James A. Dix

Crowding Effects on Diffusion in Solutions and Cells

We review the effects of molecular crowding on solute diffusion in solution and in cellular aqueous compartments and membranes. Anomalous diffusion, in which mean squared displacement does not increase linearly with time, is predicted in simulations of solute diffusion in media crowded with fixed or mobile obstacles, or when solute diffusion is restricted or accelerated by a variety of geometric or active transport processes. Experimental measurements of solute diffusion in solutions and cellular aqueous compartments, however, generally show Brownian diffusion. In cell membranes, there are examples of both Brownian and anomalous diffusion, with the latter likely produced by lipid-protein and protein-protein interactions. We conclude that the notion of universally anomalous diffusion in cells as a consequence of molecular crowding is not correct and that slowing of diffusion in cells is less marked than has been generally assumed.

The aqueous pore in the red cell membrane: band 3 as a channel for anions, cations, nonelectrolytes, and water.

This article develops arguments for the existence of an aqueous pore in the red cell membrane as the principal route for passive flux of ions, water, and small nonelectrolytes and proposes a molecular model for the pore. In principle, such an aqueous pore would provide easy passage into and out of the cell for all solutes small enough to enter the channel. The red cell membrane, however, regulates the fluxes of cations and anions closely and discriminates carefully among other small solutes. These constraints have been incorporated into the model, which visualizes the channel and its associated regulatory system as governing passive transport of ions of either sign, as well as water and small nonelectrolytes into and out of the cell. The model, which was formulated to consolidate a number of observations already in the literature, has caused us to look for new interrelations between inhibitors specific to cation, anion, and nonelectrolyte transport. The results of these experiments, presented below, demonstrate that interrelations d o exist and provide evidence that supports the view that a common aqueous channel provides primary access to the red cell cytoplasm.

Mapping of fluorescence anisotropy in living cells by ratio imaging. Application to cytoplasmic viscosity

Steady-state and time-resolved fluorescence properties of probes incorporated into living cells give information about the microenvironment near the probe. We have extended studies of spatially averaged fluorescence anisotropy (r) by using an epifluorescence microscope, equipped with excitation and emission polarizers and an image analysis system, to map r of nonoriented fluorophores incorporated into cultured cells. With this imaging system, r for reflected light or glycogen scattering solutions was greater than 0.98. Measurement of r over the range 0.01-0.35 for fluorophores in bulk solution and in thin capillary tubes placed side-by-side gave values equivalent to r measured by cuvette fluorometry. Cytoplasmic viscosity (eta) in Madin-Darby canine kidney (MDCK) cells and Swiss 3T3 fibroblasts was examined from anisotropy images and time-resolved fluorescence decay of the cytoplasmic probes 2,7-bis-carboxyethyl-5 (and 6)-carboxy-fluorescein (BCECF) and indo-1. Nanosecond lifetimes and anisotropy decay were measured using a pulsed light source and gated detector interfaced to the epifluorescence microscope. Anisotropy images of BCECF in MDCK cells revealed two distinct regions of r: one from the cytoplasm (r = 0.144 +/- 0.008) and a second appearing at late times from the interstitial region (r = 0.08 +/- 0.03), representing BCECF trapped beneath the tight junctions. Anisotropy values, taken together with intracellular life-times and the calibration between r and eta/tau f for water/glycerol mixtures, gave eta values of 10-13 cP at 23 degrees C. These values assume little fluorophore binding to intracellular components and are therefore upper limits to cytoplasmic viscosity. These data establish a new methodology to map anisotropy in intact cells to examine the role of fluidity in cellular physiology.

Fluorescence measurement of chloride transport in monolayer cultured cells. Mechanisms of chloride transport in fibroblasts.

