Pfli.igers Archiv

Pflfigers Arch. 375, 87-95 (1978)

EuropeanJournal of Physiok)gy 9 by Springer-Verlag 1978

Asymmetry of the Chloride Transport System in Human Erythrocyte Ghosts* K l a u s F. Schnell, E l i s a b e t h Besl, a n d A n n e t t e M a n z Institut ffir Physiologie, Universitfit Regensburg, Postfach 397, D-8400 Regensburg, Federal Republic of Germany

A b s t r a c t . The c o n c e n t r a t i o n d e p e n d e n c e o f the unidi-

rectional chloride flux a n d the i n h i b i t i o n o f the unidirectional chloride flux by sulfate were studied in h u m a n red cell ghosts. The c o n c e n t r a t i o n d e p e n d e n c e o f the u n i d i r e c t i o n a l chloride flux a n d its inhibition by sulfate were asymmetric. T h e u n i d i r e c t i o n a l chloride flux can be s a t u r a t e d f r o m the inner a n d f r o m the outer m e m b r a n e surface. F o r the inner m e m b r a n e surface, lower chloride h a l f - s a t u r a t i o n c o n s t a n t s were o b t a i n e d t h a n for the o u t e r m e m b r a n e surface. T h e i n h i b i t i o n o f the u n i d i r e c t i o n a l chloride flux by sulfate is c o m p e t i tive. In c o n t r a s t to the chloride h a l f - s a t u r a t i o n constants, the i n h i b i t i o n c o n s t a n t s o f sulfate for the inner m e m b r a n e surface were higher t h a n the inhibition c o n s t a n t s o f sulfate for the o u t e r m e m b r a n e surface. Either there are fixed a n i o n b i n d i n g sites at the inner a n d at the o u t e r m e m b r a n e surface which c o n t r o l the access o f a n i o n s to a pore, o r there is a mobile carrier which is in c o n t a c t with b o t h m e m b r a n e surfaces. The a s y m m e t r y o f the c o n c e n t r a t i o n response a n d o f the i n h i b i t i o n o f the u n i d i r e c t i o n a l chloride flux suggest t h a t the a n i o n b i n d i n g sites at the inner a n d at the outer m e m b r a n e surface differ with respect to their affinities for chloride a n d for sulfate. Alternatively, the a s y m m e try o f the chloride t r a n s p o r t system could indicate an a s y m m e t r i c d i s t r i b u t i o n o f a mobile a n i o n carrier across the e r y t h r o c y t e m e m b r a n e . Key words: Chloride -

-

Sulfate -

15] or by a dielectric p o r e the acess to which is c o n t r o l l e d by superficial a n i o n a d s o r p t i o n sites [23, 24, 28]. The u n i d i r e c t i o n a l chloride flux exhibits a s a t u r a t i o n kinetics i n d i c a t i n g t h a t chloride on its way across the red cell m e m b r a n e interacts with a limited n u m b e r o f m e m b r a n e sites [ 3 , 6 - 8 , 1 4 , 2 9 ] . In these experiments the chloride c o n c e n t r a t i o n s at the outer a n d the inner m e m b r a n e surface were s i m u l t a n e o u s l y increased. T h u s it is impossible to decide whether the m e m b r a n e sites are l o c a t e d on the o u t e r or on the inner m e m b r a n e surface, o r w h e t h e r the a n i o n carrier is d i s t r i b u t e d s y m m e t r i c a l l y or a s y m m e t r i c a l l y across the erythrocyte membrane. In o r d e r to characterize the a n i o n t r a n s p o r t system o f the red b l o o d cell, the u n i d i r e c t i o n a l chloride flux in h u m a n red cell ghosts was studied u n d e r c o n d i t i o n s where the intracellular a n d the extracellular chloride c o n c e n t r a t i o n s c o u l d be separately varied. F u r t h e r more, the sidedness o f the i n h i b i t o r y action o f sulfate on the chloride t r a n s p o r t was investigated. The results o f o u r studies indicate t h a t the a n i o n t r a n s p o r t system o f the red b l o o d cell m e m b r a n e is asymmetric. Either there are fixed a n i o n t r a n s p o r t sites at b o t h m e m b r a n e surfaces which differ in their affinities for the different a n i o n species, o r there is a m o b i l e a n i o n - c a r r i e r which is a s y m m e t r i c a l l y d i s t r i b u t e d across the red b l o o d cell membrane.

E r y t h r o c y t e ghosts

Transport.

