J. Phy8iol. (1976), 262, pp. 679-698 With 7 text-figures Printed in Great Britain

679

CHLORIDE TRANSPORT IN HUMAN ERYTHROCYTES AND GHOSTS: A QUANTITATIVE COMPARISON

BY J0RGEN FUNDER* AND JENS OTTO WIETHt From the tDepartment of Biophysics and the *Departmeent of Medical Physiology A, University of Copenhagen, 2100 Copenhagen, Denmark

(Received 26 April 1976) SUMMARY

1. Homogeneous preparations of resealed ghosts with intracellular KCl concentrations between 15 and 900 mm could be prepared. Virtually all ghosts sealed to chloride. The chloride transport system was found not to be damaged: a quantitative comparison of the self-exchange of 38C1across intact and resealed membranes showed that both the transport capacity and a number of characteristic properties were identical (saturation kinetics, temperature dependence and the effect of inhibitors). 2. Due to the absence of intracellular titratable buffers intracellular chloride concentration in ghosts vary only slightly between pH 5 and 11. The unidirectional exchange flux was constant between pH 7 and 11, showing that the transport system does not have a functionally important titratable group in the alkaline range, as previously assumed. The decrease of transport below pH 7 is similar in intact erythrocytes and ghosts. 3. Mean cellular volume of the resealed ghosts was a function of the amount of KCl added at 'reversal', before the ghosts are sealed. The ghosts shrank by osmosis when KCl was added to the suspension of 'unsealed' ghosts. The reflexion coefficient of sucrose (and therefore the osmotic effect) is larger than that of KCl. It was, therefore, possible to demonstrate that volume changes do not affect the chloride transport across the human red cell membrane. Unidirectional chloride fluxes at a KCl concentration of 165 mm were independent of ghost volume (10040 #m3). INTRODUCTION

Isolated cell membranes of erythrocytes (ghosts) provide a valuable tool in the characterization of membrane transport. Qualitative studies have proved that the resealed membranes of haemolysed erythrocytes retain a number of specialized transport systems, e.g. the active transport of Na and K (Gardos, 1954; Hoffman, 1962), the passive facilitated diffusion of

JORGEN FUNDER AND JENS OTTO WIETH 680 glucose (LeFevre, 1961) as well as the mechanism responsible for the exchange of anions across the membrane (Lepke & Passow, 1973; Schnell, Gerhardt, Lepke & Passow, 1973). So far no attempt has been made to make quantitative comparisons between the transport properties of erythrocytes and their resealed ghosts. A number of technical innovations of the ghost preparation procedure (Bodemann & Passow, 1972; Schwoch & Passow, 1973) have now removed those obstacles for quantitative transport studies, which were mainly due to the considerable inhomogeneity of ghost populations prepared by earlier techniques (Hoffman, 1958). In this article we present a quantitative comparison of chloride transport in human erythrocytes and their resealed ghosts. It has been suggested that anion transport in red cells is crucially dependent on the integrity of a membrane protein (mol. wt. 95,000) (Rothstein, Cabantchik & Knauf, 1976). Further studies have also suggested that lysis and resealing of red blood cells do not lead to detectable changes of the reaction of this membrane protein with surface labels or proteolytic enzymes (Cabantchik, Balshin, Breuer, Markus & Rothstein, 1975). Our study in addition shows that the capacity and the functional properties of the chloride transport system are not changed by the lysis and resealing of human red cell membranes. METHODS

All experiments were carried out by determining the rate of 36Cl-efflux from human erythrocytes or from resealed ghosts.

Preparation of resealed ghosts The technique for preparing a uniform population of resealed ghosts was based on the directions given by Schwoch & Passow (1973, Table 4). Freshly drawn heparinized blood was washed thrice in a 165 mM-KCI solution. The red cells were resuspended at a haemlatocrit of 40 00. and cooled to 0° C. Lysis and preparation of ghosts for resealing was carried out at 00 C ( 0. 1) according to the following scheme. I. Lysis. The volumes employed for the treatment of 1 ml. cell suspension were multiplied according to the amount of ghosts needed. One ml. of the erytlirocyte suspension was lysed in 10 ml. of the haemolysing solution: 3 8 rnM acetic acid, 4 mM MgSO4. After lysis pH was 5 8-6 0. Five minutes later 1 ml. of a buffered salt solution containing 2 M-KCl and 25 mM Trizma base (Sigma) was added (reversal solution), changing the pH of the haemolysate to 7-1-7-3. In order to prepare ghosts with a varied chloride concentration (Fig. 3) the volume, KCl and Tris concentrations of the salt solution were varied in these experiments to obtain the desired KCI concentrations in the final haemolysate. After addition of reversal solution, the haemolysate was left at 00 C for 10 min. The ghosts are still unsealed at this stage, but nevertheless, as demonstrated in the Results section, they shrink acutely by osmosis when salt is added. The haemoglobin accordingly becomes concentrated in the ghost but equilibrates again with the external solution during the 10 min at 00 C as illustrated by the experiment shown in Fig. 5. II. Resealing. The lysate was now transferred to a 380 C bath. Sealing is not an all-or-none response: the ghosts reseal rapidly to 13IJ-labelled albumin when the

