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Role of polyamine structure in inhibition of K⁺-Cl^- cotransport in human red cell ghosts

J. R. Sachs and D. W. Martin

MS 9402 Received 19 March 1999; accepted after revision 13 August 1999.

	ABSTRACT

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1. K⁺-Cl^- cotransport in human red cell ghosts is inhibited by divalent inorganic cations, soluble polycations and amphipathic organic cations. These findings suggest a common mechanism of inhibition, namely, binding of the cations to negative charges at the surface of a hydrophobic structure.

2. We have characterized the inhibitory capacity of a number of polyamines in order to obtain information about the nature of the charges with which they interact. Neomycin inhibited swelling-stimulated cotransport. The diquaternary amines dimethonium and decamethonium were relatively ineffective inhibitors. These compounds are thought to shield negative charges, but not bind to them.

3. Comparison of a homologous series of polyamines indicated that primary amines were better inhibitors than secondary amines, that inhibition increased with the charge of the polyamine, and that inhibition increased as the distance separating the amines increased.

4. The results indicate that the negative charges to which polycations bind are multiple and mobile. Since they must be associated with a hydrophobic environment, it is likely that they are negatively charged phospholipids located in the inner leaflet of the bilayer membrane.

5. Heating red cells or ghosts to 49 °C denatures spectrin. Heating markedly increased K⁺ uptake in swollen ghosts but not in shrunken ghosts. The increase in uptake was reversed when swollen ghosts were shrunk even though denaturation of spectrin was not reversed. Polyamines, which inhibited swelling-activated K⁺ uptake in control ghosts, similarly inhibited the increased uptake in heated ghosts.

6. We speculate that spectrin, which is closely associated with the inner bilayer leaflet, shields negative charges in a volume-dependent manner and so regulates volume-sensitive K⁺ transport.

	INTRODUCTION

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When animal cells swell or shrink, due either to exposure to anisosmotic external solutions or to uptake or loss of osmolytes along with osmotically obligated water, they are able to restore their volume towards normal, in the short run, by activating appropriate ion transporters (Sachs, 1996). When cells swell, K⁺ is lost along with either Cl^- or an organic anion. In many cells, loss occurs through independently controlled K⁺ and Cl^- channels, but in red cells, and in some other cells, swelling activates K⁺-Cl^- cotransporters (Lauf et al. 1992). Human red cell ghosts demonstrate swelling-activated K⁺-Cl^- cotransport with all the characteristics of the same process in the intact cells from which they are derived (Dunham & Logue, 1986; Sachs, 1988; O'Neill, 1989).

Although much is known about the characteristics of the K⁺-Cl^- cotransporter, less is known about how volume change is sensed, or how the signal of volume change is transmitted to the cotransporter. In red blood cells, cotransport activation is believed to result from dephosphorylation of a serine/threonine group by volume-insensitive protein phosphatase I (Krarup & Dunham, 1996), and deactivation on cell shrinkage from phosphorylation of the same serine/threonine by a volume-sensitive protein kinase (Jennings & Al-Rohil, 1990; Jennings & Schulz, 1991). In red blood cells of some species, there is evidence that volume sensing results from a change in the concentration of intracellular proteins (Colclasure & Parker, 1991). An elegant theory that macromolecular crowding regulates protein kinases which control the activity of the cotransporter has been developed (Minton et al. 1992).

In human red cell ghosts, however, swelling activates K⁺-Cl^- cotransport even though there are neither macromolecules to crowd nor to do the crowding (Sachs & Martin, 1993). Moreover, although dephosphorylation by protein phosphatase I is necessary for activation of K⁺-Cl^- cotransport in both intact cells and ghosts, even if dephosphorylation of the serine/threonine group occurs before swelling takes place, cotransport in ghosts can be activated by swelling by a direct pathway independent of phosphorylation-dephosphorylation events (Sachs & Martin, 1993). Similar conclusions were drawn from experiments with sheep red blood cells (Dunham et al. 1993) and inside-out vesicles derived from them (Kelley & Dunham, 1996). No known secondary messenger system which transmits the signal of volume change from a volume sensor to the cotransporter has been identified (Sachs, 1988; Sachs & Martin, 1993).

A great deal of attention has been paid to the phosphorylation pathways which regulate K⁺-Cl^- cotransport, but almost nothing is known about the direct pathway which operates independently of phosphorylation events (Sachs, 1998). We demonstrated that cationic amphiphiles such as sphinogosine and soluble polycations such as spermine inhibit K⁺-Cl^- cotransport in a concentration-dependent manner (Sachs, 1994). Here we report some experiments which show that the polyamine neomycin inhibits swelling-stimulated K⁺-Cl^- cotransport, that inhibition depends on the binding of the amines to negative charges, and that the negative charges must be mobile and relatively close together. We also show that heat denaturation of spectrin, which is known to interact with bilayer membranes containing negatively charged phospholipids, reversibly activates cotransport in swollen ghosts.

	METHODS

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Ethylenediamine, 1,3-diaminopropane, 1,10-diaminodecane, methylaminopropylamine, decamethonium, iodomethane and N,N'-tetramethylenediamine were obtained from Fluka. Spermine, spermidine, N⁸-acetylspermidine and neomycin were supplied by Sigma. Dimethonium was synthesized by reacting N,N'-tetramethylenediamine with iodomethane in ethanol at room temperature overnight (McLaughlin et al. 1983). Dimethonium iodide was washed with ethanol and converted to the chloride form by exchange on a Dowex 1 ion exchange column in the hydroxide form and titration to pH 7·0 with HCl. All other reagents were obtained from commercial sources and were the highest quality available.

Venous blood anticoagulated with heparin or citrate-phosphate-dextrose solution was obtained from the New York Blood Service. Cells were stored at 4°C and used within 1 week of collection. Resealed red cell ghosts were prepared by a gel filtration procedure which has been fully described (Sachs, 1994). Resealing was also performed as described previously (Sachs, 1994), except that the resealing solution always contained a small amount of ⁵⁴Mn²⁺. In previous studies, the ghosts were resealed in solutions made up so that the ghosts after resealing all contained the same concentration of solutes when suspended in a solution of 295 mosmol (kg H₂O)^-1, even though their volumes varied. In some of the studies reported here, however, all ghosts were resealed at the same osmolality (295 mosmol (kg H₂O)^-1) and the volume was altered by modifying the osmolality of the influx solution. Therefore the intracellular solute concentration varied with ghost volume. Such experiments are indicated in the legends to the figures and tables.

