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B Prod'hom

Publications and source records attributed to B Prod'hom.

17 recordsLinked to original sources
PubMed1996

Characterization of a whole-cell Ca2+-blockable monovalent cation current in isolated ectodermal cells of chick embryo.

The presence of a Ca2+-blockable monovalent cation current is demonstrated in isolated ectodermal cells of the chick embryo using the whole-cell patch-clamp method. In the absence of any stimulation, the whole-cell current is time independent and rectifies outwardly at membrane potentials higher than +40 mV. The outward current is neither carried by Cl- channels nor by K+ channels. Application of a Ca2+-free solution containing 1 mmol/l ethylenediaminetetraacetic acid (EDTA) elicits a large inward current and increases the outward current. The inward current can be carried by extracellular Li+, Na+, K+ and Cs+, but not N-methyl-D-glucamine. The Ca2+-blockable monovalent cation channel discriminates very poorly among these cations. The estimated number of channels per cell is around 2000. Extracellular protons block the inward Na+ current in the absence of extracellular Ca2+. The apparent negative logarithm of the dissociation constant for proton (pKH) at -100 mV is 5.8. Among 12 potential channel modulators, including verapamil and nifedipine, only quinine decreases the current. Quinine blocks this current with a dissociation constant, Kd, equal to 0.18 mmol/l, independent of the membrane potential. This study demonstrates the presence of a whole-cell Ca2+-blockable monovalent cation current in dissociated chick ectodermal cells with permeation properties similar to those observed at the single-channel level. Contrary to studies made of other tissues, we did not observe any blocking effect of verapamil and nifedipine on the Ca2+-blockable monovalent cation current.

Animals↗
PubMed1995

Extracellular protons modulate the Ca2+ block of a Ca(2+)-blockable monovalent cation channel in chick embryo.

Some epithelial nonselective cation channels although in an open conformation are impermeable because they are blocked by extracellular Ca2+ ions. Such a channel is present in the apical membrane of the ectodermal cells of the chick gastrula. This single-channel study shows that protonation of a site located inside the pore of this embryonic channel reduces the Ca2+ block thus allowing Na+ to pass. The apparent pK for Ca2+ at -50 mV shifts from 5.7 at pH 7.4 to 3.5 at pH 4.5. Unexpectedly, the change in Ca2+ affinity does not originate from a destabilization of the binding but comes from a large reduction of the association rate.

Animals↗
PubMed1995

Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy.

The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions.

Binding Sites↗
PubMed1995

Permeation properties of a Ca(2+)-blockable monovalent cation channel in the ectoderm of the chick embryo: pore size and multioccupancy probed with organic cations and Ca2+.

A Ca(2+)-blockable monovalent cation channel is present in the apical membrane of the ectoderm of the gastrulating chick embryo. We used the patch clamp technique to study several single-channel permeation properties of this channel. In symmetrical conditions without Ca2+, the Na+ current carried by the channel rectifies inwardly. The channel has an apparent dissociation constant for extracellular Na+ of 115 mM at 0 mV and a low density of negative surface charge (-0.03 e/nm2) at its extracellular entrance. The minimal pore diameter is approximately 5.8 A, as calculated from the relative permeabilities of 10 small organic cations. Extracellular application of six large organic cations decreased the inward Na+ current in a voltage-dependent manner, which strongly suggests an intrachannel block. The presence of at least two ion binding sites inside the pore is inferred from the Na+ dependence of the block by the organic cations. This hypothesis is strengthened by the fact that the extracellular Ca2+ block is also modified by the Na+ concentration. In particular, the rise of the unblocking rate with increased Na+ concentrations clearly suggests the presence of an interaction between Ca2+ and Na+ inside the channel. A low probability of double occupancy at physiological ionic conditions is implied from the absence of an anomalous mole fraction effect with mixtures of extracellular Li+ and K+. Finally, the absence of inward current at very strong hyperpolarizations and in the presence of 10 mM extracellular Ca2+ demonstrates the absence of significant Ca2+ current through this channel. It is argued that this embryonic epithelial Ca(2+)-blockable monovalent cation channel is related to both L-type Ca2+ channel and cyclic nucleotide-gated channels.

Animals↗
PubMed1994

Cation channel blocked by extracellular Ca2+ in the apical membrane of the chick embryonic ectoderm.