Introduction

The chloride t r a n s p o r t across the red b l o o d cell m e m b r a n e is m e d i a t e d either by a m o b i l e a n i o n carrier [ 1 2 * A part of the results was presented at the 48th meeting of the German Physiological Society at Regensburg, March 15-18, 1977 [26] and at the XXVIIth International Congress of Physiological Sciences at Paris, July 18-23, 1977 [27]

M a t e r i a l s and M e t h o d s

The experiments were conducted with resealed red blood cell ghosts which were prepared according to the methods of Bodemann and Passow [1] and of Lepke and Passow [17]: Blood from healthy adult donors was withdrawn and stored for maximally 4 days at 4~C. Coagulation was prevented by the addition of an acid-citratedextrose solution. A 50 ~ (w/v) cell suspension was made in isotonic KC1 solutions (165mM). The red blood cells were osmotically hemolysed (5 min; pH 6.2; 0~C) by adding 2 ml of the suspension to 20 ml of a hypotonic MgSO~/acetic acid solution (4mM MgSO4 + 3.5 mM CHsCOOH ). The resealing of the ghosts was performed in a double isotonic KCI/K-citrate solution (660 mosm/1; 45 min; pH 7.2;

0031-6768/78/0375/0087/$1.80

88

PfliJgers Arch. 375 (1978)

37~ C). The chloride concentrations were varied by substituting the K-citrate solutions (244mM) with KCI solutions (330 mM) of the same tonicity. Under these conditions at least 90 % of the ghosts reseal. After resealing, the ghosts were washed and resuspended in KC1/K-citrate solutions which had the same composition as the solutions during the resealing period. If sulfate had to be incorporated into the ghosts, resealed ghosts were incubated in double isotonic KCl/K2SOJK-citrate solutions (660mosm/l). A K2SO 4 solution (264 mM) was added to the incubation solution either in exchange for isoosmolar KC1 solution (330 mM) or in exchange for isoosmolar Kcitrate solution (244mM). The samples were divided into three portions and were loaded with 36C1, 3sSO4 and '4C-L-arabinose, respectively. The equilibration and the labeling of the ghosts with radioactive isotopes took 60 min at 37~C, pH 7.2. A portion of the 36Cl-labeled ghosts was withdrawn, stored at 4~ C and used later on for determining the rate constant of the 36Cl-back-exchange. The radioactively labeled ghosts were spun down, the supernatant was removed, 36C1, 3sSO~ and 14C-L-arabinose within the snpernatant were counted and the chloride concentration of the supernatant was measured by coulometric titration. Subsequently the ghosts were washed at 0~C in approximately 50 volumes of a non-radioactive solution that additionally contained either 2mM SITS (4Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) or 2 mM phtorhizin. After washing, the ghosts were lysed and a6C1, 3sSO4 and 14C-L-arabinose were counted. The amounts ofintracellular chloride and of intracellular sulfate were calculated from the specific activities of chloride and of sulfate. The intracellular volume was calculated from the equilibrium distribution of *4C-L-arabinose. The 36Cl-back-exchange was measured under asymmetric conditions at 0~ C, pH 7.1 - 7.2. Approximately 0.4 g of tightly packed, 36Cl-labeIed ghosts were injected into 40 ml of a non-radioactive solution of appropriate composition. Serial samples were withdrawn from the suspension at suitable time intervals by using the filtration techniques of Dalmark and Wieth [9] and 36C1 in the filtrate was counted. Usually an equilibrium distribution of 36C1 is reached within less than 60 s. In addition, 5 min after the injection of the ghosts, duplicate samples were withdrawn from the suspension and the 36C1 concentration in the extracellular solution at isotopic equilibrium was determined. Attempts were made to measure the chloride net-flux at 0 ~C, pH7.2 from 36Cl-labeled ghosts that were incubated in double isotonic Na-citrate/sucrose solutions (660 mosm/1). Citrate concentrations ranging from 1 0 - 244mM were used. The ghosts concentration was 1% (w/w). Samples were withdrawn over a period of 15 min; 36C1 and K + in the extracellular solution were determined either by liquid scintillation counting or by flamephotometry. We could not measure a significant increase of the concentrations of radioactive chloride and of potassium in the extracellular solution. With regard to the chloride self-exchange, the ghosts behave as a homogeneous population. This is in accordance with the results of Funder and Wieth [11]. The tracer back-exchange from the 36Cllabeled red cell ghosts follows a single exponential equation which can be written as: In (Yo~-Y,) = - k t + in (y~ - Yo)(t) Y, Yo, and y~ (CPM), are the radioactivity of the outside solutions at time t, at zero time and at infinite time, respectively; k (min- 1) is the rate constant and t (min) is time. k was determined by fitting the CPM/tirr~ curves to Eq. (1). Values of y, close to the equilibrium value y~ were rejected. The unidirectional chloride flux -~l was then calculated by using Eq. (2): J~CI --