To estimate K⁺ uptake, 0·1 ml of ghosts was added to 1 ml of appropriate choline chloride or choline nitrate solution made up to 295 mosmol (kg H₂O)^-1 that contained 2 mM KCl and a trace amount of ⁸⁶Rb⁺ (Rb⁺ substitutes for K⁺ in K⁺-Cl^- cotransport). When the ghosts contained a diamine to which the ghost membrane was judged partially permeable, the influx solution contained the diamine at a concentration equal to that of the intracellular solution and the choline concentration was reduced to maintain solution osmolality at 295 mosmol (kg H₂O)^-1. The suspensions were mixed and incubated at 37°C for 30 or 45 min. The uptake measurement was terminated by transferring the tubes to an ice bath and adding 1 ml of ice-cold 107 mM MgCl₂ solution. The suspensions were mixed and centrifuged, and the supernatants poured off. The ghosts were washed twice with 107 mM MgCl₂ solution and then added to scintillation solution and counted for ⁸⁶Rb⁺ and ⁵⁴Mn²⁺. Samples of the resealing solutions were counted for ⁵⁴Mn²⁺ and samples of the uptake solutions were counted for ⁸⁶Rb⁺. The volume of ghosts used in the uptake measurement, at the time of resealing, was calculated by dividing the ⁵⁴Mn²⁺ content of the ghosts by the ⁵⁴Mn²⁺ concentration of the resealing solution. The concentration of ⁵⁴Mn²⁺ in the ghosts which resealed was, therefore, taken to be the same as the concentration of the resealing solution (which assumes that resealing to ⁵⁴Mn²⁺ is an all-or-none phenomenon). K⁺ uptake was calculated from the amount of ⁸⁶Rb⁺ accumulated by the ghosts during the uptake measurement, the ⁸⁶Rb⁺ concentration of the uptake solution, and the K⁺ concentration of the uptake solution. In all cases, 2 × 10^-4 M ouabain was present in resealing solutions, washing solutions and uptake solutions, so that all values reported are ouabain-resistant uptakes.

To calculate the concentration of Mg²⁺ necessary to yield the concentration of Mg²⁺ indicated in the legends, the dissociation constant for MgATP was taken to be 50 µM, for magnesium creatine phosphate 25 mM, and for MgEGTA 6·17 mM (Sachs, 1994).

The uptake determinations shown in the figures and tables are representative of two or more experiments each using blood from a different donor. Each point in the figures and tables is the mean of four determinations, and the standard error of the mean is indicated unless it is smaller than the symbols. The equations were fitted to the data by a non-linear least-squares program.

	RESULTS

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Inhibition of K⁺-Cl^- cotransport by neomycin

Neomycin is a large, soluble polycation with six secondary amino groups. It interacts strongly with phospholipid vesicles composed of polyphosphoinositides (Gabev et al. 1989), and interferes with polyphosphoinositide metabolism (Schacht, 1980). It also binds strongly to phospholipid vesicles made up of phosphatidylserine. We found previously that neomycin inhibits K⁺-Cl^- cotransport when resealed in red cell ghosts (Sachs, 1988). Figure 1 shows the results of an experiment in which neomycin inhibited the K⁺-Cl^- cotransport rate in ghosts that were swollen to 1·6 times their volume at resealing. Neomycin was present within the ghosts, but not in the extracellular solution. Half-maximal inhibition (KI) occurred at 0·3 mM neomycin. Even at a concentration of neomycin of 4 mM, K⁺-Cl^- cotransport was not completely inhibited.

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Figure 1. Cl--dependent K+ uptake vs. concentration of neomycin within the ghosts Ghosts were prepared to contain, during the uptake measurement, 143 mM K+, 2 mM ATP, 0·1 mM Mg2+ and the indicated concentrations of neomycin. Ghosts were resealed by incubation for 45 min at 37 °C in a 472 mosmol (kg H2O)-1 solution. K+ uptake was measured over a 45 min period in buffered choline chloride or choline nitrate solution containing 1·94 mM K+ and set at 295 mosmol (kg H2O)-1 so that ghost volume during the uptake measurement was 1·6 times its value at resealing. Curves are v = Vm/(1 + [I]/KI), where v is velocity, Vm is maximum velocity and [I] is the concentration of the inhibitor. Vm was 2·5 mmol (l ghosts)-1 h-1 and KI was 0·3 mM.

Figure 2 gives the results of an experiment in which K⁺-Cl^- cotransport was measured as a function of ghost volume, with and without 4 mM neomycin. In the control ghosts, there was an 8-fold increase in cotransport rate when the value at the highest relative ghost volume (1·6) and the lowest relative volume (0·4) were compared. Neomycin inhibited K⁺-Cl^- cotransport at all ghost volumes. Inhibition was nearly complete at relative volumes less than 1·0, but, as in the experiment shown in Fig. 1, was less complete at higher relative volumes. In the neomycin ghosts, the cotransport rate was 5-fold higher when the ghosts at the highest relative volume and the lowest relative volume were compared. Neomycin inhibits cotransport at all ghost volumes, and inhibits nearly completely the increase in cotransport rate which occurs when the ghosts are swollen.

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Figure 2. Cl--dependent K+ uptake vs. relative ghost volume Ghosts were prepared to contain, during the uptake measurement, 141 mM K+, 2 mM ATP, 0·1 mM Mg2+ and either no () or 4 mM () neomycin. Ghosts were resealed by incubation for 45 min at 37 °C at osmolalities which varied from 118 to 472 mosmol (kg H2O)-1. K+ uptake was measured over a 45 min period in a buffered choline chloride or choline nitrate solution set to 295 mosmol (kg H2O)-1 which contained 2·27 mM K+. The curves were drawn to describe the data and are without theoretical significance.

In the remainder of this section, we describe the results of some experiments in which we sought information about the nature of the membrane constituents with which the polycations interact by examining the effects of polyamines with different structures.

The inhibitory effect of diquaternary amines

It seemed appropriate to determine whether polyamines decrease swelling-stimulated K⁺-Cl^- uptake by 'shielding' negative charges or by 'binding' to negative charges. To do this, we made use of an observation reported by McLaughlin et al. (1983). These authors demonstrated that dimethonium, in which two quaternary ammonium groups are separated by about 4·5 Å (1 Å = 0·1 nm), collapsed the surface potential but did not bind with the negative charges in the bilayer (the Debye length, at which the potential adjacent to a charged surface falls to 1/e of its value at the surface, is about 7 Å for a phospholipid membrane containing about 40 % phosphatidylserine and suspended in a 160 mM solution, like the inner phospholipid bilayer of ghost membranes (McLaughlin, 1977)). Inorganic divalent cations, such as Ca²⁺, also collapse the surface potential, but are directly absorbed to the negative charges in the bilayer (McLaughlin et al. 1983).

Figure 3 shows the results of an experiment in which we measured the effect of dimethonium on swelling-activated K⁺-Cl^- uptake. The calculated concentration for half-maximal inhibition was greater than 1 M. For comparison, we examined the effect of the analogous compound ethylenediamine in which the quaternary amines are replaced by primary amines. Half-maximal inhibition occurred at 3·8 mM. Red cell membranes are permeable to ethylenediamine at pH 7·4 so it is likely that it accumulates in the hydrophobic phospholipid bilayer (red cells are essentially impermeable to dimethonium) and the measurement is not comparable to that with dimethonium, but it does show that if the small diprimary amine is able to bind directly to negatively charged membrane groups, swelling-stimulated K⁺-Cl^- uptake is inhibited. The dimethonium experiment can also be compared with the experiment shown in Fig. 5 in which the effect of diaminopropane, to which red cells are relatively impermeable, on swelling-stimulated K⁺-Cl^- uptake was measured; half-maximal inhibition occurred at 16 mM.