Cl- TRANSPORT IN HUMAN ERYTHROCYTES

681

temperature is increased from 0 to 380 C. Labelled albumin added to the lysate in the last minute of the incubation at 00 C was found to have reached 25 % equilibration with the intracellular phase in the resealed ghosts. In contrast equilibration was 90-100 % accomplished when the labelled albumin was added 10 min earlier together with the concentrated salt solution. When the albumin was added immediately after the transfer to 380 C the ghosts were almost impermeable to albumin (the intracellular concentration reached only 3 % of the concentration in the haemolysate). A longer heating period is needed to seal the membrane to smaller molecules and ions. As described below virtually all ghosts seal to chloride ions after incubation for 45 min at 380 C. It was, therefore, not necessary to remove leaky ghosts by- the sucrose cushion technique described by Schwoch & Passow (1973). TABLE 1. Composition of resealed ghosts. The analyses were performed on ghost specimens prepared at pH 7-2 with a KCl concentration of 165 mm and packed by centrifugation at 50,000g. All results were corrected for the amount of extracellular medium trapped between the packed ghosts, determined with 14C-labelled inulin. Cli- and Cl7- are the chloride concentrations in the intra- and extracellular water phases Trapping of inulin MCV between Haemoglobin Chloride dismean packed (gfl. ghost Water tribution ratio volume ghosts No. % (wjw) water) (g/kg ghosts) Cli- /Cl('sm3) 1 9-6 968 1-003 100 8.1 2 10*7 969 1.018 89 4 10.9 3 15-3 963 1-000 86-9 6-0 4 13-1 963 0-991 86-8 6-7 5 972 8-0 0-986 101-7 6-8 6 79 975 1P017 110-2 7-8 7 9-8 962 0-983 61 90-2 8 8-2 971 0-986 87-5 13-1 Mean 10-1 969 0-998 94.1 8-2 + S.D. + 2-7 +5 +0-014 + 8-8 + 2-5

III. Labelling and packing of resealed ghosts for experiments. The pink resealed ghosts were isolated from the haemolysate by centrifugation (20,000 9), and resuspended and washed repeatedly in the electrolyte medium (pH 7-2, 00 C) used for the efflux experiment (see below). The osmolality of the electrolyte medium was always within 10% of that of the haemolysate to minimize volume changes in the ghosts. Ghosts with a pH different from 7-2 (Fig. 6) were titrated with 0-1 M solutions of KOH or HCl dissolved in 0-165 M-KCl. After titration these ghosts were washed twice with electrolyte medium of the desired pH. After washing the ghosts were resuspended at a haematocrit of 30-40 %, and 36CI was added (0.2-0.6 ,c/ml. suspension). Equilibration of radioactive chloride between medium and ghosts is completed in a few seconds at room temperature and was always checked by measuring the distribution of radioactive chloride between ghosts and medium. A sample of the suspension was saved for ghost counting (Coulter Counter Model DN) and the remaining portion was centrifuged for 20 min at 50,000g in nylon tubes (i.d. 3 mm). The cytocrit of the packed ghosts was measured and the ghosts and

682

JORGEN FUNDER AND JENS OTTO WIETH

supernatant isolated by cutting the tube just below the interface. Mean volume of the ghosts was calculated from the counts and the cytocrit after correcting the latter for the amount of extracellular medium trapped between the packed ghosts. The trapped extracellular volume was determined in separate samples by means of 3Hlabelled inulin (0 1 uc/ml. suspension). Trapping is more pronounced between ghosts than between intact erythrocytes: the mean value of eight samples (the ghosts and cells prepared for the experiments shown in Table 2) was: ghosts 8-2 % (range 6-0131 %), red cells 1-6% (range 1-4-1-8%). The typical composition of the resealed ghosts is shown in Table 1.

Experimental procedure The rate of tracer efflux was determined at 0° C by injecting packed ghosts or erythrocytes into a stirred isotope-free electrolyte medium buffered with 2 mm Tris and containing KCl as specified in text and figures. The media with pH values below 7-2 (Fig. 6) in addition contained 0-5 mM-KH2PO4. The efflux suspension contained 0-5-- % labelled cells or ghosts, and samples of cell-free medium were serially isolated for analysis using the filtration technique described in detail by Dalmark & With (1972), who also describe the analytical methods employed.

Calculations The approach of the isotope concentration towards equilibrium as a function of time was followed by determining the activity of the cell-free filtrates. The following equation describes the time-dependence of the concentration of -"Cl- in the medium: at = (a,- a) (I1-e-kt) + ao, where t is time of sampling (min), k is the rate coefficient of chloride exchange (min-), ao (cpm/ml.) is the concentration of 3Cl- in the medium at time = zero (caused by the presence of trapped extracellular medium between the packed cells or ghosts), at is the concentration at the time of sampling, and a., is the equilibrium concentration of the isotope. The results showed that the exchange kinetics was well described by this closed two-compartment model with well mixed compartments of constant volume. The rate coefficient (k) is dependent on the compartment sizes, except when the extracellular compartment is very large compared to the intracellular phase, as was the case in the present experiments. The rate coefficient of chloride exchange is equal to the numerical value of the slope of the graph relating In (1 - at/a.) to time. The value was found by linear

regression analysis of duplicate runs (viz. Fig. 2). The intercept of the graph with the ordinate equals ln (1 -ao/a,,) and the difference of the intercept from unity expresses, therefore, the fraction of isotope located in the extracellular phase at the beginning of the experiment. To be comparable with our previous calculations of chloride transport in human red cells (expressed in in-mole/kg cell solids x min) we have presented the unidirectional chloride fluxes in ghosts in m-mole/3- 1 x 10'3 cells x min, that number of normal cells containing one kilogram of solids (determined by drying cells to constant weight). The chloride flux per unit membrane area (J4) is given by the equation:

JA

=

k(V/A) Cli (mole.cm-2.min-'),

where k (muin-) is the rate coefficient of chloride exchange, V(cm3) is the solvent volume in cells or ghosts, A is the volume independent surface area (assumed to be 1-42 x 106 cm2) and Cl, (mole/cm3 intracellular water) is the intracellular chloride concentration. The apparent permeability (cm/sec), which is found to vary with

Cl- TRANSPORT IN HUMAN ERYTHROCYTES

683

chloride concentration, is defined as the product k(VIA). The flux per 3 1 x 1013 cells or ghosts (J) was found by multiplying JA with the factor 4-4 x 107 cm2 per 3 1 x 1013 cells. We have previously based our calculations of surface area on Ponders value of 163 /sm2 (Ponder, 1948). Realizing that this value is too high (Westerman, Pierce & Jensen, 1961; LaCelle, 1972) we have based our calculations on a surface area of 142 /sm2 and a normal red cell volume of 87 /zm3. This change of values does not affect fluxes calculated in m-mole/kg cell solids x min (Dalmark & Wieth, 1972; Gunn, Dalmark, Tosteson & Wieth, 1973) although the change of values alters the calculated area from 4-9 x 107 to the new value of 4-4 x 107 cm2/kg cell solids. Homogeneity of the ghost preparation The fact that the 36C1- efflux from resealed ghosts was well described by a single exponential shows that the ghosts behave as a reasonably homogeneous population with

10 08

-6 0_6 0

04 0.2

0

0

20 40 60 80 Volume (arbitrary units)

100

Fig. 1. Volume distribution of human erythrocytes (x) and their resealed ghosts (0) as determined by gradually increasing the discrimination level of the Coulter Counter (Model DN). The ghosts were resealed at a KCl concentration of 165 mm, and the graph demonstrates that the size distributions of erythrocytes and their ghosts were identical. The mean cellular volumes (determined from the total number of cells or ghosts, the cytocrit, and corrected for the amount of inulin trapped extracellularly in the sample of packed cells or ghosts) were: erythrocytes 89-6 /m3, ghosts 87-3 /Sm3. The ordinate of the graph is the fraction of red cells or ghosts (N) as a fraction of the total number (Ntotl). The abscissa shows the setting of the discriminator (divisions in arbitrary units). 1 division 2-1 /Zm3. regardto chloride transport, as is the case with intact erythrocytes (Dalmark & Wieth, 1972). Our results demonstrate that practically all cells seal to chloride: the fraction of 36Ch- which is extracellularly located in the sample of packed ghosts can be calculated from the value of the intercept = ln (1 - ao/aOO) (cf. Fig. 2). By analysis of the results obtained with ghosts resealed to an extra- and intracellular KCl concentration of

F~ I

JORGEN FUNDER AND JENS OTTO WIETH

684

165 mm we found that the value of ao/a, varied within the range 0-06-0 12 corresponding to the trapped inulin space found between the packed ghosts (6-13%). The chloride concentrations in ghosts and medium are identical, so this finding implies that all ghosts that seal to inulin (mol. wt. 5000) are also sealed to 36C0-. The size distribution of ghosts measured by the Coulter Counter was found to be the same as that found in the original blood sample. Fig. 1 shows a volume distribution curve of erythrocytes and ghosts (165 mM-KCl). The two populations had the same mean cellular volume, and the graph shows that the distribution curves obtained by successively increasing the lower threshold of the Coulter Counter are almost identical in cells and ghosts.

10 0-5-

R.B.C.

30 45 60 Time (sec) Fig. 2. The rate of M6l efflux from erythrocytes and ghosts at pH 7x2 (00 C). The ordinate has a logarithmic scale; a is the concentration of MCl- in the medium at the time of sampling and a. is the concentration after isotopic equilibrium has been attained. The rate of tracer efflux from the red cells (k = 3*6 min-) was twice the rate of OCl- efflux from the ghosts (k = 1-7 min-'). However, as explained in the text the rates of tracer movement do not alone provide a measure of relative permeabilities. The fluxes were almost the same in the two sets of experiments shown in the Figure. Ghosts 855 and erythrocytes 924 m-mole/(3.1 x 10" cells x min). 0

15

RESULTS

Rate of "Cl-efflux from erythrocytes and ghosts Fig. 2 shows the rate of MCl-efflux from erythrocytes and ghosts under conditions where intra- and extracellular chloride concentrations were identical and constant. The rate of chloride exchange in the red cells is almost twice as rapid as in the ghosts. However, the rate coefficient alone does not provide a measure of the permeability as explained in the Methods section. The fluxes calculated from the experiments presented in the Figure were in fact almost the same (see below and legend of Fig. 2).

