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R Grygorczyk

Publications and source records attributed to R Grygorczyk.

35 records · Page 2Linked to original sources
PubMed1994

Cloning and expression of a cDNA for the human prostanoid FP receptor.

A cDNA clone coding for a functional human prostanoid FP receptor has been isolated from a uterus cDNA library. The human FP receptor consists of 359 amino acid residues with a predicted molecular mass of 40,060, and has the seven putative transmembrane domains characteristic of G-protein-coupled receptors. Challenge of Xenopus oocytes expressing the FP receptor with 10 nM of either prostaglandin (PG) F2 alpha or the selective FP-receptor agonist fluprostenol resulted in an elevation in intracellular Ca2+. Radioreceptor binding studies using membranes prepared from mammalian COS cells transfected with the FP receptor cDNA showed that the rank order of potency for prostaglandins and prostaglandin analogs in competition for [3H]PGF2 alpha specific binding sites was as predicted for the FP receptor, with PGF2 alpha approximately fluprostenol > PGD2 > PGE2 > U46619 > iloprost. In summary, we have cloned the human prostanoid FP receptor which is functionally coupled to the Ca2+ signalling pathway.

Amino Acid Sequence↗
PubMed1993

Cloning and expression of a cDNA for the human prostaglandin E receptor EP1 subtype.

A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2 alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.

Amino Acid Sequence↗
PubMed1992

Whole-cell chloride conductances in cultured brushed human nasal epithelial cells.

Human airway epithelial cells were obtained by nasal brushing, thus avoiding the use of proteolytic enzymes for cell isolation. Whole-cell Cl- conductances were studied in these cells by means of the patch-clamp technique. During whole-cell recordings, cell swelling activated a Cl- conductance that was blocked by indanyloxyacetic acid (48 +/- 10% inhibition at 50 microM). The swelling-induced current outwardly rectified and showed inactivation at depolarizing voltages (> or = +60 mV) and activation at hyperpolarizing voltages (< or = -30 mV). The voltage sensitivity of current activation was approximately twice that of inactivation. Another Cl- current with different kinetics was observed when nonswollen airway cells were stimulated with ionomycin (2 microM) in the presence of 1 mM Ca2+. The Ca(2+)-induced current exhibited activation during depolarizing voltage steps (> or = +40 mV) and inactivation during hyperpolarizing voltage steps (< or = -40 mV). In contrast to the swelling-induced current, the activation of Ca(2+)-induced current was less sensitive to voltage compared with its inactivation. Tail current analysis suggested that Cl- channels having a linear current-voltage relation mediate the response to Ca2+. This study indicates that brushed human nasal epithelial cells possess Cl- conductances that are regulated by cell swelling and Ca2+ and that they represent a useful in vitro model for studying ion transport in epithelia.

Calcium↗
PubMed1991

The nucleotide sequence of a voltage-gated chloride channel from the electric organ of Torpedo californica.

The cDNA encoding the voltage-gated chloride channel from the electric organ of Torpedo californica has been isolated and sequenced. The 2.7 kilobase pair cDNA encodes an 810 amino acid polypeptide which is highly homologous at both the DNA (97%) and amino acid (97%) levels to the voltage-gated chloride channel from the electric organ of T. marmorata. The majority of the 24 amino acid differences between the T. californica and T. marmorata voltage-gated chloride channels are clustered in two putative cytoplasmic domains with six differences located between residues 10-92 and 14 differences occurring between residues 576 to 708. Only one amino acid difference occurs in one of the predicted transmembrane domains. The most dramatic difference is an insertion of Asp-Val-Pro-Gly in a large cytoplasmic domain at amino acid residue 627 of the T. californica channel.

Amino Acid Sequence↗
PubMed1991

Ba(2+)-induced action potentials in osteoblastic cells.