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Figure 3. Cl--dependent K+ uptake vs. the concentration of dimethonium () or ethylenediamine () within the ghosts Ghosts were prepared to contain, during the uptake measurement, 85 mM K+, 2 mM ATP, 0·1 mM Mg2+ and the indicated concentrations of dimethonium or ethylenediamine. When the concentration of the inhibitor was less than 40 mM, it was replaced with choline chloride to maintain constant osmolality. Ghosts were resealed by incubation for 45 min at 37 °C and 472 mosmol (kg H2O)-1. K+ uptake was measured over a 45 min period in buffered choline chloride or choline nitrate solution containing 2 mM K+ and set at 295 mosmol (kg H2O)-1 so that ghost volume during the uptake measurement was 1·6 times its value at resealing. In solutions to which ghosts containing ethylenediamine were added, ethylenediamine at the concentrations indicated replaced an equal amount of choline. Curves are v = Vm/(1 + [I]/KI). For the experiment with dimethonium, Vm was 4·6 mmol (l ghosts)-1 h-1 and KI was 1221 mM. For the experiment with ethylenediamine, Vm was 3·8 mmol (l ghosts)-1 h-1 and KI was 3·8 mM.

The results of a similar experiment with decamethonium, in which the quaternary amines are separated by about 17 Å, are shown in Fig. 4; half-maximal inhibition of swelling-stimulated K⁺-Cl^- uptake was calculated to occur at 67 mM. This result is more difficult to interpret than that with dimethonium. The quaternary amines of decamethonium are much further apart than the Debye length of 7 Å. The compound cannot therefore be considered a divalent point charge and its ability to shield the surface potential by accumulating in the diffuse double layer cannot be expected to equal that of dimethonium, unless decamethonium preferentially aligns parallel to the membrane surface. On the other hand, decamethonium has many more methylene groups than dimethonium and its increased inhibitory capacity when compared with dimethonium may result from an increased tendency to accumulate in hydrophobic membrane areas. The inhibitory effect of decamethonium may be compared with that of the diprimary amine diaminodecane. Red cells are permeable to diaminodecane (although less so than to ethylenediamine). Diaminodecane half-maximally inhibited at 2 mM, much less than the value of 67 mM at which half-maximal inhibition by decamethonium occurred.

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Figure 4. Cl--dependent K+ uptake vs. the concentration of decamethonium () or diaminodecane () within the ghosts Ghosts were prepared to contain, during the uptake measurement, 122 mM K+ and the indicated concentrations of decamethonium or diaminodecane. When the concentration of inhibitor was less than 20 mM, it was replaced by an equal volume of isosmotic choline chloride. ATP was omitted from the resealing solutions since we found that addition of diaminodecane to solutions containing ATP produced a precipitate. Ghosts were resealed by incubation for 45 min at 37 °C and 472 mosmol (kg H2O)-1. K+ uptake was measured over a 45 min period in buffered choline chloride or choline nitrate solution containing 2·0 mM K+ and set at 295 mosmol (kg H2O)-1 so that ghost volume during the uptake measurement was 1·6 times its volume at resealing. In the uptake solutions, diaminodecane at the concentration indicated replaced an equal amount of choline. Curves are v = Vm/(1 + [I]/KI). For the experiment with decamethonium, Vm was 0·8 mmol (l ghosts)-1 h-1 and KI was 67 mM. For the experiment with diaminodecane, Vm was 0·8 mmol (l ghosts)-1 h-1 and KI was 1·7 mM.

From these experiments we concluded that diquaternary amines which only shield negative charges are much less effective inhibitors of swelling-stimulated K⁺-Cl^- uptake than analogous diprimary amines which both shield and bind to negative charges.

Inhibition of K⁺-Cl^- uptake by soluble polyamines

We hoped to obtain insight into the nature of the membrane components which interact with the soluble polycations. For this purpose we compared inhibition by diaminopropane with inhibition by N-methyldiaminopropane (Fig. 5), inhibition by spermidine with inhibition by N⁸-acetylspermidine (Fig. 6), and inhibition by spermine with inhibition by N-methyldiaminopropane (Fig. 7). The structures of the probes are shown in the figures, and the results of the experiments are summarized in Table 1.

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Figure 5. Cl--dependent K+ uptake vs. the concentration of diaminopropane () or N-methyldiaminopropane () within the ghosts Ghosts were prepared to contain, during the uptake measurement, 94 mM K+, 2 mM ATP, 0·1 mM Mg2+ and the indicated concentrations of diaminopropane or N-methyldiaminopropane. When the concentration of inhibitor was less than 20 mM, it was replaced by an equal volume of isosmotic choline chloride. Ghosts were resealed by incubation for 30 min at 37 °C and 531 mosmol (kg H2O)-1. K+ uptake was measured over a 30 min period in buffered choline chloride or choline nitrate solution containing 2·14 mM K+ and set at 295 mosmol (kg H2O)-1 so that ghost volume during the uptake measurement was 1·8 times its volume at resealing. Diaminopropane and N-methyldiaminopropane at the concentrations indicated replaced an equal amount of choline in the uptake solutions. Curves are v = Vm/(1 + [I]/KI). For the experiment with diaminopropane, Vm was 3·2 mmol (l ghosts)-1 h-1 and KI was 16 mM, and for the experiment with N-methyldiaminopropane, Vm was 2·4 mmol (l ghosts)-1 h-1 and KI was 54 mM.

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Figure 6. Cl--dependent K+ uptake vs. the concentration of spermidine () and N8-acetyl spermidine () within the ghosts Ghosts were prepared to contain, during the uptake measurement, 94 mM K+, 2 mM ATP, 0·1 mM Mg2+ and the indicated concentrations of spermidine or N8-acetylspermidine. When the concentration of the inhibitor was less than 20 mM, it was replaced by an equal volume of isosmotic choline chloride. Ghosts were resealed by incubation for 30 min at 37 °C and 531 mosmol (kg H2O)-1. K+ uptake was measured over a 30 min period in buffered choline chloride or choline nitrate solution containing 2·06 mM K+ so that ghost volume during the uptake measurement was 1·9 times its volume at resealing. Curves are v = Vm/(1 + [I]/KI). For the experiment with spermidine, Vm was 5·4 mmol (l ghosts)-1 h-1 and KI was 5·0 mM. For the experiment with N8-acetylspermidine, Vm was 5·9 mmol (l ghosts)-1 h-1 and KI was 16 mM.

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Figure 7. Cl--dependent K+ uptake vs. the concentration of spermine () or N-methyldiaminopropane () within the ghosts Ghosts were prepared to contain, during the uptake measurement, 132 mM K+, 2 mM ATP, 0·1 mM Mg2+ and the indicated concentrations of spermine or N-methyldiaminopropane. When the concentration of inhibitor was less than 8 mM, it was replaced by an equal volume of isosmotic choline chloride. Ghosts were resealed by incubation for 45 min at 37 °C and 472 mosmol (kg H2O)-1. K+ uptake was measured over a 45 min period in buffered choline chloride or choline nitrate solution containing 1·99 mM K+ and set at 295 mosmol (kg H2O)-1 so that ghost volume during the uptake measurement was 1·6 times its volume at resealing. Curves are v = Vm/(1 + [I]/KI). For the experiment with spermine, Vm was 3·4 mmol (l ghosts)-1 h-1 and KI was 1·2 mM; for the experiment with N-methyldiaminopropane, Vm was 3·8 mmol (l ghosts)-1 h-1 and KI was 67 mM.