Cl- TRANSPORT IN HUMAN ERYTHROCYTES

685

Comparison of unidirectional chloride fluxes in erythrocytes and ghosts Red cell chloride content varies with pH and temperature. At 0° C and pH 7*20 the ratio between intra- and extracellular chloride concentration is close to unity. It was, therefore, possible to make a quantitative comparison of chloride transport in erythrocytes and ghosts at that pH. Table 2 shows the results obtained with cells from eight normal persons. TABLE 2. Comparison of the unidirectional self-exchange flux in human red cells and their resealed ghosts. All experiments were carried out at 0° C, pH 7-2 in a medium containing 165 mM-KC1, Tris 2 mm. The chloride distribution ratios (C1I/C1O) were: erythrocytes 0 90 (± 004), ghosts 0-998 (+ 0-014). Cl1 and 1CIO are respectively the chloride concentrations in the intra- and extracellular water phase

Chloride selfexchange flux, m-mole/(3*1 x 1013 cells) x min

Rate coefficient of 36CI exchange (min-)

Erythro- Red cell ErythroNo. 1 2 3 4 5 6 7 8 Mean + S.D.

cytes 711 784 754 795 917 924 956 817 832 +89

ghosts 717 760 910 875 855 855 918 713 826 +83

MCV mean volume

cytes 2-39 2-78 2-64 2-88 3-24 3-64 3-42 3-18 3-02

±0-42

Red cell Erythro- Red cell ghosts ghosts cytes 85-8 1-61 100 89-4 1-72 89-8 1-94 86-9 91*3 86-8 86-4 1-87 101-7 1-74 108-7 110-2 1-68 90.1 90-2 95-0 2-09 87-5 1-62 87-7 94-1 91-9 1-78 + 7-4 + 8-8 +0-17

The average rate coefficient found in ghosts was 60 % of that found in red cells, but the mean values of the unidirectional chloride fluxes in cells and ghosts were identical, suggesting that the resealed ghosts regain the capacity for chloride transport quantitatively. The following sections demonstrate that not only the capacity, but also the functional properties of the chloride transport system are similar in ghosts and red cells.

Concentration dependence of chloride transport Chloride exchange in human red cells shows saturation kinetics (Gunn et al. 1973; Dalmark, 1976). Fig. 3 demonstrates that this is also the case in ghosts: the shape of the graph is the same as that found by Cass & Dalmark (1973, Fig. 3) who loaded intact cells (pH 7.20) with KCl. The chloride transport reaches

a

maximum at a concentration of 100-150 mM,

686 J0RGEN FUNDER AND JENS OTTO WIETH ' self-depression' of transport is apparent at higher concentrations, and the transport is half maximal at a chloride concentration of 20 mM. During our studies of the dependence of transport on chloride concentration we observed that the volume of the resealed ghosts were dependent on the magnitude of the concentration increase induced by adding a concentrated KCI solution to the 'open' ghosts before the subsequent resealing at 380 C (cf. Methods section). Therefore, it was necessary to determine the mean cellular volume of each ghost preparation to obtain the results shown in Fig. 3.

,1 X~~

900

700

-

-c '4

0

U

IV300 E 100 l

0

100

l

300

500 KCI (mM)

700

900

Fig. 3. Dependence of chloride self-exchange flux of resealed ghosts on intra- and extracellular chloride concentrations (pH 7-2, 0° C). Halfmaximal flux was reached at a chloride concentration of 20 mm and the flux was depressed at chloride concentrations above 200 mm, similar to results obtained with KCl loaded intact red cells by Cass & Dalmark (1973).

Ghost volume as a function of KCl added before resealing We have investigated the course of events leading to the volume changes and report the results because the phenomenon is of importance for work involving quantitative determination of transport parameters in resealed ghosts. Fig. 4 shows the dependence of ghost volume on KCl concentration in the range between 15 and 900 mM-KCl. Further investigation showed that this variation of cell volume is due to an osmotic effect of KCl on the unsealed ghost membrane: although the unsealed membranes are still permeable to haemoglobin at the moment when the

687 Cl- TRANSPORT IN HUMAN ERYTHROCYTES salt concentration of the haemolysate is increased by adding KCl, the reflexion coefficient of the membrane towards KC1 is still significantly different from zero. Therefore, the sudden increase of extracellular KC1 concentration causes an osmotic flow of water out of the intracellular compartment until KCl has reached diffusion equilibrium. The Figure also illustrates that ghosts prepared without the addition of KCl (by sealing the

160

-

Lytic volume

" 120 E

o>

80

-

0

I.. 40

100

300

500

700

900

KCI (mm)

Fig. 4. Mean cellular volume of resealed ghosts as a function of their final KCl concentration. Ghosts which were resealed in the haemolysate without the addition of reversal solution had a volume equal to the haemolytic volume of human erythrocytes (- 140 flm3). The cellular volumes of the ghost preparations decreased, when the amount of KCl added with the reversal solution was increased. Further explanation is found in the text and in Fig. 5. The results of Tables 3 and 4 show that chloride transport was not affected when volume Variations took place at a fixed KCl concentration.

ghosts at the 'native' KCl concentration of the haemolysate 15 mM) have a volume of 140 ,tm3 which is the critical haemolytic volume of the red cell. When preparation includes the addition of salt the ghosts shrink by osmosis and the final volumes of the ghosts vary as illustrated in the Figure. The unsealed ghosts behave by no means as perfect osmometers to KCl. By applying van't Hoffs law to the results of Fig. 3 the reflexion coefficient to KCl may be estimated to be 0 3. Nevertheless, the 'holes' in the unsealed membrane must be very narrow, since KCl diffusion is restricted. This notion was confirmed by the finding that haemoglobin is concentrated

