The transport of chloroquine across human erythrocyte membranes is mediated by a simple symmetric carrier

The kinetic properties of the mediated transport of chloroquine in human erythrocytes are investigated. The high rates of translocation across the cell membrane and high adsorbance properties to glass surfaces have led to the development of new techniques for measuring initial rates of transport. Three different methodological procedures are used to accomplish a complete kinetic characterization of the system. All measurements were done at 25°C. Under zero-trans conditions the system displays complete symmetry, the Michaelis constants being 39.2±2.4 μM for influx and 36.6±5.6 μM for efflux. The respective maximal velocities are 206.4±36.0 μM·min^?1 and 190.0±7.8 μM·min^?1. Under equilibrium-exchange conditions the Michaelis constant is 108.6±15.6 μM and the maximal velocity is 630.3±50.4 μM·min^?1. This 3-fold increase in both K and V over the zero-trans values indicates that the rate-limiting step in the transport of chloroquine is the movement of the unloaded carrier. The kinetic data are consistent with the prediction of a simple carrier model.     

The binding of complement by complexes formed between a rabbit antibody and oligosaccharides of increasing size.

Immune complexes formed between a homogeneous rabbit antibody to type III pneumococcal polysaccharide and a series of oligosaccharides of varying size derived from it were prepared and tested for their ability to fix guinea pig hemolytic complement. Antibody and either tetra-, hexa-, or octasaccharide formed only monomeric antibody-hapten complexes and did not show any complement binding. A dodecasaccharide and a 16-sugar residues oligomer formed dimer and trimer immune complexes. These complexes were also unable to fix complement. However, as the size of the sugar oligomers was increased to about 21 sugar residues per oligosaccharide molecule or more, the resulting complexes exhibited substantial complement binding, concomitant with the formation of antigen-antibody aggregates higher than trimers. On the other hand, an independent study carried out with the same material suggested changes in the conformation of the Fc moiety in the antibody molecule upon addition of oligosaccharide ligands as small as a 16-residue unit. Since the resulting complexes hardly ehibited any complement binding, ligand-induced conformational changes in the Fc part of the antibody molecule appears to be an insufficient condition per se for triggering complement fixation.     

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: II. Kinetic properties of NBD-taurine transfer in asymmetric conditions

The transport of inorganic anions across human red blood cell membranes is accomplished by a carrier-like mechanism which involves an electroneutral and obligatory one-for-one anion exchange. The transport kinetics were described by models that involve alternation of single transport sites between the two membrane surfaces. These models predict that each carrier shows either an inward-facing Ei or an outward-facing Eo, conformation, each capable of binding either a monovalent anion or a divalent anion + a proton, to yield an electroneutral translocating complex. Unidirectional transport rates provide, therefore, a measure for the relative concentration of carriers at a given membrane surface. In the present work we assessed how modulation of the transmembrane distribution of carriers by the anion composition of cells and media, and by pH, affect the anion transport system. We have set the system in asymmetric conditions with respect to anions, so that a fast transportable anion (e.g., chloride) was present in one side of the membrane and slow transportable anions (e.g., sulfate, phosphate, oxalate, isethionate, gluconate, HEPES) were present on the other side of the membrane. The skewed distribution of carriers induced in these conditions were assessed by two methods: 1) NBD-taurine transfer which provided a measure for [Ei], the monovalent inward-facing form of the carrier, and 2) inhibition of NBD-taurine transfer by the specific impermeant and competitive inhibitor 4,4'-dinitro-2,2'-stilbene disulfonic acid (DNDS), which provided a measure for the availability of the carrier at the outer membrane surface. In the various symmetric and asymmetric conditions, we found marked differences in transport rates and transport profiles as well as in the susceptibility of the system to inhibition by DNDS. Direct binding studies of DNDS to cells in the various asymmetric conditions supported the conclusion derived from transport studies that transport sites can be recruited towards the membrane surface facing the slow transportable anions.     

The mechanism of anion transport across human red blood cell membranes as revealed with a fluorescent substrate: I. Kinetic properties of NBD-taurine transfer in symmetric conditions

Summary The molecular mechanism of anion exchange across the human red blood cell membrane was assessed with the fluorescent substrate analog NBD-taurine and the method of continuous monitoring of transport by fluorescence. The efflux of NBD-taurine was studied under a variety of experimental conditions such as temperature, pH and anion composition of cells and media. The temperature profile of NBD-taurine transfer from Cl-loaded cells into Cl media resembled that of Cl self-exchange, whereas that of NBD-taurine transfer from sulfate-loaded cells into sulfate media resembled that of sulfate self-exchange. Although the pH profiles of NBD-taurine transfer from Cl-loaded cells into Cl media and that of Cl self-exchange resembled each other, the analogous transfer with sulfate replacing Cl was markedly different. These and other data were analyzed and found to be consistent with a model which comprises the following: (a) a H⁺-titratable group in the carrier mechanism; (b) alteration of transport sites between the two sides of the membrane (i.e., ping-pong kinetics); and (c) transmembrane distribution of transport sites which is modulated by pH. It is shown that NBD-taurine transfer represents a tracer flux of a fluorescent substrate which gives a measure for the presence of monovalent transport sites at the inner surface of the membrane. The latter is markedly affected by the relative concentrations of anions and H⁺ on both sides of the red blood cell membrane.     