Table 1. Inhibition of swelling-stimulated K⁺-Cl^- cotransport by soluble polyamines

Several conclusions can be drawn. Comparison of inhibition by diaminopropane with inhibition by N-methyldiaminopropane, and inhibition by spermidine with inhibition by N⁸-acetylspermidine suggests that primary amines are more effective inhibitors than secondary amines. Combined with the findings in the previous section, the results suggest that the effectiveness of an amine on inhibition is in the order primary > secondary > quaternary. Inhibition may depend on direct interaction of a positively charged amino group with negative charges, and that interaction between the charges is impeded as the number of bulky substituents attached to the N atom increases.

Comparison of the results in Fig. 5 with those in Fig. 6 indicate that inhibition increases with the number of charges. It is expected from classic electrostatics (McLaughlin, 1977) that the concentration of a point charge in the diffuse double layer, and therefore its effect, increases by an order of magnitude for each increase in the number of charges. This is clearly not the case for the probes considered here. The concentration of spermidine which half-maximally inhibited was about one-third of the half-maximal inhibitory concentration of diaminopropane, and the concentration of N⁸-acetylspermidine which half-maximally inhibited was about one-third of the half-maximal inhibitory concentration of N-methyldiaminopropane. The discrepancy between the observed and expected increase in effectiveness as the number of positive charges increased by 1 (3-fold rather than 10-fold) may be rationalized by considering that, while the distance between the primary amine in spermidine and the secondary amine is 6 Å, just less than the Debye length of 7 Å, the distance between the two primary amines is 14 Å. The compound cannot be considered a point source, and some fractional charge between 1 and 3 related to such variables as the orientation of the probe relative to the membrane surface should be used in estimating its orientation in the diffuse double layer (Denisov et al. 1998). A comparison of the results in Figs 5 and 6, then, suggests that, in a homologous series of polyamines, replacing a primary amine with a secondary amine reduces the affinity for the probe 3-fold, and adding a third charge to a diamine increases the affinity 3-fold.

Figure 7 compares the inhibitory effect of spermine, a tetravalent cation, with the inhibitory effect of N-methyldiaminopropane, which is almost exactly half the symmetrical spermine molecule. The distance between the primary amine and the secondary amine in N-methyldiaminopropane is about 6 Å, the same as the distance between one of the primary amines of spermine and the nearest secondary amine. On the other hand the distance between the primary amines in spermine is 20 Å and the distance between a primary amine and the second secondary amine is 14 Å. K_I for N-methyldiaminopropane was 56 times that for spermine. If one assumes that the effect of one extra positive charge on the K_I of the compounds is a 3·3-fold decrease calculated from Figs 5 and 6, and that both extra charges in spermine have the same effect (probably not true since the second extra charge is even further beyond the Debye length than the first), only an 11-fold decrease in K_I would be expected. There must be many more spermine molecules adjacent to the membrane than would be expected from the surface potential effects alone.

Binding of the first primary amine in spermine reduces its translational mobility by one degree of freedom since it can only move in the plane of the membrane (reduction in dimensionality) and its rotational mobility by two degrees of freedom. The same consideration holds for diaminopropane. However, the two primary amines in spermine are much further apart than the two primary amines in diaminopropane. Theoretical treatments of the interaction of a polyvalent ligand with a receptor mobile in a membrane predict that the observed affinity of the ligand for the receptor increases as the distance between the binding sites increases since the number of receptors available for binding increases as the radius of the circle which the ligand can sweep increases (Reynolds, 1979; Dwyer & Bloomfield, 1981). This effect may account for the greater reduction in the inhibitory K_I for spermine due to the addition of the extra charges than expected from the comparison of the diaminopropane-spermidine and the N-methyldiaminopropane-N-acetylspermidine pairs.

Heating modifies the response of the K⁺-Cl^- cotransporter to swelling

Heating rat red cells to 49°C modified the response of the Na⁺-H⁺ exchanger and the Na⁺-K⁺-2Cl^- cotransporter and of the K⁺-Cl^- cotransporter to volume change (Orlov et al. 1993). Heating did not alter Na⁺-H⁺ exchange or Na⁺-K⁺-2Cl^- cotransport in cells suspended in isosmotic solutions (normal sized cells), but prevented an increase in their activities when the cells were shrunk. On the other hand, heating did not alter K⁺-Cl^- cotransport in the swollen cells, but greatly increased it in cells at normal cell volume.

When red cells or their membranes are heated to 49°C, the only detectable alteration is the irreversible denaturation of spectrin (Lysko et al. 1981). There is a large and controversial literature addressing the question of whether spectrin binds weakly and reversibly with phospholipid membranes containing negatively charged phospholipids such as phosphatidylserine (for references see McKiernan et al. 1997). In view of our findings with organic cations, we examined the effect of heating on volume-stimulated K⁺-Cl^- cotransport in ghosts.

Figure 8 shows that heating red cell ghosts to 49°C increased ouabain-insensitive K⁺ uptake in swollen ghosts, but not in shrunken ghosts. Both Cl^--sensitive and -insensitive K⁺ uptake was stimulated, the latter more than the former. A significant part of the swelling-stimulated K⁺ uptake in heated ghosts is not K⁺-Cl^- cotransport. We have found that even in ghosts which have not been heated a significant fraction of swelling-activated K⁺ uptake is independent of Cl^- (Sachs, 1988), and that the amount varies from experiment to experiment. Ouabain-insensitive K⁺ uptake in trout red cells is increased by catecholamines and by hypotonic cell swelling; calmodulin-stimulated K⁺ uptake is Cl^- dependent but K⁺ uptake in swollen cells is not (Guizouarn et al. 1993). In both cases K⁺ uptake is electroneutral, but K⁺ uptake in swollen cells is not K⁺-H⁺ exchange. The authors conclude that in both cases K⁺ uptake is K⁺ anion cotransport, but nitrate or some other anion replaces Cl^- when uptake is increased by swelling. When ghosts are heated, the Cl^- insensitivity of the activated K⁺ uptake may have the same explanation; cotransport in heated ghosts may lose its specificity for Cl^-, and Cl^- may be replaced by nitrate.

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Figure 8. Ouabain-insensitive K+ uptake in swollen and shrunken ghosts vs. time that the ghosts were incubated at 49 °C Ghosts were resealed at 295 mosmol (kg H2O)-1 and then incubated at 49 °C for the times indicated. They were then washed 3 times in buffered choline chloride (, ) or choline nitrate (, ) solutions set at 295 mosmol (kg H2O)-1. Ghosts were suspended in the same solutions at 168 mosmol (kg H2O)-1 (, ) so that they were 1·6 times their volume at resealing, or 738 mosmol (kg H2O)-1 (, ) so that they were 0·4 times their volume at resealing. The solutions contained 1·87 mM K+, and K+ uptake was measured at 37 °C for 45 min. The curves were drawn to guide the eye and are without theoretical significance.

In Fig. 9, the results of an experiment in which Cl^--dependent K⁺ uptake was measured in heated ghosts and control ghosts as a function of relative ghost volume are shown. Heating somewhat decreased K⁺ uptake at low volumes. At large volumes, K⁺ uptake increased much more rapidly with volume in the heated ghosts than in the control ghosts. In control ghosts, there was not much sign of a set point; K⁺ uptake increased monotonically with ghost volume without a break in the curve. In heated ghosts, however, uptake increased with volume much more steeply at the high end of the curve, and the shape of the curve was similar to that of curves obtained with red cells of other species which were not heated (Parker, 1993).