Trains of long-duration "action potentials" were induced by Ba2+ in osteoblast-like rat osteosarcoma cells (ROS 17/2.8), under current clamp and voltage clamp. Large depolarizing pulses were seen in microelectrode measurements at 37 degrees C following the addition of 10 or 20 mM Ba2+ to physiological bathing medium. Application of BAY K 8644 resulted in the onset of the pulses at earlier times and at more negative potentials. The pulses were blocked by nifedipine and Cd2+, but not by Ni2+. Large inward current pulses were seen in whole-cell patch technique voltage-clamp measurements at 37 degrees C in the presence of from 10 to 110 mM Ba2+ in the bathing medium. The current pulses were not seen at 22 degrees C in the presence of 110 mM Ba2+, but could be induced by BAY K 8644. These pulses were not blocked by TTX, but were blocked by nifedipine, Cd2+, Zn2+, Co2+, and by an increase in bathing [Ca2+]. The shape and frequency of the current pulses were the same as for voltage pulses under current clamp. A model that can explain these observations involves opening of L-type Ca2+ channels in a voltage-independent manner by cytosolic Ba2+ via a screening of Ca2+ from sites that produce either inactivation or a lower probability of opening in the activated state. There would be a closing of these channels at higher [Ba2+] as Ba2+ is forced onto these sites. A refractory period is also required to give repeated pulses of openings.

Action Potentials↗
PubMed1990

A large, multiple-conductance chloride channel in normal human T lymphocytes.

Chloride (Cl) channels have been proposed to play roles in lymphocyte functions including volume regulation and cellular cytotoxicity; however, direct studies of such channels in normal human lymphocytes are lacking. In the present study we describe a large conductance Cl channel observed in about 50% of excised, inside-out patches from normal human peripheral T lymphocytes. The channel has multiple conductance states with linear single-channel current-versus-voltage relationships in symmetrical Cl solutions. The most prevalent state is the largest, which has a conductance of about 365 pS. The channel closes in a voltage-dependent manner at both negative and positive potentials, but does not show voltage-dependent inactivation. The probability of opening is maximal between -15 mV and +15 mV and the voltage dependence is well described by two Boltzmann equations with half-maximal probabilities at -22.8 mV and +18.0 mV. The slopes of the voltage dependence suggest two gates in series with 5.7 and 9.6 equivalent charges. The channel was about 30 times more selective for Cl- than for Na+ or K+ under balanced osmolarity but less selective (approx. 11:1) under a large osmotic gradient. The single-channel conductance increased with Cl concentration with an apparent saturation at about 581 pS and a Michaelis-Menten constant of about 120 mM. The selectivity sequence among anions, determined from changes in reversal potential was: I- greater than NO3- greater than Br-, Cl- greater than F-, isethionate, HCO3- greater than SO4(2-) greater than gluconate, propionate greater than aspartate much greater than Na+, K+ and was apparently the same for subconductance states.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1989

Modulation of the Ca2+- or Pb2+-activated K+-selective channels in human red cells. II. Parallelisms to modulation of the activity of a membrane-bound oxidoreductase.

Modulation of Ca2+-activable K+ permeability was compared with modulation of a membrane-bound oxidoreductase activity in human erythrocytes. Changes in the K+ permeability were monitored by flux measurements and single-channel recordings. The enzyme activity was detected by measuring reduction of ferricyanide. Pb2+, Atebrin and menadione had parallel effects on the channel protein and the enzyme. In contrast, propranolol stimulates K+ permeability, but is without effect on enzyme activity. The results demonstrate that the K+ channel and the enzyme are distinct membrane proteins but that the enzyme activity may influence channel gating.

Calcium↗
PubMed1989

Osteoblastic cells have L-type calcium channels.