CD59 functions as a signal-transducing molecule for human T cell activation.

The CD59 Ag is a 20-kDa protein that is widely expressed on most leukocytes and RBC, is coupled to the membrane by a phosphatidylinositol-glycan anchoring structure, plays a role in cell interaction between monocytes and T cells, and also functions as an inhibitor of cytolysis by the terminal C components C5b-9. Because this molecule is structurally related to the murine Ly-6 family of Ag, we have investigated whether anti-CD59 mAb might be capable of activating human T lymphocytes in a manner similar to that described for antibodies to the murine Ly-6 Ag. In the presence of the appropriate co-stimulators, mAb to one of the two epitopes on CD59 were capable of inducing both a rise in intracytoplasmic free Ca2+, inositol phosphate production, IL-2 production, and T cell proliferation. Anti-CD59-induced inositol phosphate turnover and IL-2 production were dependent on co-expression of the CD3/TCR complex. CD59-loss mutants of the Jurkat cell line were completely responsive to stimulation by anti-CD3 thereby demonstrating that CD59 does not play a role as a signal transducer downstream from the TCR. Taken together, these results demonstrate that the CD59 Ag can play multiple distinct roles in the regulation of the immune response.     

Polyol permeability of the human red cell. Interpretation of glucose transport in terms of a pore

The kinetic equations describing transport through a pore that has a binding site and that undergoes a conformational change are identical to those of a carrier model. Therefore, in order to distinguish between the two models it is necessary to test specific predictions based on detailed mechanistic models. A pore model is described in which the substrate (glucose) is able to reach the single binding site only from the outside when the pore is in conformation I and only from the inside when it is conformation II. On the basis of this model it is predicted that solutes which do not have any specific affinity for the binding site should still have a finite permeability via the glucose transport system if they are the same size or smaller than glucose. This permeability should be proportional to the volume of distribution of the solute in the pore and should therefore decrease with increasing molecular size. A geometric pore volume can be estimated from this size dependence. In order to test these predictions, the glucose-dependent permeability of a series of 4-carbon (erythritol), 5-carbon (d-arabitol, l-arabitol and xylitol) and 6-carbon (d-mannitol, d-sorbitol and $\text{myo-}\text{inositol}$) polyols was measured. The permeability of all the polyols is decreased by the presence of glucose and the $\text{K}{}_{\mathrm{<mtext>I</mtext>}}$ of this “inhibitable” component is similar to that of d-sorbose, suggesting that this component is associated with the glucose transport system. Since these observations could be explained entirely in terms of a specific affinity for a carrier binding site, they do not exclude a carrier mechanism. However, as predicted for the pore model, this “inhibitable” permeability decreased with increasing molecular size and the calculated geometric pore volume was of a size that would be expected for a cell membrane pore.     

Quantitative predictions of a noncarrier model for glucose transport across the human red cell membrane

There is an increasing amount of experimental data on transport across biological membranes which cannot be readily accommodated by classical mobile carrier models. We propose models for membrane transport based upon current concepts in molecular enzymology, in which the membrane component involved in transport is an oligomeric protein which undergoes substrate-induced conformational changes. A number of paradoxical observations on glucose transport in the human erythrocyte are explained if the protein involved is a tetramer possessing two classes of binding sites with different affinities for glucose. We develop in detail a particular model of this type, the internal transfer model, in which transport occurs by transfer of substrate from one subunit to another of the protein. The fit of the predictions of the internal transfer model with most of the experimental data is very good. Those data which cannot be fitted by the model cannot be accounted for by any presently available model. We extend our model qualitatively to include the sodium-activated cotransport systems for sugars and amino acids.     

The role of Ca2+ transport across the plasma membrane for cell migration.

Cell migration plays a central role in many physiological and pathophysiological processes. On a cellular level it is based on a highly coordinated restructuring of the cytoskeleton, a continuous cycle of adhesion and de-adhesion as well as on the activity of ion channels and transporters. The cytoplasmic Ca2+ ([Ca2+]i) concentration is an important coordinator of these intracellular processes. Thus, [Ca2+]i must be tightly controlled in migrating cells. This is among other things achieved by the activity of Ca2+ permeable channels, the plasma membrane Ca2+-ATPase (PMCA) and the Na+/Ca2+ exchanger (NCX) in the plasma membrane. Here, we wanted to determine the functional role of these transport proteins in cell migration. We therefore quantified the acute effect of inhibitors of these transport proteins (Gd3+, vanadate, KB-R7943) on migration, [Ca2+]i, and intracellular pH (pHi) of MDCK-F cells. Migration was monitored with computer-assisted time-lapse video microscopy. [Ca2+]i and pHi were measured with the fluorescent indicators fura-2 and BCECF. NCX expression in MDCK-F cells was verified with ion substitution experiments, and expression of PMCA was tested with RT-PCR. All blockers lead to a rapid impairment of cell migration. However, the most prominent effect is elicited by NCX-inhibition with KB-R7943. NCX-blockade leads to an almost complete inhibition of migration which is accompanied by a dose-dependent increase of [Ca2+]i and an intracellular alkalinisation. We show that inhibition of NCX and PMCA strongly affects lamellipodial dynamics of migrating MDCK-F cells. Taken together, our results show that PMCA and in particular NCX are of critical importance for cell migration.     

