A Hundred-Year Researching History on the Low Ionic Strength in Red Blood Cells: Literature Review

This review article provides a critical survey of work from 1904 to 2003 on the effects of low ionic strength in Red Blood Cells (RBCs) incubated in media with impermeable sugars such as sucrose. In 1904 Gürber A washed RBCs of different species with isotonic sucrose solution to eliminate the outside ions in order to better analyse their intracellular ionic composition; however, this approach was not feasible because of a substantial salt efflux from the cells. A prominent feature of the salt loss is the shrinking of the RBCs. A central role in the understanding of the ionic movements is thereby the new Donnan equilibrium of the anions. Experimental evidence has been given by Jacobs MH and Parpart AK in 1933. In the sucrose medium two phases could be predicted: 1) a very rapid anionic shift resulting in an unequal distribution of chloride and hydroxyl anions on both sides of the membrane and 2) a leakage of salts from the RBCs. In 1940 Wilbrandt W assumed that a positive membrane potential is in line with the salt loss at low ionic strength in RBCs. In 1977 Knauf PA, Fuhrmann GF, Rothstein S and Rothstein A observed in RBCs an inhibition of both, anion exchange and also of net anion efflux, by incubation with disulfonic stilbene derivates. At low ionic strength the Donnan equilibrium is immediately obtained by the Anion Exchanger Protein (AEP). The resulting positive membrane potential opens at least two new types of cation pores or channels. Thereby is the conductivity pathway for the anions, namely the AEP, in charge of the net anion loss at low ionic strength. The AEP pathway is extensively blocked by disulfonic stilbene compounds. The permeability ways for cations through these pores or channels are not yet explored.

Computational simulations determining disulfonic stilbene derivative bioavailability within human serum albumin

First structural insights into disulfonic acid stilbene derivatives interacting with the most abundant carrier protein, human serum albumin.

Sat1 is dispensable for active oxalate secretion in mouse duodenum

Mice deficient for the apical membrane oxalate transporter SLC26A6 develop hyperoxalemia, hyperoxaluria, and calcium oxalate stones due to a defect in intestinal oxalate secretion. However, the nature of the basolateral membrane oxalate transport process that operates in series with SLC26A6 to mediate active oxalate secretion in the intestine remains unknown. Sulfate anion transporter-1 (Sat1 or SLC26A1) is a basolateral membrane anion exchanger that mediates intestinal oxalate transport. Moreover, Sat1-deficient mice also have a phenotype of hyperoxalemia, hyperoxaluria, and calcium oxalate stones. We, therefore, tested the role of Sat1 in mouse duodenum, a tissue with Sat1 expression and SLC26A6-dependent oxalate secretion. Although the active secretory flux of oxalate across mouse duodenum was strongly inhibited (>90%) by addition of the disulfonic stilbene DIDS to the basolateral solution, secretion was unaffected by changes in medium concentrations of sulfate and bicarbonate, key substrates for Sat1-mediated anion exchange. Inhibition of intracellular bicarbonate production by acetazolamide and complete removal of bicarbonate from the buffer also produced no change in oxalate secretion. Finally, active oxalate secretion was not reduced in Sat1-null mice. We conclude that a DIDS-sensitive basolateral transporter is involved in mediating oxalate secretion across mouse duodenum, but Sat1 itself is dispensable for this process.

Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle

Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2′-disulfonic acid (DBDS), and 4,4′-dinitrostilbene-2,2′-disulfonic acid (DNDS)] was investigated in planar bilayers using SO[Formula: see text] as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1–3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 μM for DBDS and 3.4 and 55 μM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14–24 Å and that the pore constriction is ∼10 Å in diameter.

The Noncompetitive Inhibitor Ww781 Senses Changes in Erythrocyte Anion Exchanger (Ae1) Transport Site Conformation and Substrate Binding

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl−. Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl− seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl− does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl− binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl−-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl−-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure–function relationships.

A thermodynamic study of electroneutral K-Cl cotransport in pH- and volume-clamped low K sheep erythrocytes with normal and low internal magnesium.

Swelling-induced human erythrocyte K-Cl cotransport is membrane potential independent and capable of uphill transport. However, a complete thermodynamic analysis of basal and stimulated K-Cl cotransport, at constant cell volume, is missing. This study was performed in low K sheep red blood cells before and after reducing cellular free Mg into the nanomolar range with the divalent cation ionophore A23187 and a chelator, an intervention known to stimulate K-Cl cotransport. The anion exchange inhibitor 4,4'diisothiocyanato-2,2'disulfonic stilbene was used to clamp intracellular pH and Cl or NO3 concentrations. Cell volume was maintained constant as external and internal pH differed by more than two units. K-Cl cotransport was calculated from the K effluxes and Rb (as K congener) influxes measured in Cl and NO3, at constant internal K and external anions, and variable concentrations of extracellular Rb and internal anions, respectively. The external Rb concentration at which net K-Cl cotransport is zero was defined as flux reversal point which changed with internal pH and hence Cl. Plots of the ratio of external Rb concentrations corresponding to the flux reversal points and the internal K concentration versus the ratio of the internal and external Cl concentrations (i.e., the Donnan ratio of the transported ions) yielded slopes near unity for both control and low internal Mg cells. Thus, basal as well as low internal Mg-stimulated net K-Cl cotransport depends on the electrochemical potential gradient of KCl.