Cin!)in' CexVex

(2)

CinVin 4- Cex!~ex

c~. and c~x (moles/ml) are the intracellular and the extracellular chloride concentrations, v~ and vex(dimensionless) are the intracellu-

lar and the extracellular fractional volumes. The dimension of the flux is moles, min- 1. ml suspension- 1. Since the concentration of the ghost suspension is known, the fluxes can be converted to moles, min 1 9g cells - 1. The term "g cells" refers to the wet weight of tightly packed red blood cells (5000 x g centrifugation; 10 min; 20~ C; pH 7.3) from which the red cell ghosts were prepared.

Results I n o r d e r to c h a r a c t e r i z e the a n i o n t r a n s p o r t s y s t e m o f the red b l o o d cell m e m b r a n e , the s y m m e t r y o f t h e c o n c e n t r a t i o n d e p e n d e n c e o f the c h l o r i d e t r a n s p o r t a c r o s s the red cell m e m b r a n e was studied. F o r this p u r p o s e , the c h l o r i d e c o n c e n t r a t i o n at t h e c i s - m e m b r a n e side was c h a n g e d w h i l e the c h l o r i d e c o n c e n t r a t i o n at the t r a n s - m e m b r a n e side was k e p t c o n s t a n t . T h e e x p e r i m e n t s w e r e c o n d u c t e d w i t h red cell g h o s t s p r e p a red in d o u b l e i s o t o n i c K C 1 / K - c i t r a t e s o l u t i o n s o f v a r y i n g ratios. Since the v o l u m e o f the r e d cell g h o s t s c h a n g e s if t h e r e is an o s m o t i c g r a d i e n t a c r o s s the red cell m e m b r a n e , it is n e c e s s a r y to c o u n t e r b a l a n c e the v a r i a t i o n s in the c h l o r i d e c o n c e n t r a t i o n s by the a d d i t i o n o f a suitable substitute. T h e c h l o r i d e c o n c e n t r a tions o f the e x t r a c e l l u l a r s o l u t i o n s w e r e raised by s u b s t i t u t i n g KC1 for e i t h e r sucrose, K - c i t r a t e o r K 2 S O 4. T h e c h l o r i d e c o n c e n t r a t i o n s o f the i n t r a c e l l u l a r s o l u t i o n s w e r e i n c r e a s e d b y s u b s t i t u t i n g KC1 f o r e i t h e r K - c i t r a t e o r K 2 S O 4. T h e c h l o r i d e c o n c e n t r a t i o n s at the t r a n s - m e m b r a n e side was k e p t c o n s t a n t at 300 m M . T h i s c o n c e n t r a t i o n is sufficiently h i g h to exert a m a x i m a l a c t i v a t i o n o f the c h l o r i d e t r a n s p o r t system. In all e x p e r i m e n t s a b a c k g r o u n d c o n c e n t r a t i o n o f 10 m M K - c i t r a t e b u f f e r was present. T h e u n i d i r e c t i o n a l chloride fluxes w e r e m e a s u r e d at 0 ~ C by u s i n g the f i l t r a t i o n t e c h n i q u e s o f D a l m a r k a n d W i e t h [9]. T h e 36C1 b a c k e x c h a n g e at 0 ~ C is u s u a l l y c o m p l e t e d w i t h i n a p e r i o d o f less t h a n I rain. U n d e r o u r e x p e r i m e n t a l c o n d i t i o n s (0~ p H 7 . 2 ) n o c h l o r i d e n e t - f l u x c o u l d be d e t e c t e d o v e r a p e r i o d o f 15 m i n . T h u s the c h l o r i d e i n w a r d flux u n d e r o u r e x p e r i m e n t a l c o n d i t i o n s is e q u a l to the c h l o r i d e o u t w a r d flux a n d is m o n i t o r e d by the t r a c e r efflux f r o m the r e d cell ghosts. F o r e x p e r i m e n t a l details the r e a d e r is r e f e r r e d to t h e m e t h o d s section. F i g u r e 1 s h o w s t h a t the u n i d i r e c t i o n a l c h l o r i d e flux c a n be s a t u r a t e d f r o m b o t h m e m b r a n e surfaces. T h e u n i d i r e c t i o n a l c h l o r i d e flux increases p r o g r e s s i v e l y as e i t h e r the i n t r a c e l l u l a r o r the e x t r a c e l l u l a r c h l o r i d e c o n c e n t r a t i o n s were increased. F i n a l l y , in all experim e n t s a p p r o x i m a t e l y t h e s a m e m a x i m a l flux is a t t a i n e d . T h e a n i o n t r a n s p o r t system, h o w e v e r , is a s y m m e t r i c w i t h r e s p e c t to its c o n c e n t r a t i o n d e p e n d e n c i e s . A t the i n n e r m e m b r a n e surface, l o w e r c h l o r i d e c o n c e n t r a t i o n s are r e q u i r e d for a h a l f - s a t u r a t i o n o f the c h l o r i d e t r a n s p o r t t h a n at the o u t e r m e m b r a n e surface. F u r t h e r m o r e , in K2NO 4 s o l u t i o n s h i g h e r h a l f - s a t u r a t i o n c o n -