688 JORGEN FUNDER AND JENS OTTO WIETH in the ghosts as a consequence of the shrinking, and that a subsequent release of haemoglobin from the unsealed ghosts proceeds over a period of several minutes. Fig. 5 shows this gradual equilibration of haemoglobin between ghosts and haemolysate during 10 min incubation at 00 C following the addition of KCl (increasing the chloride concentration of the haemolysate from 15 to 175 mM). Preliminary investigations of the membrane leakiness to proteins had shown that the ghosts seal very slowly to [1311]albumin after 6.0

pH

Lysis

-2 Reversal

20 K

to -o

I

=15

t

\

_

0

Ir

I be

10_

0

~~~~~im

0

(

in

5

10

15

Time (min)

Fig. 5. Volume variation in ghosts following the addition of reversal solution as reflected by the intracellular haemoglobin concentration of the resealed ghosts. The six ghost samples were prepared as described in the Methods section with the only exception, that the incubation period at 00 C after reversal but before resealing at 380 C was varied between 5 sec and 10 min. The Figure shows the haemoglobin concentration in the haemolysate (continuous line) and in six samples of resealed ghosts (O - -- 0) prepared by transferring samples of the haemolysate to 380 C at the following times after addition of reversal solution: (1) 5 sec, (2) 25 sec, (3) 1 min, (4) 3 min, (5) 5 min, and (6) 10 min. The trapping of inulin between the packed ghosts was between 4-6 and 744%. MCV of the ghost samples increased slightly from sample 1 (80 /%m3) to sample 6 (90 /%m3). The chloride concentration of ghosts and haemolysate was 175 mm after reversal.

the addition of KCl, if the haemolysate is kept at 0° C, as in the procedure of Schwoch & Passow (1973). In contrast the ghosts seal rapidly to albumin (in the matter of seconds), if the haemolysate is heated to 380 C. This rapid sealing of the membrane to proteins made it possible to determine the intracellular haemoglobin concentration as a function of time (Fig. 5).

Cl- TRANSPORT IN HUMAN ER YTHROC YTES 689 Samples were successively transferred from 0 to 380 C, during a period of 10 min after the addition of KCl 5 min after lysis (Fig. 5). Haemoglobin concentration, mean cellular volume, and water content were determined in each of the six ghost preparations. The graph shows that on addition of KCl the haemoglobin concentration of the ghosts rose instantaneously from 11 g/l. (the concentration of the haemolysate) to 19 g/l. We thus found perfect agreement between the increase in haemoglobin concentration and the decrease of ghost volume from the critical volume (- 140 ,/m3) to a new volume of 80 pm3. During the subsequent period the haemoglobin concentration of the ghosts fell, and reached diffusion equilibrium after about 5 min. The moderate 're-swelling' of the ghosts in this period (from 80 to 90 jtm3) is only responsible for a small fraction of the decrease in haemoglobin concentration. TABLE 3. Independence of chloride self-exchange flux on ghost volume (00 C, pH 7 2). The volume of resealed ghosts was varied by addition of sucrose together with KCl in the reversal solution. The self-exchange flux was found to be constant in spite of the large volume variations shown Sucrose concentration in haemolysate and Chloride selfghosts after exchange flux, Cl, intracellular addition of MCV chloride Rate coefficient m-mole/(3-1 x reversal solution mean volume concentration of 36C1 exchange, 1013 ghosts x (mM) k (min-) (mM) min) (#m3) 0 94 165 1-283 610 53 100 165 2-174 576 41 200 165 3-013 604 TABLE 4. Independence of chloride self-exchange flux on ghost volume (O° C, pH 7.2). The volume of the resealed ghosts decreased when reversal solution was added at temperatures higher than 00 C. As in the experiments shown in Table 3, the chloride exchange flux was found to be independent of volume variations Chloride selfCli, intracellular Temperature of exchange flux, chloride Rate coefficient m-mole/(3-1 x haemolysate at MCV, mean volume concentration of 36Cl-exchange, 1013 ghosts x addition of reversal solution k (m -1) (mM) min) (#m3) 0 102 165 1-374 717 89 5 165 1-559 706 10 78 165 1-853 735

The osmotic properties of the unsealed ghost are further illustrated by the results shown in Table 3. In this experiment sucrose was added to the concentrated KCl solution to obtain final sucrose concentrations of 100 and 200 mm in the haemolysate. The addition of sucrose had a pronounced

JORGEN FUNDER AND JENS OTTO WIETH 690 effect on ghost volume (reducing it from 94 to 41 um3 when sucrose concentrations was varied from 0 to 200 mM). The reflexion coefficient to sucrose must, therefore, be higher than that of KCl. The osmotic properties of the unsealed membrane also depended on the temperature at the time, when the reversal solution was added. Table 4 shows the results of three experiments where the temperature of the haemolysate was varied between 0 and 100 C during reversal. It is seen that the osmotic effect of the same KCl concentration on final ghost volume increased with increasing temperature. Chloride transport was determined in the experiments reported in Tables 3 and 4. The results showed that the unidirectional flux was independent of cellular volume, and the rate coefficient of chloride efflux was, therefore, inversely proportional to cell volume. This finding is important because it shows that changes of cell volume in itself has no effect on the chloride transport capacity, meaning that the saturation kinetics (Fig. 3) is not caused by the volume variations of the ghosts (Fig. 4). The pH dependence