The cardiac Na-H exchanger: a key downstream mediator for the cellular hypertrophic effects of paracrine, autocrine and hormonal factors.

The major mechanism by which the heart cell regulates intracellular pH is the Na(+)-H(+) exchanger (NHE) with the NHE-1 isoform as the primary cardiac subtype. Although NHE-1 has been implicated in mediating ischemic injury, more recent evidence implicates the antiporter as a key mediator of hypertrophy, which is produced by various autocrine, paracrine and hormonal factors such as endothelin-1, angiotensin II, and alpha(1) adrenoceptor agonists. These agonists activate the antiporter via phosphorylation-dependent processes. NHE-1 inhibition is likely conducive to attenuating the remodelling process after myocardial infarction. These effects probably occur independently of infarct size reduction and involve attenuation of subsequent postinfarction heart failure. As such, inhibitors of NHE offer substantial promise for clinical development that will attenuate acute responses to myocardial postinfarction and chronic pos t infarction, which evolve toward heart failure. The regulation of NHE-1 is discussed as is its potential role in mediating cardiomyocyte hypertrophy.     

The inactivation by fluorodinitrobenzene of glucose transport across the human erythrocyte membrane The effect of glucose inside or outside the cell

The inactivation of glucose transport in human red cells by fluorodinitrobenzene is accelerated by 120 mM glucose outside the cell but retarded at least 50% by 120 mM glucose inside the cell. This suggests that the transport system is predominantly in one conformation when there is glucose inside the cell, and in another conformation when there is glucose outside the cell.     

Mathematical modelling of the transport of low molecular weight solutes across biological membranes. The transport of Leu, His and Glu into human blood platelets

A model describing the transport of low molecular weight solutes across cell membranes is presented. The model accounts for many different systems which may mediate the fluxes of various solutes, for the effect of Na+ ions, and for time dependence of the processes. It generalizes the classical three-parameter equation for transport. Solutions to the model were employed to interprete experimental data obtained for the uptake of DL-leu, L-his and L-glu by human blood platelets.     

The terminal membrane C5b-9 complex of human complement. Evidence for the existence of multiple protease-resistant polypeptides that form the trans-membrane complement channel

C5b-9(m) complexes were incorporated into lecithin liposomes and subjected to proteolysis in the presence of DTT to remove the externally oriented annulus. Liposomes were recovered that selectively carried the membrane-bound, thin-walled cylindrical portion of the C5b-9(m) complex. The presence of DTT during proteolysis enhanced peptide bond cleavage in the C5b-9(m) complex. All C5-C9 components were degraded to lower m.w. fragments. A protease-resistant, but hydrophilic 85 to 86,000-dalton polypeptide derivative of C5, possibly representing the C5 beta-chain, was recovered in the fluid phase. This component is not intimately associated with the target lipid bilayer. Immunochemical analyses yielded evidence for the existence of minor C5-C9 antigenic determinants on the membrane-bound C5b-9(m) residue. SDS polyacrylamide gel electrophoreses of liposomes carrying the C5b-9(m) residues revealed the persistence of at least six major polypeptides of approximately m.w. 50,000, 45,000, 40,000, 38,000, 20,000, and 16,000. The data are interpreted to indicate that multiple protease-resistant polypeptide chains derived from several terminal C components participate in formation of the trans-membrane C channel.     

The unidirectional flux transient as a tool for quantifying parallel diffusional pathways through membranes. Exact solution for two pathways

If a plane membrane consists of patches, each with a given area and a given diffusion coefficient, then the transient of the total unidirectional flux of a diffusing substance (as defined experimentally by Ussing) is predictable. Here the inverse problem is studied: given only the observed transient of the total unidirectional diffusion flux, the unknown membrane heterogeneity transverse to the flux is to be quantified. The ratio of the arithmetic and of the harmonic means (both area-weighted) of the diffusion coefficients, evaluated over the membrane, is expressed in terms of the observed transient alone and is used to characterize the heterogeneity. A unique exact solution of the inverse problem for two kinds of patches is obtained in closed form. A singular limit of this solution pertains to currently postulated models of endothelial membranes, for which a characteristically shaped transient is predicted.