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Figure 9. Cl--dependent K+ uptake in heated () and control () ghosts as a function of relative ghost volume Ghosts were resealed at 295 mosmol (kg H2O)-1. Half were then heated for 5 min at 48 °C, and half incubated at room temperature. Ghosts were washed with buffered choline chloride or choline nitrate solution at the same osmolality. Ghosts were then distributed to solutions whose osmolalities were set so that the final ghost volumes were as indicated. K+ uptake was measured at 1·95 mM K+ for 45 min at 37 °C. The curves were drawn to guide the eye and are without theoretical significance.

We performed several experiments in an effort to characterize the K⁺ uptake which is activated by heating.

Denaturation of spectrin by heating is irreversible. On the other hand, swelling-stimulated K⁺ uptake is reversible when the ghosts are reshrunk (Sachs & Martin, 1993). Table 2 shows that the increased swelling-activated K⁺ uptake in heated ghosts was equally reversible when the ghosts were reshrunk. Ghosts were resealed and either heated at 49°C for 7 min or maintained at room temperature. Heated and non-heated ghosts were each separated into two sets. The ghosts in one set were swollen to 1·8 times their volume at resealing, and the ghosts in the second set were shrunk to 0·4 times their volume at resealing. The ghosts were incubated for 30 min at 37°C in these solutions while K⁺ uptake was measured. As expected, activation of K⁺ influx by swelling was much greater in the heated than in the control ghosts. In each group, K⁺ uptake was measured during a second 30 min period; half of the swollen ghosts in each group were shrunk and half of the shrunken ghosts were swollen. When swollen ghosts were shrunk, K⁺ uptake returned to the level seen in the shrunken ghosts in the first incubation, and when shrunken ghosts were swollen, uptake approached the level seen in swollen ghosts in the first incubation. Even though heat damage is irreversible, the increased response of K⁺ uptake to ghost swelling is not. Protein denaturation does not directly activate K⁺ uptake stimulated by ghost swelling.

Table 2. Stimulation of ouabain-insensitive K⁺ uptake by swelling is reversible in both heated and control ghosts

We compared the effect of some inhibitors of K⁺-Cl^- cotransport on swelling-stimulated K⁺ uptake in control and heated ghosts, and the results are shown in Table 3. Both the total ouabain-resistant K⁺ uptake and the Cl^--dependent uptake are given. The ghosts used in the experiments were all swollen to about 1·8 times their volume at resealing before uptake was measured. As usual, heating activated Cl^--independent swelling-stimulated K⁺ uptake as well as Cl^--dependent uptake. The results in Table 3 show that polyamines inhibited both Cl^--dependent and Cl^--independent K⁺ uptake in the control swollen ghosts, the former more completely than the latter. Heating increased K⁺ uptake in the swollen ghosts, and the fractional inhibition by the polyamines was about as great in the heated ghosts as in the control ghosts. Polyamines reduced the signal of ghost swelling about as much in the heated ghosts as in the control ghosts.

Table 3. Effect of inhibitors on ouabain-insensitive K⁺ uptake and K⁺-Cl^- cotransport in swollen control and heated ghosts

	DISCUSSION

Top Abstract Introduction Methods Results Discussion References

The nature of the sensor of cell volume change, and of the signal transduction pathway which communicates between the sensor and the K⁺-Cl^- cotransporter, is, at best, uncertain (Sachs, 1998). In red cells, it is widely believed that swelling inactivates a protein kinase, and dephosphorylation of the relevant serine/threonine group by volume-insensitive protein phosphatase I activates K⁺-Cl^- cotransport (Jennings & Al-Rohil, 1990; Jennings & Schulz, 1991). Phosphorylation of the same group by the volume-sensitive kinase inactivates K⁺-Cl^- cotransport when the cells shrink. Although it is not known for certain what regulates the volume-sensitive kinase, there is evidence in dog red cells that the volume sensor is macromolecular crowding (Minton et al. 1992), and it is supposed that crowding, which occurs when cells shrink, increases the thermodynamic activity of the kinase (Parker, 1993). The theory is elegant, and it provides an intellectually satisfying explanation for many of the observations about volume regulation in the red cells of many species. However, it is not the whole story (Sachs & Martin, 1993; Dunham et al. 1993; Ortiz-Carranza et al. 1997).

The observation that the serine/threonine phosphatase inhibitors okadaic acid and calyculin inhibit K⁺-Cl^- cotransport and its response to swelling is the crucial piece of evidence for the proposed mechanism of swelling-stimulated cotransport regulation outlined above. A typical protocol for an experiment with ghosts involves resealing them by incubating at a specific osmolality and with solutes at a fixed concentration, and then changing the osmolality of the solution to swell or shrink them while K⁺ uptake is measured. Okadaic acid inhibits K⁺-Cl^- cotransport and its response to ghost swelling if it is added at the beginning of the resealing period, but is largely ineffective if it is added after resealing is complete (Sachs & Martin, 1993). The inescapable conclusion is that serine/threonine dephosphorylation, which must occur before K⁺-Cl^- cotransport is activated, is completed in the resealed ghosts before the changes in cotransport rate in response to volume change occur. We believe that dephosphorylation occurs during resealing because the volume-sensitive kinase is largely cytoplasmic and lost during haemolysis (or the kinase is inoperative because the macromolecules which regulate it are lost), but the volume-insensitive phosphatase remains associated with the ghost membrane and is able to act unopposed during the resealing period (Sachs & Martin, 1993). In ghosts there is a mechanism for detecting volume change and for signalling the cotransporter that is independent of phosphorylation state and macromolecular crowding since the ghosts are nearly free of haemoglobin and other proteins.

The K⁺-Cl^- cotransport rate in ghosts resealed without ATP is less at any ghost volume than it is in ghosts at the same volume resealed with MgATP (Sachs & Martin, 1993); MgATP stimulates K⁺-Cl^- cotransport in ghosts just as it does in intact red cells (Lauf et al. 1992). Stimulation of cotransport by MgATP is abolished by genistein, a tyrosine kinase inhibitor, and, in the absence of ATP, the cotransport rate is increased in the presence of the tyrosine phosphatase inhibitor MgVO₄ (Sachs & Martin, 1993). Together, these findings suggest that stimulation by MgATP is due to phosphorylation of a tyrosine group. Since the effect of both genistein and vanadate can be demonstrated in ghosts, both the kinase and phosphatase must be associated with the cell membrane. Tyrosine phosphorylation might increase the cotransport rate by activating protein phosphatase I described above, but that cannot be the case here since resealing ghosts with MgATP increases cotransport even though dephosphorylation of the serine/threonine phosphate goes to completion in ATP-free ghosts. In mouse red cells, phosphorylation of the phosphatase by a tyrosine kinase inhibits rather than activates K⁺-Cl^- cotransport (DeFranceschi et al. 1997). The tyrosine kinase may be a volume-sensitive enzyme, stimulated by cell swelling, which regulates the phosphorylation state of a tyrosine group somewhere in the cotransport mechanism in response to volume change. The tyrosine kinase would, then, transmit the signal of volume change from the sensor to the cotransporter. There is some evidence against this: when the intracellular Mg²⁺ concentration is rapidly reduced in intact cells before volume is changed, further phosphorylation by the tyrosine kinase is prevented, although the phosphorylation state of the relevant tyrosine is not likely to change very much over the short term. Swelling still stimulates the cotransport rate in these cells to about the same extent as it does in control cells in which phosphorylation is still possible (Dunham et al. 1993; Ortiz-Carranza et al. 1997). While phosphorylation of a tyrosine may be necessary for maximal activation of cotransport rate, these results suggest that phosphorylation does not occur in response to cell swelling. If the tyrosine kinase is volume sensitive, molecular crowding cannot be responsible for its regulation; swelling can only dilute macromolecules, and only macromolecular concentration can increase the thermodynamic activity of enzymes.