In the chick embryo (20 h incubation, gastrula stage), the apical membrane of the ectodermal cells shows a high density of a non-selective cation channel which is blocked by very low extracellular Ca2+ concentrations. Properties of this channel were studied at the single-channel level using the patch-clamp technique in the cell-attached mode. With 1 mmol/l Ca2+ in the pipette, only outward current was present and the channel conductance measured at +120 mV was 25.5 pS. In the absence of Ca2+, also inward current through the channel was observed. The conductances measured at -50 mV were 49.5 pS with Na+ as the charge carrier, 72.5 pS with K+, 49.1 pS with Cs+, and 18.5 pS with Li+. The conductance measured at +80 mV was around 23 pS in all four cases. The reversal potential was similar (around 25 mV) for all four ions, which indicates a poor selectivity of the channel. In the absence of Ca2+ and the presence of 1 mmol/l ethylenebis(oxonitrilo)tetraacetate (EGTA), the kinetics of the channel were characterized by bursts of the order of seconds. During a burst, the channel flickered between one open and one closed level. The open time was constant between -30 mV and -80 mV, while the closed time decreased with hyperpolarization. The open time varied according to the permeant ion (K+ < Na+ = Cs+ < Li+). Extracellular Ca2+ blocked the inward current in a voltage-dependent manner. The Kd values, 1 mumol/l at -30 mV and 3.2 mumol/l at -80 mV, indicate that Ca2+ ions exit the channel toward the intracellular side. A weak voltage dependency of the association rate constant suggests that the Ca(2+)-binding site is close to the outside mouth. Extracellular Ca2+ was much less efficient at blocking the outward current (Kd about 1 mmol/l at 80 mV). Tetracaine, but not uraniumdioxide, decreased the opening probability of the channel. The embryonic channel shows similarities with the Ca(2+)-blockable, poorly selective channel described in the epithelium of toad urinary bladder.

Animals↗
PubMed1993

The nature of the ion binding interactions in EF-hand peptide analogs: free energy simulation of Asp to Asn mutations.

The binding of the La3+ ion to a tridecapeptide, which is a model for the EF-hand in calcium-binding proteins, is studied in solution by free energy simulations. The calculations analyze the effect on the La3+ ion binding of the mutation of Asp to Asn for side chains that interact directly with the ion. The results are compared with the measurements of Marsden,B.J., Hodges,R.S. and Sykes,B.D. (1989) Biochemistry, 28, 8839, on the same system. They found that the Asp to Asn mutation has only a small effect on the binding; the observed differences in the free energies on changing one Asp to an Asn are between -0.3 and 1.8 kcal/mol. This result is analyzed by alchemical simulations for the tridecapeptide in the bound (loop) structure and free (extended) form. The free energy changes due to the mutation of an Asp to an Asn are large and positive for both the bound and free forms. However, since the values of the free energy changes are calculated to be similar in the two forms, the difference in the binding free energy of Asp and Asn peptides is found to be small, in agreement with experiment. By use of thermodynamic integration, the various contributions to the free energy changes are estimated. In the complexed form, the Asp to Asn mutation is favored by the reduction in the repulsive interaction with other charged residues of the peptide; it is disfavored by the reduction of the stabilization of the ion and the surrounding water has a small effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1989

Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions.

We studied the pH dependence of the proton-induced current fluctuations that appear in single open L-type Ca channels when monovalent ions are the charge carriers. We used different methods of analysis to obtain kinetic measurements even under conditions where the individual transitions were too fast to be resolved directly as discrete current steps between two conductance levels. The reciprocal of the dwell times at the high conductance level increased linearly with the pipette proton activity, with a slope that was similar for Cs, K, and Na as permeant ions. Contrary to the expectation for a simple model in which the high and low conductances represent the unprotonated and protonated states of the channel, respectively, the dwell times at the low conductance level were also pH dependent and lengthened with increasing proton activity. At all pH values the dwell times at the low conductance level were longest with Cs as permeant ion and shortened in the order Cs greater than K greater than Na. We introduce a more general model of the protonation cycle in which the channel is represented by four states and can be protonated and deprotonated both at the high and low conductance levels. The conductance change is represented by a conformational change of the channel protein. We discuss the validity of this model and its implications for the mechanism by which protons interact with ion permeation through L-type Ca channels.

Animals↗
PubMed1989

Interactions of protons with single open L-type calcium channels. Location of protonation site and dependence of proton-induced current fluctuations on concentration and species of permeant ion.