of chloride transport The intracellular chloride concentration and the membrane potential of erythrocytes change with pH. These variations are not found in a study of the effect of pH on chloride transport in red cell ghosts. Our ghost preparation contained about 10 g haemoglobin per litre ghosts and the ratio between chloride in the intra- and extracellular water phases (i/0Ce) was found only to decrease from 1-05 to 0 97 when pH was increased from 5to 11. Fig. 6 shows the chloride self-exchange flux as a function of pH. Below pH 7 the flux decreased steeply with pH, and at pH 5-25 the flux was reduced by 90 %. This is similar to previous findings on intact erythrocytes (Gunn et al. 1973). A conspicuous difference from the results obtained with intact red cells is observed in the alkaline range: there was no decrease of chloride transport in ghosts, when pH was increased from 7 to 11, there the self-exchange flux of intact erythrocytes decreases steeply. This finding excludes the existence at 00 C of a functionally important titratable group in the alkaline pH range, as has previously been suggested (Gunn, 1972; Gunn et al. 1973).

Temperature dependence of chloride transport Fig. 7 is an Arrhenius diagram showing the relation between the natural logarithm of the rate coefficient of chloride exchange vs. the reciprocal absolute temperature (corresponding to the temperature interval 0-10° C). The Arrhenius activation energy, calculated from the slope of the graph,

Cl- TRANSPORT IN HUMAN ERYTHROCYTES

691

was 31 kcal/mole in agreement with the value found in intact cells (Dalmark & Wieth, 1972). These results were obtained at pH 7-2-7-4. Temperature activation of chloride transport in red cells has been found to decrease below pH 6 (Gunn, Wieth & Tosteson, 1975). This was also the case in our studies on ghosts. Table 5 shows that the Q10 (0-10 C) of chloride transport in ghosts decreased from 7 to 3-8, when pH was lowered from 7-2 to 5*3. C

800

0

-

E0

0

0

x

0

6X'W200I 5

I 6

7

8 pH

9

10

11

Fig. 6. The pH dependence of chloride exchange flux in human ghosts at 00 C. T'he continuous line describes the relation: J = 830/(1 + K~P) m-molel(3*1 x 1013 cells x mmn).

K - 10-62 is the dissociation constant of a single He-binding group in the transport system and it is assumed that chloride transport is inhibited noncompetitively when the group is titrated with a single H+ (cf. Discussion section).

The effect of inhibitors on chloride transport in ghosts We have tested the effect of a potent non-specific inhibitor of chloride transport (phloretin) and of the most specific inhibitor known, the amino reagent DIDS (4,4-diisothiocyano-2,2-stilbene disulphonic acid, Cabantchik & Rothstein, 1974). Phloretin inhibits a number of facilitated transport processes in red cells (Wieth, Funder, Gunn & Brahm, 1974), whereas DIDS has so far been found to inhibit only the chloride transport system 24

PHY

262

JORGEN FUNDER AND JENS OTTO WIETH

692

6

5a 0

x £

\

RB\

4S Ghosts

3 mra a II

I

3.5

I

I

3*7

3-6 I

x103

Fig. 7. Arrhenius diagram of the relation between the natural logarithm of the rate coefficient of chloride exchange in ghosts (k sec') and the reciprocal absolute temperature (0-10 C). The continuous line shows the results obtained with ghosts, and the interrupted line shows the relation found in intact red cells by Dalmark- & Wieth (1972). The activation energy calculated from the slope of the relation was 30 7 + 1-5 kcal/mole in ghosts to be compared with 33-2 ± 1-2 kcal/mole in the red cells. TABLE 5. Decreasing temperature activation of chloride exchange at low pH values. The experiments were performed with ghosts and medium containing 165 mM-KCl at 0 and 10° C (pH 7.2). Duplicate experiments were made at the two temperatures with resealed ghosts from the same donor. Q10 is the ratio between the rates of exchange at 10 and 0° C. The activation energy presented is the one which corresponds to the QLO in the interval 0 to 100 C

Rate coefficient of 36Cl-exchange, k (min-')

pH

000C

7-2 6-9 6-5 6-0

1-77

5.3

2-00 1-85 1*01 0 18

10C 12*4 11-5 10-8 4-99 0-73

Arrhenius activation energy

Q1o

(keal/mole)

70 5X8 5-8 4.9 3-8

29-9 26-8 27-0 24-5 21*7

693 Cl- TRANSPORT IN HUMAN ERYTHROCYTES (Rothstein et al. 1976). The results shown in Table 6 demonstrate that both agents under favourable conditions inhibit the chloride exchange of resealed ghosts more than 99 %. TABLE 6. The effect of phloretin and of DIDS (4,4-diisothiocyano-2,2-stilbene disulphonic acid) as inhibitors of the chloride self-exchange flux in resealed ghosts (00 C, pH 7-2, KCl concentration of ghosts and medium 165 mM). The ghosts were equilibrated with 0-25 mn phloretin before the experiment was performed in a medium containing 0-25 m phloretin. DIDS is an irreversible inhibitor of chloride transport (Cabantchik & Rothstein, 1974). The ghosts were treated with DIDS for 20 min at 380 C by adding 250 jel. 1 mm-DIDS solution to 5 ml. of a ghost suspension with a cytocrit of 30 %. After the incubation the ghosts were isolated by centrifugation, and 3Clexchange was determined in a DIDS-free medium Rate coefficient of 3Cl-exchange, k (min-')

Control Phloretin 0-25mM DIDS-treated

Chloride self-exchange flux, m-mole/(3- 1 x 1013 Flux as % of control ghosts x min)

1-60 (± 0-08 S.D., n = 6) 0-0049

801 ( ± 24 S.D., n = 6) 2-1

1.9

0-0042

100 0-26

0-24

ghosts DISCUSSION