Whether or not tyrosine phosphorylation stimulates K⁺-Cl^- cotransport in response to ghost swelling, ghost swelling activates cotransport even when the ghosts are resealed in the complete absence of ATP (Sachs & Martin, 1993). The direct pathway of activation does not require ATP.

Although there must be a volume sensor and signal transduction pathway independent of phosphorylation- dephosphorylation events, little is known about what it may be; most studies of volume-sensitive transport in red blood cells have focused on the activity of protein kinases and phosphatases. Both the sensor and the signalling mechanism of the direct pathway must exist entirely in the cell membrane since the pathway can be demonstrated in ghosts. We have tried to demonstrate participation of a known signal transduction pathway in the regulation of cotransport rate in response to volume change without success (Sachs, 1988; Sachs & Martin, 1993).

It has long been known that divalent cations inhibit K⁺-Cl^- cotransport in red cells and ghosts (Lauf et al. 1992). While evaluating the possibility that protein kinase C (PKC) or calmodulin is involved in signal transduction between a volume sensor and the cotransporter in ghosts, we found that the soluble polyvalent cation neomycin and the amphipathic organic cation trifluoperazine inhibit K⁺-Cl^- cotransport at relatively low concentrations (Sachs, 1988). For other reasons we later concluded that neither PKC nor calmodulin is involved in signal transduction. However, the observation that both divalent inorganic cations and organic cations inhibit K⁺-Cl^- cotransport suggested to us that intracellular negative charges might be involved in volume sensing or in signal transduction. We examined the effects of two other soluble polycations, spermine and methylglyoxal, and two other amphipathic organic cations, sphingosine and tetracaine, on the cotransport rate (Sachs, 1994). We found that each of the four organic cations tested inhibit the cotransport rate in both swollen and nominally shrunken ghosts. There have been reports that organic cations also inhibit cotransport in sheep red cells (Adragna & Lauf, 1994; Ortiz-Carranza et al. 1997).

In the experiment reported here, neomycin inhibited swelling-stimulated cotransport at all ghost volumes. However, there is significant K⁺-Cl^- cotransport even in the smallest ghosts. It is conceivable that the mechanism which monitors cell swelling is operative at all ghost volumes and that the volume sensor in ghosts is more a rheostat than an on-off switch, so that it would not be surprising if an agent which interferes with signal transduction reduces the cotransport rate to some extent at all volumes.

Since both soluble polycations and amphipathic organic cations, such as sphingosine, which are known to preferentially insert in hydrophobic regions such as phospholipid bilayers, inhibit the K⁺-Cl^- cotransport rate, we assumed that the most likely site of the relevant negative charges is the surface of the bilayer membrane. We examined the effects of a variety of soluble polyamines on swelling-stimulated K⁺-Cl^- uptake in an attempt to obtain some insight into the nature of the sites at which they inhibit. The soluble polycations inhibit when present in the intracellular solution only, so the charges must be present on the inner membrane surface. We presented our results with the polyamines in terms of studies which have been performed with model systems of charged phospholipid membranes. The results with the diquaternary amines dimethonium and decamethonium showed that these compounds were ineffective as inhibitors of cotransport compared with the analogous compounds ethylenediamine and diaminodecane in which the quaternary amines are replaced by primary amines. The hydrophobicity of ethylenediamine and diaminodecane (both penetrate intact red cell membranes, the former more rapidly than the latter) may be in part responsible for their increased effectiveness, but the ineffectiveness of the quaternary amines is more likely to be due to their inability to bind to negative charges, as demonstrated in model vesicular preparations (McLaughlin et al. 1983).

Comparison of a homologous series of polyamines shows that the effectiveness of the amines as inhibitors of K⁺-Cl^- cotransport decreases in the order primary > secondary > quaternary. In theoretical models of negatively charged membranes in electrolyte solutions, a decrease in the positive charge of a cation decreases the concentration of the cation next to the membrane and therefore increases the inhibitory constant, K_I. The pK values of amines, however, are 8·4 or greater and these experiments were done at pH 7·4 so that variation of charge of primary, secondary, and quaternary amines is not likely to account for the difference in their effectiveness. It seems more likely that the increase in the number of substituents on the N atom structurally impedes the ability of the amine to approach and 'bind' to negatively charged structures.

On the other hand, we found that an increase in the number of positive charges in a homologous series of polyamines decreases the inhibitory K_I by an amount qualitatively (but far from quantitatively) in accord with predictions from model systems. Part of the discrepancy between theoretical predictions and our experimental results may be explained by the fact that polyamines are not point sources, and positive charges are separated by distances large in comparison with the Debye length. This point becomes important when comparing spermine with N-methyl-diaminopropane which represents approximately half the spermine molecule. Spermine has two more positive charges than N-methyldiaminopropane, and if one applied the same multiple (about 3·3) calculated above from comparison of the KI for diaminopropane with that for spermidine and the KI for N-methyldiaminopropane with that for N⁸-acetylspermidine to predict the effect of each additional positive charge of spermine on its inhibitory K_I, then, from the inhibitory K_I for N-methyldiaminopropane, one would predict that the K_I for spermine should be about 6·2 mM, higher than the value of 1·2 mM that we measured. The contribution of the two extra charges on spermine to the reduction in K_I may not result solely from increased concentration of the compound in the diffuse double layer, but also from the reduction in dimensionality of its translational movement and the limitation of its rotational mobility resulting from the binding of the first amine of spermine with a negative surface charge (Reynolds, 1979; Cutsforth et al. 1989). Binding of additional amines of the same molecule will occur with a higher affinity than binding of the first amine, and therefore the overall inhibitory K_I will decrease. This formulation is based on the assumption that the relevant negative charges are plentiful and mobile or, if fixed, that they are nearly exactly complementary to the arrangement of the amines in the probe.