We further investigated the rapid fluctuations between two different conductance levels promoted by protons when monovalent ions carry current through single L-type Ca channels. We tested for voltage dependence of the proton-induced current fluctuations and for accessibility of the protonation site from both sides of the membrane patch. The results strongly suggest an extracellular location of the protonation site. We also studied the dependence of the kinetics of the fluctuations and of the two conductance levels on the concentration of permeant ion and on external ionic strength. We find that saturation curves of channel conductance vs. [K] are similar for the two conductance levels. This provides evidence that protonation does not appreciably change the surface potential near the entry of the permeation pathway. The proton-induced conduction change must therefore result from an indirect interaction between the protonation site and the ion-conducting pathway. Concentration of permeant ion and ionic strength also affect the kinetics of the current fluctuations, in a manner consistent with our previous hypothesis that channel occupancy destabilizes the low conductance channel conformation. We show that the absence of measurable fluctuations with Li and Ba as charge carriers can be explained by significantly higher affinities of these ions for permeation sites. Low concentrations of Li reduce the Na conductance and abbreviate the lifetimes of the low conductance level seen in the presence of Na. We use whole-cell recordings to extrapolate our findings to the physiological conditions of Ca channel permeation and conclude that in the presence of 1.8 mM Ca no proton-induced fluctuations occur between pH 7.5 and 6.5. Finally, we propose a possible physical interpretation of the formal model of the protonation cycle introduced in the companion paper.

Animals↗
PubMed1988

Conformational changes associated with ion permeation in L-type calcium channels.

The mechanism by which ions deliver their message to effector proteins involves a change in the protein conformation which is induced by the specific interaction of the ion with its binding site on the protein. In the case of an ion-channel protein, conformational changes induced by permeant ions and the consequences for channel function have received little attention. Here we report that binding of permeant cations to an intra-channel binding site of the dihydropyridine (DHP)-sensitive (L-type) Ca2+ channel leads to a conformational change which destabilizes the protonated state of a group on the external channel surface, and can shift its apparent pK value by more than 2 pH units. The lifetime of the protonated state correlates with the occupancy of an intra-channel binding site by the permeant cation. The demonstration of such conformational changes in a channel protein induced by the permeant ion has important implications for realistic models of the mechanism of ion permeation.

Animals↗
PubMed1987

Voltage-dependent cooperative interactions of calcium channel ligands in intact cardiac cells.

Voltage-dependent Ca channels were studied in living, tissue cultured rat heart cells using patch clamp analysis of single channels and radioligand binding studies at different membrane potentials. Ca channel activating and blocking dihydropyridines (DHP) show cooperative interaction and, therefore, bind to at least two different binding sites on the channel protein. Cooperative interactions between activating and blocking DHP, between DHP and verapamil and between DHP and diltiazem are all voltage dependent. The type of interaction in polarized cells and hence, the possible effects in vivo cannot be predicted from studies in cell homogenates.

Allosteric Site↗
PubMed1986

Properties and modulation of cardiac calcium channels.

Voltage-dependent calcium channels are widely distributed in excitable membranes and are involved in the regulation of many cellular functions. These channels can be modulated by neurotransmitters and drugs. There is one particular type of calcium channel in cardiac cells (L-type) whose gating is affected in different ways by beta-adrenoceptor and 1,4-dihydropyridine agonists. We have analysed single calcium channel currents (i) in myocytes from rat hearts in the absence and presence of isoproterenol or 8-bromo-cAMP. We have found that both compounds have similar effects on calcium channel properties. They increase the overall open state probability (po) of individual calcium channels while i remains unaffected. Analysis of the gating kinetics of calcium channels showed: a slight increase in the mean open times of calcium channels, a reduction in time intervals between bursts of channel openings, an increase in burst length and a prominent reduction in failures of calcium channels to open upon depolarization. These kinetic changes caused by isoproterenol and 8-bromo-cAMP can account for the increase in po. Since the macroscopic calcium current, ICa, can be described by ICa = N X po X i, the increase in po accounts for the well-known increase in ICa by beta-adrenergic catecholamines. Cyclic AMP-dependent phosphorylation of calcium channels is a likely metabolic step involved in this modulation. Another class of drug that modulates calcium channel gating is the 1,4-dihydropyridines which can either enhance or reduce ICa, either by prolonging the open state of the channels or by facilitating the inactivated state. Both effects depend strongly on membrane potential and are independent of cyclic AMP-dependent phosphorylation reactions.

Adrenergic beta-Agonists↗
PubMed1986

Studies on Ca channels in intact cardiac cells: voltage-dependent effects and cooperative interactions of dihydropyridine enantiomers.