It is the conclusion of this study that it is possible to prepare a homogeneous population of ghosts, which all seal to chloride and possess a quantitatively intact chloride transport system. Chloride transport is dependent on the integrity of an integral membrane protein with a molecular weight of 95,000 (Cabantchik & Rothstein, 1974; Zaki, Fasold, Schuhmann & Passow, 1975). Quantitative studies of chloride transport may, therefore, contribute to characterize the functional state of the 'anion transport protein' in isolated membrane preparations. Although no definite conclusions were drawn with regard to this protein, Staros, Haley & Richards (1974) concluded from protein labelling studies that several membrane proteins re-orient, when cells are lysed and their membranes resealed. Cabantchik et al. (1975) found no detectable changes in the protein labelling of the 95,000 mol. wt. protein in cells and ghosts, but the functional property of the ghosts is presumably an even more sensitive sensor of the state of the membrane protein. Our ghost preparation technique was based on the important innovations following the discovery of the critical role of pH and temperature during lysis, reversal, and resealing as described by Schwoch & Passow (1973). It is likely that the ghost preparation technique is critical for the integrity of membrane proteins and their transport function. We found a quantitative agreement between the chloride transport 24-2

J0RGEN F UNDER AND JENS OTTO WIETH function in red cells and ghosts under conditions where intra- and extracellular chloride concentrations in the red cells were equal. This statement applies to the transport capacity (Table 2), the saturation kinetics (Fig. 3), the temperature dependence (Fig. 7), as well as to the response to inhibitors (Table 6). It must be noted that the quantitative recovery of chloride exchange through the resealed ghosts cannot be extrapolated to other membrane functions. Indirect evidence shows that chloride conductance in human red cells is very low. Electrophysiological examinations of Amphiuma erythrocytes have confirmed that the chloride exchange mechanism has an apparent permeability, which is 4-5 orders of magnitude larger than the chloride ion conductance (Lassen, Pape & VestergaardBogind, 1975), and even a considerable increase of the latter would be undetectable in a study of the magnitude of chloride self-exchange. The only difference between chloride transport in red cells and ghosts was found in the study of the pH dependence of chloride self-exchange. In contrast to the independence of chloride transport on pH in the alkaline range found in ghosts (Fig. 6) it has been demonstrated repeatedly that the self-exchange flux at a physiological ionic strength of 0. 15 at 00 C in erythrocytes has a broad pH maximum between pH 7 and 8, decreasing steeply both below and above this pH interval (Gunn et al. 1973; Dalmark, 1975). The decrease at low pH is similar in red cells and ghosts. It may be ascribed to a non-competitive effect of hydrogen ions on a group in the transport system with a pK near 6 in general agreement with the results of Dalmark (1975), although it was not necessary to assume that more than a single H+ reacts with the group (viz. legend of Fig. 6). We do not think that the difference found between chloride transport at alkaline pH values is due to different properties of the transport system in red cells and ghosts. We rather believe that it reflects the different effects of pH on intracellular chloride concentration and membrane potential in cells and ghosts respectively. The reduction of the chloride flux at alkaline pH values in red cells disappears, when transport is studied in erythrocytes whose KCl content has been drastically increased during a reversible nystatin induced permeability increase to KCl (Dalmark, 1975). The important similarity between the KCl-enriched cells and the ghosts studied here is that the effect of pH on intracellular chloride concentration and on membrane potential has been efficiently weakened in both preparations: in the cells by increasing the chloride contents so that the chloride shift following titration of intracellular buffers only causes minor relative concentration changes at levels where the transport system is already saturated. In the ghosts the removal of 97 % of the intracellular buffers creates a situation where intracellular pH can be decreased from 11 to 5 with only a minor increase of the intracellular chloride content. 694

695 Cl- TRANSPORT IN HUMAN ERYTHROCYTES It was our hope that an analysis of the effect of pH on chloride exchange in red cells and ghosts would make it possible to decide whether change of intracellular chloride concentration, increase of membrane potential or both these factors were responsible for the decrease of exchange at alkaline pH values. The results of Fig. 6 show that chloride permeability is constant between pH 7 and 11. In red cells where intracellular chloride decreases and the negative membrane potential increases with increasing pH the exchange flux of chloride is reduced by a factor of two to four at pH 9 5. If the anion concentration of one side of the membrane was the limiting factor for the reduced flux (a possibility mentioned by Passow & Wood, 1974) one would from the results of Fig. 3 only expect a reduction of the exchange of flux by 33 % at an intracellular chloride concentration of 33 mm, the intracellular chloride concentration at pH 9-5 calculated from the results of Gunn et al. (1973). If on the other hand chloride ions are subjected to the force of an electrical field during their transport through the membrane it can be calculated (with the assumption