Whole cell patch clamp studies on osteoblast-like rat osteosarcoma cells (ROS 17/2.8) show the existence of L-type calcium channels in the cell membrane. Measurements were carried out at both 21 and 37 degrees C. With isotonic CsCl in the pipette and a bathing medium containing either 110 or 10 mM Ba2+, a strong depolarizing pulse was required to activate an inward current. The current-voltage relationship (I-V) of this inward current showed a maximum amplitude near +30 mV at 21 and 37 degrees C, with 110 mM Ba2+ in the bathing medium, and near +10 mV at 37 degrees C with 10 mM Ba2+. At both 21 and 37 degrees C the dihydropyridine, BAY K 8644 (2 microM), increased this current and shifted the I-V maximum to less positive potentials, while nifedipine (5 microM) reduced the current. Cd2+ (50 microM) and Co2+ (100 microM) blocked the current. At 21 degrees C the measured inward current showed a slow inactivation, with a time constant of some hundreds of milliseconds. At 37 degrees C, inactivation was considerably faster. The current was suppressed by holding the membrane potential more positive than -30 mV. These data are strong evidence that ROS 17/2.8 cells have a significant number of 'L-type' calcium channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Temperature dependence of Ca2+-activated K+ currents in the membrane of human erythrocytes.

The currents through single Ca2+-activated K+ channels were studied in excised inside-out membrane patches of human erythrocytes. The effects of temperature on single-channel conductance, on channel gating and on activation by Ca2+ were investigated in the temperature range from 0 up to 47 degrees C. The single-channel conductance shows a continuous increase with increasing temperature; an Arrhenius plot of the conductance gives the activation energy of 29.6 +/- 0.4 kJ/mol. Reducing the temperature alters channel-gating kinetics which results in a significant increase of the probability of the channel being open (Po). The calcium dependence of Po is affected by temperature in different ways; the threshold concentration for activation by Ca2+ is not changed, the Ca2+ concentration of half-maximal channel activation is reduced from 2.1 mumol/l at 20 degrees C to 0.3 mumol/l at 0 degrees C, the saturation level of the dependence is reduced for temperatures higher then about 30 degrees C. The relevance of the obtained data for the interpretation of the results known from flux experiments on cells in suspensions is discussed.

Calcium↗
PubMed1987

Potential dependence of the "electrically silent" anion exchange across the plasma membrane of Xenopus oocytes mediated by the band-3 protein of mouse red blood cells.

Mouse erythroid band-3 protein was incorporated into the plasma membrane of Xenopus oocytes by microinjection of poly(A)+-mRNA from spleens of anemic mice. Subsequently, the efflux of microinjected 36Cl was continuously followed in single oocytes in a perfusion chamber the bottom of which was formed by the window of a Geiger-Müller tube. During the flux measurements, the membrane potential was clamped to different holding potentials. The efflux increased over the voltage range of -10 to -100 mV by a factor of about 1.5. Since the membrane potential cannot act as a driving force of anion exchange, it is suggested that the observed slight potential dependence is related to a recruitment of the anion-loaded transport protein by the electrical field, thereby changing the steady-state distribution between inwardly and outwardly facing anion binding sites of the transport molecules. The experimental data are discussed in terms of ping-pong kinetics, assuming that the potential dependence is primarily due to an effect of the electrical field in the membrane on the rate-limiting interconversion of inwardly and outwardly oriented anion binding sites. The results are compatible with the assumption that in the oocyte membrane the substrate-loaded band-3 molecules are preferentially inwardly oriented, and that the transition from the inwardly to the outwardly oriented conformation is associated with a reorientation of an effective charge of 0.1 elementary charge. During progesterone-induced maturation of the oocytes, several endogenous transport systems change their activity drastically. The mouse band-3 protein in the oocyte membrane also undergoes activity changes; however, these changes do not seem to involve direct regulation by specific metabolic processes. They can be explained as a consequence of the depolarization of the membrane potential associated with the maturation process.

Animals↗
PubMed1985

Lead-induced activation and inhibition of potassium-selective channels in the human red blood cell.

The selective increase of net K+ permeability in human red cells brought about by either Ca2+ or lead was studied using a light scattering technique to measure net K+ fluxes in cell suspensions and the patch-clamp technique to study K+ transport in individual K+-selective channels of the red cell membrane. Using ultrapure solutions it was demonstrated that the effect of lead is neither the indirect consequence of a lead-induced increase of the accessibility of the receptor sites of the K+-selective channels to traces of Ca2+ that are present as contamination in analytical grade reagents nor to the release of Ca2+ from intracellular Ca2+ stores. It is further shown that in cell-free membrane patches low concentrations of lead (10 microM) in Suprapur solutions evoke the same single-channel events as added Ca2+ and that this activity can be inhibited by high concentrations of lead (100 microM), similar to the net KCl efflux measured by means of the light scattering technique. It is concluded, therefore, that both Ca2+ and lead independently activate the same K+-selective channels in the red cell membrane.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1985