We have investigated the effects of two oppositely acting enantiomers of the 1,4-dihydropyridine derivative 202-791 on voltage-dependent Ca channels by combining electrophysiological techniques and binding studies. The (S)-enantiomer of 202-791 promoting prolonged openings of single Ca channels, and thereby increasing transmembrane Ba currents, was classified as channel activator. The (R)-enantiomer favoring a closed state of the channel, and thereby reducing Ba currents, was classified as a channel blocker. Both compounds shifted the steady state current inactivation curve toward more negative potentials. At holding potentials positive to -20 mV, the Ca channel-activating effect of the (S)-enantiomer turned over into a blocking effect. In cells with normal resting potential the combination of the two enantiomers revealed a possible positive cooperative effect resulting in an enhancement of the open state probability of the channels. At depolarized holding potentials the activator enhanced the inhibitory effect of the blocker. Binding studies in intact cells were performed by using the radiolabeled channel-blocking dihydropyridine 3H-(+)-PN 200-110. The results showed a strong increase in binding affinity but no change in binding capacity when the cells were depolarized. Analysis of the interactions of (S)- and (R)-202-791 with this radioligand indicated stimulation of 3H-(+)-PN 200-110 binding by the (S)-enantiomer in polarized cells (membrane potential -38 +/- 4 mV). This effect could be attributed to an increase in binding affinity. The (R)-enantiomer had no such positive cooperative effect, but acted as a purely competitive ligand. Depolarization to 0 mV increased the apparent affinity of both enantiomers by factors of 38 (blocker) and 12 (activator), but abolished the cooperative effect of (S)-202-791 on the binding of the radioligand. Ca ions had little effect on the binding of 3H-(+)-PN 200-110 in polarized cells. However, in the presence of the activating (S)-enantiomer, Ca transformed the usual hyperbolic binding isotherm of the radioligand into a strongly sigmoid curve. Sigmoidicity was minimal with 3-5 microM Ca and maximal with 0.5 mM Ca. Together these data demonstrate homotropic and heterotropic cooperative interactions between channel activator and channel blocker. They indicate that at least two high affinity binding sites for dihydropyridines are associated with voltage-dependent Ca channels. Voltage dependence of both--binding affinity and cooperativity--suggests that these binding sites are located close to a structural component of the channel which is involved in the potential-sensitive gating process.

Aging↗
PubMed1986

The voltage-dependent effect of 1,4-dihydropyridine enantiomers on Ca channels in cardiac cells.

We studied voltage-dependent binding and action of 1,4-dihydropyridine enantiomers (Sandoz (+)-(S) & (-)-(R)-202-791) in intact cardiac cells. Drug action was studied by patch clamp method. (+)-enantiomer primarily prolonged open time of Ca channel, thus enhancing Ca currents, while (-)-enantiomer predominantly favored closed state of the channel, reducing the currents. Electrophysiological study also revealed that steady-state inactivation was greatly enhanced by both enantiomers. Therefore, it seems that both enantiomers have a capability of activating and blocking effects. Strongly voltage-dependent binding affinity of these enantiomers was revealed by displacement of 3H-(+)-PN200-110 with each enantiomers. Binding affinity of both enantiomers in depolarized cells was much higher than in polarized cells. These results indicate that both enantiomers have higher affinity when Ca channels are inactivated.

Animals↗
PubMedDate not supplied

Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration.

A rapid, transient rise in the free cytosolic Ca2+ concentration ([Ca2+]i) is one of the earliest events in neutrophil activation and is assumed to be involved in many of the subsequent cellular reactions. Both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space contribute to the rise in [Ca2+]i. In an attempt to assess the relative importance of these pools and the sequences leading to the rise in [Ca2+]i, we have studied the time course of changes in [Ca2+]i after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet-activating factor (PAF) using the Ca2+ indicators quin-2 and fura-2. We observed a time lag of 1-3 s between stimulation and rise in [Ca2+]i. This lag depends on the agonist concentration but is independent of extracellular Ca2+. Thus Ca2+ release from intracellular stores is rate limiting for the rise in [Ca2+]i. After this, cation channels in the plasma membrane (measured with the patch clamp method) are opened. These non-selective channels, which also pass Ca2+, are activated by the initial rise in [Ca2+]i, but by neither fMLP nor inositol 1,4,5-trisphosphate (IP3) directly.

Calcium↗
PubMedDate not supplied

Direct measurement of proton transfer rates to a group controlling the dihydropyridine-sensitive Ca2+ channel.

Protons participate in most biologically important reactions, as substrates, products, cofactors and modulators, and proton transport is an essential step in energy transduction. The dynamics of protonation reactions have been studied extensively in solution and in model systems involving lipid-water interfaces, but have never been resolved at the timescale of the elementary molecular event. Here we show that, under appropriate conditions, binding and unbinding reactions of single protons and deuterium ions to a single site on the L-type calcium channel can be resolved and the protonation and deprotonation rates quantified. The protonation rate constant considerably exceeds previous estimates obtained in simpler systems. The functional consequences of channel protonation is a threefold reduction of the channel conductance, independent of the applied voltage. The data are consistent with the presence of a single protonatable group with pK in the physiological pH range, close to the external mouth of the channel. The two conductance levels of the open channel might be explained by greatly differing local potentials associated with the protonated and deprotonated state of the group.

Animals↗