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. REVIEW OF EVIDENCE FOR THE AQUEOUS POREIn 1957, Paganelli and Solomon' measured water diffusion across the human red cell membrane and proposed that the membrane was traversed by a n "equivalent pore" of 3.5 A radius. The pore dimensions were established from the ratio of the osmotic permeability coefficient, PI, measured by Side1 and Solomon,2 to the diffusional permeability coefficient, P d . Subsequently, Goldstein and Solomon' measured the reflection coefficient of a number of small hydrophilic solutes and found that these conformed t o expectations for an aqueous pore whose dimensions could be computed from the equations governing steric hindrance put forward by Pappenheimer et a/.' and R e n k h s Drawing primarily on these results, as well as a subsequent determination of P d by Barton and Brown,' Solomon' calculated that Pf/Pd = 3.4 and concluded that the red cell equivalent pore had a radius of 4.2-4.6 A.Further support for the concept of an aqueous pore was provided by Vieira et aLB who measured the apparent activation energy for water diffusion into the human red cell and found it to be 6 kcal/mol, slightly greater than the 4.5 kcal/mol characteristic of water diffusion in bulk s o l~t i o n .~ Vieira et a/. found the activation energy* Supported in part by National Institutes of Health grants 5R01 GM15692 and 2R01Annals New York Academy of Sciences for water diffusion into the dog red cell, whose equivalent pore radius was 6 A, to be 4.5 kcal/mol, the same as in bulk solution, and ascribed the slightly larger value in human red cells to interactions between the water molecule and the hydrophilic walls of the smaller pore. Wang9 measured water diffusion in bulk solution and computed the product D w~w / T (in which DW is the diffusion coefficient and q~, the viscosity of bulk water), which contains all the temperature-dependent terms in the Sto...

We have previously proposed that a membrane transport complex, centered on the human red cell anion transport protein, band 3, links the transport of anions, cations and glucose. Since band 3 is specialized for HCO3-/Cl- exchange, we thought there might also be a linkage with carbonic anhydrase (CA) which hydrates CO2 to HCO3-. CA is a cytosolic enzyme which is not present in the red cell membrane. The rate of reaction of CA with the fluorescent inhibitor, dansylsulfonamide (DNSA) can be measured by stopped-flow spectrofluorimetry and used to characterize the normal CA configuration. If a perturbation applied to a membrane protein alters DNSA/CA binding kinetics, we conclude that the perturbation has changed the CA configuration by either direct or allosteric means. Our experiments show that covalent reaction of the specific stilbene anion exchange inhibitor, DIDS, with the red cell membrane, significantly alters DNSA/CA binding kinetics. Another specific anion exchange inhibitor, benzene sulfonate (BSate), which has been shown to bind to the DIDS site causes a larger change in DNSA/CA binding kinetics; DIDS reverses the BSate effect. These experiments show that there is a linkage between band 3 and CA, consistent with CA interaction with the cytosolic pole of band 3.

When 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 Solomon's 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 much less than 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.