Mediation of anion transport in oocytes of Xenopus laevis by biosynthetically inserted band 3 protein from mouse spleen erythroid cells.

mRNA from the spleens of anemic mice was purified by oligo(dT)-cellulose chromatography and fractionated by density gradient centrifugation. After injection into oocytes of Xenopus laevis, two of the four fractions obtained led, after 16 h of incubation at 20 degrees C, to the expression of mouse band 3 protein, as demonstrated by immunoprecipitation with polyclonal antibodies against mouse band 3. Flux measurements showed an approximately 2- to 4-fold increment of 36Cl- uptake, which could be abolished by two different stilbene disulfonates, specific inhibitors of band 3-mediated anion transport in red blood cells.

Animals↗
PubMed1985

Ca2+-activated K+ permeability in human erythrocytes: modulation of single-channel events.

Elevated levels of intracellular Ca2+ activate a K+-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K+ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs+, tetraethylammonium and Ba2+ and of single-channel activity by quinine and Ba2+ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na+ and Rb+) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.

Calcium↗
PubMed1984

Ca2+-activated K+ channels in human red cells. Comparison of single-channel currents with ion fluxes.

Exposure of the inner surface of intact red cells or red cell ghosts to Ca2+ evokes unitary currents that can be measured in cell-attached and cell-free membrane patches. The currents are preferentially carried by K+ (PK/PNa 17) and show rectification. Increasing the Ca2+ concentration from 0 to 5 microM increases the probability of the open state of the channels parallel to the change of K+ permeability as observed in suspensions of red cell ghosts. Prolonged incubation of red cell ghosts in the absence of external K+ prevents the Ca2+ from increasing K+ permeability. Similarly, the probability to find Ca2+-activated unitary currents in membrane patches is drastically reduced. These observations suggest that the Ca2+-induced changes of K+ permeability observed in red cell suspensions are causally related to the appearance of the unitary K+ currents. Attempts to determine the number of K+ channels per cell were made by comparing fluxes measured in suspensions of red cells with the unitary currents in membrane patches as determined under comparable ionic conditions. At 100 mM KCl in the external medium, where no net movements of K+ occur, the time course of equilibration of 86Rb+ does not follow a single exponential. This indicates a heterogeneity of the response to Ca2+ of the cells in the population. The data are compatible with the assumption that 25% of the cells respond with Pk = 33.2 X 10(-14)cm3/s and 75% with Pk = 3.1 X 10(-14)cm3/s. At 100 mM external K+ the zero current permeability of a single channel is 6.1 X 10(-14)cm3/s (corresponding to a conductance of 22 pS). Using appropriate values for the probability of a channel in the open state, we estimated that 25% of the cells in the population contain 11-55, and 75% of the cells 1-5 channels per cell that are activated in the time average (20 degrees C, pH 7.6).

Calcium↗
PubMed1983

Properties of the CA2+-activated K+ conductance of human red cells as revealed by the patch-clamp technique.

Application of Ca2+ to the inner surface of red-cell membranes activates unitary currents that can be measured in cell-attached and cell-free membrane patches. Ca2+ can be replaced by Pb2+ to activate the single channels. In addition to internal Ca2+ external K+ has to be present. The channels are preferentially permeable to K+ with a selectivity ratio PK:PNa of about 15:1 as estimated from measurement of reversal potentials. The dependence of channel activity on Ca2+ is compatible with the conception that the binding of two Ca2+ is necessary to open a single channel. Both the channel activity and the single-channel conductance exhibit inward rectification. External and internal Na+ inhibit the K+ currents. The reported results suggest that the unitary current events are responsible for the Ca2+-dependent K+ permeability known from measurement on cell suspensions. Therefore, comparison of the two techniques allows calculation of the number of K+ channels per red cell, which on average is about 10.

Calcium↗