The methodology has been developed to measure Cl activity and transport in cultured cells grown on a monolayer using the entrapped Cl-sensitive fluorophore 6-methoxy-N-[3-sulfopropyl] quinolinium (SPQ). The method was applied to a renal epithelial cell line, LLC-PKI, and a nonepithelial cell line, Swiss 3T3 fibroblasts. SPQ was nontoxic to cells when present for greater than h in the culture media. To load with SPQ (5 mM), cells were made transiently permeable by exposure to hypotonic buffer (150 mOsm, 4 min). Intracellular fluorescence was monitored continuously by epifluorescence microscopy using low illumination intensity at 360 +/- 5 nm excitation wavelength and photomultiplier detection at greater than 410 nm. Over 60 min at 37 degrees C, there was no photobleaching and less than 10% leakage of SPQ out of cells; intracellular SPQ fluorescence was uniform. SPQ fluorescence was calibrated against intracellular [Cl] using high K solutions containing the ionophores nigericin and tributyltin. The Stern-Volmer constant (Kq) for quenching of intracellular SPQ by Cl was 13 M-1 for fibroblasts and LLC-PKl cells. In the absence of Cl, SPQ lifetime was 26 ns in aqueous solution and 3.7 +/- 0.6 ns in cells, showing that the lower Kq in cells than in free solution (Kq = 118 M-1) was due to SPQ quenching by intracellular anions. To examine Cl transport mechanisms, the time course of intracellular [Cl] was measured in response to rapid Cl addition and removal in the presence of ion or pH gradients. In fibroblasts, three distinct Cl transporting systems were identified: a stilbeneinhibitable Cl/HCO3 exchanger, a furosemide-sensitive Na/K/2Cl cotransporter, and a Ca-regulated Cl conductance. These results establish a direct optical method to measure intracellular [Cl] continuously in cultured cells.

Role of membrane proteins and lipids in water diffusion across red cell membranes

When human red cells are treated with the mercurial sulfhydryl reagent, p-chloromercuribenzene sulfonate, osmotic water permeability is suppressed and only diffusional water permeability remains (Macey, R.I. and Farmer, R.E.L. (1970) Biochim. Biophys. Acta 211, 104-106). It has been suggested that the route for the remaining water permeation is by diffusion through the membrane lipids. However, after making allowance for the relative lipid area of the membrane, the water diffusion coefficient through lipid bilayers which contain cholesterol is too small by a factor of two or more. We have measured the permeability coefficient of normal human red cells by proton T1 NMR and obtained a value of 4.0 X 10(-3) cm X s-1, in good agreement with published values. In order to study permeation-through red cell lipids we have perturbed extracted red cell lipids with the lipophilic anesthetic, halothane, and found that halothane increases water permeability. The same concentration of halothane has no effect on the water permeability of human red cells, after maximal pCMBS inhibition. In order to compare halothane mobility in extracted red cell membrane lipids with that in red cell ghost membranes, we have studied halothane quenching of N-phenyl-1-naphthylamine by equilibrium fluorescence and fluorescence lifetime methods. Since halothane mobility is similar in these two preparations, we have concluded that the primary route of water diffusion in pCMBS-treated red cells is not through membrane lipids, but rather through a membrane protein channel.

Test of the 'glymphatic' hypothesis demonstrates diffusive and aquaporin-4-independent solute transport in rodent brain parenchyma

Transport of solutes through brain involves diffusion and convection. The importance of convective flow in the subarachnoid and paravascular spaces has long been recognized; a recently proposed ‘glymphatic’ clearance mechanism additionally suggests that aquaporin-4 (AQP4) water channels facilitate convective transport through brain parenchyma. Here, the major experimental underpinnings of the glymphatic mechanism were re-examined by measurements of solute movement in mouse brain following intracisternal or intraparenchymal solute injection. We found that: (i) transport of fluorescent dextrans in brain parenchyma depended on dextran size in a manner consistent with diffusive rather than convective transport; (ii) transport of dextrans in the parenchymal extracellular space, measured by 2-photon fluorescence recovery after photobleaching, was not affected just after cardiorespiratory arrest; and (iii) Aqp4 gene deletion did not impair transport of fluorescent solutes from sub-arachnoid space to brain in mice or rats. Our results do not support the proposed glymphatic mechanism of convective solute transport in brain parenchyma.