of constant field (Katz, 1966)), that the unidirectional flux at constant chloride permeability will be reduced by a factor of two, when Vm changes from 0 mV at pH 7 to -35 mV at pH 9X5 (C1I/Clo being 0-22 according to Gunn et al. 1973). A third possibility is that chloride ions are transported as electroneutral ion-pairs with a mobile positively charged site (Wieth, 1972). The calculations of Dalmark (1975) show that such a liquid ion-exchange model qualitatively predicts the pH dependence of chloride exchange both in KCl-depleted, normal, and in KCl-enriched cells. According to this model the reduction of chloride flux is due both to the distribution of chloride and of the unpaired sites (S+) which are assumed to penetrate the membrane extremely slowly, but under steady-state conditions being distributed according to their equilibrium potential (Cli-/Cl- = S.+/Si+). The precision of flux determination at the most alkaline values at which the cells shrink is low, because the rate of exchange approaches the sensitivity limit of the filtration method. None of the three transport models: reaction with a fixed site, electrodiffusion of Cl- (alone or combined with an electrically neutral site), or ion-pairing with a positively charged mobile site, can, therefore, be rejected on the basis of the present results. Gunn (1973) has suggested that the decrease of flux at alkaline pH values is due to the deprotonation of a positively charged (fixed or mobile) membrane site with a pK of 8-8. Our results clearly show that there is no reason to assume that the transport system possesses a functionally important group with a pK below about 11 at 00 C. Recent experiments on ghosts have shown that the chloride exchange flux at 380 C decreases somewhat at pH 9 (Brahm, 1976). If this is due to a deprotonation of the type suggested by Gunn (1972, 1973) it means that the pK of the group decreases at least

696 J0RGEN FUNDER AND JENS OTTO WIETH 2 pH units by a temperature increase of 380 C. This would suggest a group with a very high ionization enthalpy as, for example, the guanidino group of an arginine residue or a quaternary ammonium group. There is much evidence that a 95,000 mol. wt. protein is involved in chloride transport. But as recently discussed thoroughly by Lepke, Fasold, Pring & Passow (1976) it has not been definitely proved that the protein is the transport system itself. A chloride exchange mechanism resembling that of the red cells has been found in protein-free phospholipid vesicles (Pagano & Thompson, 1968; Toyoshima & Thompson, 1975a, b). If our attention is falsely diverted from the lipids it is worth remembering that the choline group of phospholipids has a pK high enough to meet the requirements made by the results of Fig. 6. Chloride exchange in red cells closely follows the original description of a tightly coupled exchange diffusion mechanism: 'the mechanism will always take up one ion when it gives off another so that no net change in the concentrations on either side of the membrane take place' (Ussing, 1948). The exchange diffusion of chloride in phospholipid vesicles has been ascribed to ion-pairing of chloride with choline groups (Toyoshima & Thompson, 1975a, b), which can mediate an exchange diffusion by the flip-flop movements of lipids across the membrane described by Kornberg & McConnell (1971). The quantitative study of Toyoshima & Thompson (1975b) provides data for a direct comparison with our red cell results. The exchange flux in purified egg phosphatidylcholine vesicles at 6. 3 C, pH 8 and a KCI concentration of 0 1 M was 8 x 10-16 mole/cm2 sec (not 0-8 x 10-16 as misprinted in the article, Toyoshima & Thompson, 1975b). The corresponding flux in the ghosts at 6.30 C is 1-1 x 10-9 mole/cm2 x sec (calculated from a flux of 3 mole per 3x1 x 1013 cells per minute). The red cell chloride exchange is thus 6 orders of magnitude higher than the exchange found in lecithin membranes, making it likely that an additional mechanism is in fact operating in the red cell membrane, and suggesting that exchange diffusion mediated by ion-pairing of chloride with the phospholipids only contributes insignificantly to the total chloride exchange. The properties of the chloride transport system are very similar to that of a liquid ion exchange membrane (Wieth, 1972). It is beyond doubt that the bulky integral membrane protein with a molecular weight of 95,000 cannot diffuse through the membrane as a mobile carrier, but it has recently been proposed that a mobile group in the interior of the protein might carry out the ion exchange function by oscillating over a barrier of only a few Angstrom (Rothstein et al. 1976). Although 'half-way fixed', such a group would exhibit the same kinetics as a mobile carrier: no net flux of chloride ions can occur, and no electrical current can be carried by chloride ions, if the movement of the unloaded group is restricted, e.g. by

697 Cl- TRANSPORT IN HUMAN ERYTHROCYTES electrostatic forces so that it cannot move unless it becomes paired with chloride. We want to express our gratitude to Dr Klaus Schnell, University of Regensburg, Germany, who taught us the ghost preparation procedure and to Dr Z. I. Cabantchik, the Hebrew University of Jerusalem, Israel, who provided the sample of DIDS used in the inhibition study. The valued technical assistance of Hanne Christiansen, Anne Lise Mikkelsen and Helle Brinck-Lund is gratefully acknowledged. This work was supported by grant 511-3983 from the Danish State Research

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