The findings with the polyamines are at least qualitatively consistent with the findings expected for interaction of large multicharged molecules with phospholipid membranes containing many mobile negative charges. However, roughly similar findings have been reported in studies of the binding of spermine and its analogues to a soluble protein in which glutamates are located in clusters in an -helix, each separated by three amino acids (Leroy et al. 1997). The charged amino acids are located on the same face of the -helix separated by about the same distance as the amines in spermine. However, other polyamines (e.g. neomycin and methylglyoxal) in which the charges are not organized in a linear array and are separated by distances different from those in spermine and its analogues also inhibit cotransport at relatively low concentrations. It is reasonable to believe that all these inhibitors act at the same sites to inhibit cotransport, in which case a random distribution of charges such as phosphatidylserine in a phospholipid bilayer seems a much more likely target than a fixed distribution of charges in an -helix. The amphipathic cations which inhibit cotransport, especially sphingosine, preferentially insert into phospholipid bilayers. Although it is possible that sphingosine might insert into the hydrophobic core of a protein in such a way that its charged headgroup binds to a negative charge at the surface of the protein, we know of no precedent for such a mechanism, but there are many instances in which amphipathic cations insert into phospholipid bilayers and alter the surface charge. Although we cannot categorically exclude the possibility that the organic cations exert their effects by interacting with a protein somewhere in the membrane, we think it far more likely that they exert their effects by binding to negative charges in the inner leaflet of the bilayer membrane.

Some surprising observations have been reported which may be related to our findings. Lauf (1988a) replaced intracellular chloride of LK sheep red cells with thiocyanate and incubated the cells at 37°C. The cells were then removed and repeatedly washed with chloride solutions to remove intracellular thiocyanate and replace it with chloride. When Cl^--dependent K⁺ uptake was measured in these cells, it was found that it was increased at all ghost volumes. The effect of the thiocyanate gradually disappeared as incubation continued. Lyotropic anions such as thiocyanate are known to adsorb to phospholipid bilayers (McLaughlin, 1977), and therefore should increase the negative surface charge.

Our results suggest that the swelling-activated K⁺-Cl^- cotransport rate varies with the surface charge of the inner leaflet of the bilayer membrane. There have been many reports of modification of the activity of transport proteins by surface charge or by specific negatively charged lipids (Martinac et al. 1990; Shyng & Nichols, 1998), although the significance of such effects is not always clear. It is possible that surface charge serves as volume sensor in ghosts. The possibility would be more credible if some plausible relation between volume change and change in membrane surface charge could be proposed.

Many reports of the reversible association of spectrin with phospholipid membranes containing negatively charged phospholipids and with phospholipid vesicles containing phosphatidylserine have appeared (McKiernan et al. 1997). Whether or not spectrin 'binds' to negative phospholipids, spectrin, at least in the presence of protein 4.1, exists at high concentration in close proximity to the inner leaflet of the membrane bilayer where it is likely to shield the surface potential. We speculate that the degree of shielding may be volume dependent - swelling may decrease the amount of shielding and uncover negative charges which activate K⁺ uptake, and shrinkage may have the opposite effect.

The effect of heating red cells or ghosts to 49°C is highly specific: spectrin is denatured without apparent change in any other membrane component (Brandts et al. 1977). Denaturation of spectrin by heating interferes with the self association of dimers to form tetramers (Yoshino & Minari, 1987). The findings of Orlov et al. (1993), which very probably reflect the same phenomenon we have studied in human ghosts, show that heating simultaneously reduces or abolishes the sensitivity of Na⁺-H⁺ exchange and Na⁺-K⁺-2Cl^- cotransport to cell shrinkage and increases the sensitivity of K⁺ uptake to cell swelling. We have found that heating increases K⁺ uptake in swollen ghosts but not in shrunken ghosts. The inability of spectrin dimers to self associate to form tetramers and form the submembrane cytoskeleton shifts the volume set point to lower cell volumes. Diamide, which reacts with sulfhydryl groups, binds preferentially to spectrin, interferes with the self association of spectrin dimers to spectrin tetramers, and lowers the temperature threshold for heat-induced denaturation of spectrin; the effect is reversed by sulfhydryl reducing agents (Streichman et al. 1988). Diamide, like heating, increases swelling-activated K⁺ uptake in intact sheep red cells, and its effect is also reversed by exposure to sulfhydryl reducing agents (Lauf, 1988b). Denaturation of spectrin with decreased association of dimers to tetramers and inability to form cytoskeleton might result in increased exposure of negative surface charges in the phospholipid bilayer at any volume, and exposure is likely to be greater at high volume than at low. If the magnitude of K⁺ uptake is proportional to the exposed surface charge of the membrane, it would be expected that, as we found, heating increases K⁺ uptake in swollen ghosts more than in shrunken ghosts. Our finding that neomycin and spermine are as effective in inhibiting swelling-activated K⁺ uptake in heated ghosts as in control ghosts is not inconsistent with this formulation.

A cartoon of such a mechanism is shown in Fig. 10. In the shrunken ghosts, spectrin is in close contact with the phospholipid bilayer membrane and shields the negatively charged headgroups of phosphatidylserine. When the ghosts are swollen spectrin moves away from the bilayer and the negative charges become accessible to the cytoplasmic portion of the cotransporter. Association of the cotransporter with the membrane results in a conformational change which increases the cotransport rate. Although not shown, denatured spectrin may expose negative charges at high ghost volumes, but not at low volumes.

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Figure 10. A hypothesis for the regulation of K+-Cl- activity by negative charges in the inner bilayer of the ghost membrane Negative charges are indicated as phospholipids with filled head groups. In shrunken ghosts, spectrin is in close contact with the negative charges and prevents contact between them and the cytoplasmic portion of the cotransporter. In swollen ghosts, spectrin moves away from the surface charges and the cytoplasmic portion of the cotransporter is able to move into contact with them. As a result there is a conformational change in the cotransporter associated with its activation.