Osmotic properties of human red cells

SummaryWhen an osmotic pressure gradient is applied to human red cells, the volume changes anomalously, as if there were a significant fraction of “nonosmotic water” which could not serve as solvent for the cell solutes, a finding which has been discussed widely in the literature. In 1968, Gary-Bobo and Solomon (J. Gen. Physiol.52:825) concluded that the anomalies could not be entirely explained by the colligative properties of hemoglobin (Hb) and proposed that there was an additional concentration dependence of the Hb charge (zHb). A number of investigators, particularly Freedman and Hoffman (1979,J. Gen. Physiol.74:157) have been unable to confirm Gary-Bobo and Solomons experimental evidence for this concentration dependence of zHb and we now report that we are also unable to repeat the earlier experiments. Nonetheless, there still remains a significant anomaly which amounts to 12.5±0.8% of the total isosmotic cell water (P≪0.0005,t test), even after taking account of the concentration dependence of the Hb osmotic coefficient and all the other known physical chemical constraints, ideal and nonideal. It is suggested that the anomalies at high Hb concentration in shrunken cells may arise from the ionic strength dependence of the Hb osmotic coefficient. In swollen red cells at low ionic strength, solute binding to membrane and intracellular proteins is increased and it is suggested that this factor may account, in part, for the anomalous behavior of these cells.

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

SummaryThe effect of chloride on 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK1=0.67±0.16 μm,k2=1.6±0.7 sec−1,k−2=0.17±0.09 sec−1,K′1=6.3±1.7 μm,k′2=9±4 sec−1 andk′−2=7±3 sec−1. The apparent dissociation constants of chloride from band 3,KCl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.

Interaction of phloretin with the anion transport protein of the red blood cell membrane.

Phloretin is an inhibitor of anion exchange and glucose and urea transport in human red cells. Equilibrium binding the kinetic studies indicate that phloretin binds to band 3, a major integral protein of the red cell membrane. Equilibrium phloretin binding has been found to be competitive with the binding of the anion transport inhibitor, 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS), which binds specifically to band 3. The apparent binding (dissociation) constant of phloretin to red cell ghost band 3 in 28.5 mM citrate buffer, pH 7.4, 25 degree C, determined from equilibrium binding competition, is 1.8 +/- 0.1 microM. Stopped-flow kinetic studies show that phloretin decreases the rate of DBDS binding to band 3 in a purely competitive manner, with an apparent phloretin constant of 1.6 +/- 0.4 microM. The pH dependence of equilibrium binding studies show that it is the charged, anionic form of phloretin that competes with DBDS binding, with an apparent phloretin inhibition constant of 1.4 microM. The phloretin binding and inhibition constants determined by equilibrium binding, kinetic and pH studies are all similar to the inhibition constant of phloretin for anion exchange. These studies suggest that phloretin inhibits anion exchange in red cells by a specific interaction between phloretin and band 3.

Specific interaction of the water transport inhibitor, pCMBS, with band 3 in red blood cell membranes

The human red cell anion transport protein, band 3, contains six pCMBS (p-chloromercuribenzene sulfonate) reactive SH groups, five of which react with N-ethylmaleimide. We have carried out equilibrium binding experiments using N-ethylmaleimide-treated red cell ghosts and found that the sulfhydryl reactive water transport inhibitor, pCMBS, inhibits the binding to band 3 of the specific anion exchange inhibitor DBDS (4,4-dibenzoamido-2,2-disulfonic stilbene) in a non-competitive manner. Stopped-flow kinetic studies, in which DBDS is mixed with ghosts in the presence of pCMBS, show that pCMBS slows the DBDS induced conformational change in band 3. A non-competitive reaction scheme has been developed which incorporates the quantitative results of equilibrium and kinetic studies. The pCMBS effect on DBDS binding and kinetics is reversed with 5 mM cysteine suggesting a sulfhydryl bond is involved in pCMBS binding to band 3. These data suggest that pCMBS has a specific binding site on band 3, consistent with the hypothesis that band 3 mediates red cell water transport.