as a function of the external K+ concentration and the cytoplasmic K+ concentration. Symport activity is a complex function of the external K+ concentration, with first-order saturating kinetics in the micromolar range and a strong activity reduction when external K+ is in the millimolar range and the membrane depolarises. High cytoplasmic K+ levels inhibit symport activity. These responses are suggested to be part of the feedback mechanisms to maintain cellular K+ homeostasis. The general suitability of the model for analysis of carrier-mediated transport is discussed.

Antiporters↗

PubMed1993

Interaction of immunologically-active lipopeptides with membranes.

Synthetic tripalmitoyl-S-glycerylcysteinyl (Pam3Cys) peptides are derived from the N-terminal part of bacterial lipoprotein and constitute polyclonal B-lymphocyte and macrophage activators. In order to elucidate the primary events of leukocyte activation, we investigated the biophysical interaction of lipopeptides containing spin labels or fluorescent markers with phosphatidylcholine vesicles or immune cells. Utilizing fluorescence microscopy and FACS analysis we found, that the surface of cells, after incubation with a fluorescein-labelled lipopeptide, was highly fluorescent. In addition, capping and patching was observed. Furthermore, fluorescence quenching experiments and electron paramagnetic resonance studies using vesicles incubated with lipopeptides suggested, that the peptide moiety and other more polar molecules linked to the lipo-amino acid are exposed to the hydrophilic compartment. These results show that in lipopeptide conjugates the Pam3Cys moiety acts as an efficient membrane anchor for molecules covalently coupled to it. The sequestering of the fatty-acid chains of the lipopeptide within the membrane is an early step of interaction, which might induce the uptake of the lipopeptide into the cell and the stimulation of immunocompetent cells.

Amino Acid Sequence↗