	REFERENCES

Top Abstract Introduction Methods Results Discussion References

Adragna, N. C. & Lauf, P. K. (1994). Quinine and quinidine inhibit and reveal heterogeneity of K-Cl cotransport in low K sheep erythrocytes. Journal of Membrane Biology 142, 195-207	[Medline]
Brandts, J. F., Erickson, L., Lysko, K., Schwartz, A. T. & Taverna, R. D. (1977). Calorimetric studies of the structural transitions of the human erythrocyte membrane. The involvement of spectrin in the A transition. Biochemistry 16, 3450-3454
Colclasure, G. C. & Parker, J. C. (1991). Cytosolic protein concentration is the primary volume signal in dog red cells. Journal of General Physiology 98, 881-892	[Abstract]
Cutsforth, G. A., Whitaker, R. N., Hermans, J. & Lentz, B. R. (1989). A new model to describe extrinsic protein binding to phospholipid membranes of varying composition: application to human coagulation proteins. Biochemistry 28, 7453-7461
DeFranceschi, L., Fungalli, L., Olivieri, O., Carrocher, R., Lowell, C. A. & Berton, G. (1997). Deficiency of Src family kinases Fgr and Hch results in activation of erythrocyte K/Cl cotransport. Journal of Clinical Investigation 99, 220-227	[Abstract/Full Text]
Denisov, G., Wanaski, S., Luan, P., Glasner, M. & McLaughlin, S. (1998). Binding of basic peptides to membranes produces lateral domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol 4,5-bisphosphate: an electrostatic model and experimental results. Biophysical Journal 74, 731-744	[Abstract/Full Text]
Dunham, P. B., Klimczak, J. & Logue, P. J. (1993). Swelling-activation of K-Cl cotransport in LK sheep erythrocytes: a three-state process. Journal of General Physiology 101, 733-765	[Abstract]
Dunham, P. B. & Logue, P. J. (1986). Potassium-chloride cotransport in resealed human red cell ghosts. American Journal of Physiology 250, C573-578.
Dwyer, J. & Bloomfield, V. (1981). Binding of multivalent ligands to mobile receptors in membranes. Biopolymers 20, 2323-2336	[Medline]
Gabev, R., Kasianowicz, J., Abbott, T. & McLaughlin, S. (1989). Binding of neomycin to phosphatidylinositol 4,5-bisphosphate (PIP2). Biochimica et Biophysica Acta 979, 105-112	[Medline]
Guizouarn, H., Harvey, B. J., Borgese, F., Gabillat, N., Garcia-Romeu, F. & Motais, R. (1993). Volume-activated Cl--independent and Cl--dependent K+ pathways in trout red blood cells. The Journal of Physiology 462, 609-626	[Abstract]
Jennings, M. L. & Al-Rohil, N. (1990). Kinetics of activation and inactivation of swelling-stimulated K+/Cl-transport. The volume-sensitive parameter is the rate constant for inactivation. Journal of General Physiology 95, 1021-1040	[Abstract]
Jennings, M. L. & Schulz, R. K. (1991). Okadaic acid inhibition of KCl cotransport. Evidence that protein dephosphorylation is necessary for activation of transport by either cell swelling or N-ethylmaleimide. Journal of General Physiology 97, 799-817	[Abstract]
Krarup, T. & Dunham, P. B. (1996). Reconstitution of calyculin-inhibited K-Cl cotransport in dog erythrocyte ghosts by exogenous PP-I. American Journal of Physiology 270, C898-902	[Medline]
Kelley, S. J. & Dunham, P. B. (1996). Mechanism of swelling activation of K-Cl cotransport in inside-out vesicles of LK sheep erythrocyte membranes. American Journal of Physiology 270, C1122-1130	[Medline]
Lauf, P. K. (1988a). Volume and anion dependency of ouabain-resistant K-Rb fluxes in sheep red blood cells. American Journal of Physiology 255, C331-339	[Medline]
Lauf, P. K. (1988b). Thiol-dependent K:Cl transport in sheep red cells: VIII. Activation through metabolically and chemically reversible oxidation by diamide. Journal of Membrane Biology 101, 179-188	[Medline]
Lauf, P. K., Bauer, J., Adragna, N. C., Fujise, H., Zade-Oppen, A. M. M., Ryn, K. H. & Delpire, E. (1992). Erythrocyte K-Cl cotransport. Properties and regulation. American Journal of Physiology 263, C917-932	[Medline]
Leroy, D., Filhol, O., Delcros, J. G., Pares, S., Chambaz, E. M. & Cochet, C. (1997). Chemical features of the protein kinase CK2 polyamine binding site. Biochemistry 36, 1242-1250
Lysko, K. A., Carlson, R., Taverna, R., Snow, J. & Brandts, J. F. (1981). Protein involvement in structural transition of erythrocyte ghosts. Use of thermal gel analysis to detect protein aggregation. Biochemistry 20, 5570-5576
McKiernan, A. E., MacDonald, R. I., MacDonald, R. C. & Axelrod, D. (1997). Cytoskeletal binding kinetics at planar phospholipid membranes. Biophysical Journal 73, 1987-1998	[Abstract]
McLaughlin, S. (1977). Electrostatic potentials at membrane-solution interfaces. Current Topics in Membranes and Transport 9, 71-144.
McLaughlin, A., Eng, W.-K., Vaio, G., Wilson, T. & McLaughlin, S. (1983). Dimethonium, a divalent cation that exerts only a screening effect on the electrostatic potential adjacent to negatively charged phospholipid bilayer membranes. Journal of Membrane Biology 76, 183-193	[Medline]
Martinac, B., Adler, J. & Kung, C. (1990). Mechanosensitive ion channels in E. coli activated by amphipaths. Nature 348, 261-263	[Medline]
Minton, A. D., Colclasure, G. C. & Parker, J. C. (1992). Model for the role of macromolecular crowding in regulation of cellular volume. Proceedings of the National Academy of Sciences of the USA 89, 10504-10506	[Abstract]
O'Neill, W. C. (1989). Volume-sensitive, Cl-dependent K transport in resealed human erythrocyte ghosts. American Journal of Physiology 256, C81-88	[Medline]
Orlov, S. N., Kolosova, I. A., Cragoe, E. J., Gurlo, T. G., Mongin, A. A., Aksentsev, S. L. & Konev, S. V. (1993). Kinetics and peculiarities of thermal inactivation of volume-induced Na+/H+ exchange, Na+, K+, 2Cl- cotransport and K+, Cl- cotransport in rat erythrocytes. Biochimica et Biophysica Acta 1151, 186-192	[Medline]
Ortiz-Carranza, O., Adragna, N. C., Carnes, L. & Lauf, P. K. (1997). Two operational models of K-Cl cotransport in low K+ sheep red blood cells. Cellular Physiology and Biochemistry 7, 251-263.
Parker, J. C. (1993). In defense of cell volume? American Journal of Physiology 265, C1191-1200	[Medline]
Reynolds, J. A. (1979). Interaction of divalent antibody with cell surface antigens. Biochemistry 18, 261-269.
Sachs, J. R. (1988). Volume-sensitive K influx in human red cell ghosts. Journal of General Physiology 92, 685-711	[Abstract]
Sachs, J. R. (1994). Soluble polycations and cationic amphiphiles inhibit volume-sensitive K-Cl cotransport in human red cell ghosts. American Journal of Physiology 266, C997-1005	[Medline]
Sachs, J. R. (1996). Cell volume regulation. In Molecular Biology of Membrane Transport Disorders, ed. Schultz, S. G., Andreoli, T. E., Brown, A. M., Fambrough, D. M., Hoffman, J. F. & Welsh, M. J., pp. 379-406. Plenum Press, New York.
Sachs, J. R. (1998). How do red blood cells know how big they are? In Cell Volume Regulation, ed. Okada, Y., pp. 3-13. Elsevier, Amsterdam.
Sachs, J. R. & Martin, D. W. (1993). The role of ATP in swelling-stimulated K-Cl cotransport in human red cell ghosts. Phosphorylation-dephosphorylation events are not in the signal transduction pathway. Journal of General Physiology 102, 551-573	[Abstract]
Schacht, J. (1980). Inhibition by neomycin of phosphatidylinositide turnover in subcellular fractions of guinea pig cerebral cortex in vitro. Journal of Neurochemistry 27, 1119-1124.
Shyng, S.-L. & Nichols, C. G. (1998). Membrane phospholipid control of nucleotide sensitivity of KATP channels. Science 282, 1138-1144	[Abstract/Full Text]
Streichman, S., Hertz, E. & Tatarsky, I. (1988). Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association. Biochimica et Biophysica Acta 942, 333-340	[Medline]
Yoshino, H. & Minari, O. (1987). Heat-induced dissociation of human erythrocyte spectrin dimer into monomers. Biochimica et Biophysica Acta 905, 100-108	[Medline]

Acknowledgements

This work was supported by a grant from the USPHS DK19185.

Corresponding author

J. R. Sachs: Department of Medicine, State University of New York, Stony Brook, NY 11794-8151, USA.

Email: jsachs{at}mail.som.sunysb.edu

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