K. F. SchnelI et al. : Chloride Transport in Red Cell Ghosts

89 CITRATE

-'C[ x 10-4

(O~

7,2)

~'CI _L

moles

minxgcells

x10

I /~f/}[cllex: 300mM

[Cllin : 300 mM

I

I

100

200

moles

minxg cells

I

300 mM

I

I

I

100

200

300

SULFATE (O~

Fig. 1 Concentration-dependence of the unidirectional chloride flux. The unidirectional chloride flux ~CI is plotted vs. the extracellular chloride concentration [C1L~ (upper and lower left panel) or vs. the intracellular chloride concentration [Cl]~n(upper and lower right panel), respectively. The KCI concentration was increased in exchange for Kcitrate or K2SO, as indicated in the Figure. In all cases 10raM K-citrate was present as a background. The KC1 concentration at the transmembrane side was 300 mM. The points represent the mean value of three experiments, the vertical bars the standard deviation. The experiments were conducted at 0~C, pH 7.1 - 7.2

JcI

xlO -4

3"~CI

moles

minxgcells

/till

xlO -4

I

1

200 [CI]ex

c e n t r a t i o n s o f chloride were o b s e r v e d t h a n in K-citrate solutions. U n d e r symmetric conditions, when KC1 solution was substituted f o r K - c i t r a t e t h e u n i d i r e c t i o n a l c h l o r i d e fluxes reach a p l a t e a u at chloride c o n c e n t r a t i o n s o f 1 5 0 - 200 raM. O n the o t h e r h a n d , if sulfate was used as a substitute for chloride it was n o t possible to reach a p l a t e a u up to chloride c o n c e n t r a t i o n s o f 300 m M (not shown in Fig. 1). In all experiments, m a x i m a l chloride fluxes ranging f r o m 2 . 2 - 1 0 - 4 - 2 . 6 - 1 0 - 4 moles r a i n - 1 . g c e l l s - t were observed. F o r the analysis o f the kinetic data, p l o t t i n g p r o c e dures were a p p l i e d which are c o m m o n in enzyme kinetics. The L i n e w e a v e r - B u r k plots, (Fig.2) m a d e f r o m the f l u x / c o n c e n t r a t i o n curves, exhibit an a l m o s t linear relation between the reciprocal chloride flux 1/Jci a n d the reciprocal chloride c o n c e n t r a t i o n l/[C1]. The d a t a c o u l d be fitted by straight lines. The slopes o f the straight lines are significantly different and d e p e n d

7,1 ) moles

min xg cells

/•/t•

n = 300 mM

100

mM

[Cllin

[Cl ]ex

I

300 mM

[Cl]e x = 300 mM

I

300

I

200

I

300 mM

[Cllin

u p o n the m e m b r a n e side at which the c h l o r i d e concent r a t i o n s were altered and u p o n the substitutes employed. A t high chloride c o n c e n t r a t i o n s , slight deviations f r o m linearity were o b s e r v e d for the o u t e r memb r a n e surface in the presence o f sulfate. H o w e v e r , it is difficult to p r o v e w h e t h e r o r n o t these d e v i a t i o n s are really significant. Since the L i n e w e a v e r - B u r k plots tend to m a s k systematic deviations, Hofstee plots were m a d e in a d d i t i o n f r o m the f l u x / c o n c e n t r a t i o n curves (Fig. 3). F o r the inner m e m b r a n e suface a linear relation between ava a n d J a / [ C I ] was o b s e r v e d i n d i c a t i n g that the c o n c e n t r a t i o n d e p e n d e n c e o f the unidirectional chloride flux follows a simple M i c h a e l i s - M e n t e n kinetics. F o r the o u t e r m e m b r a n e surface, the Hofstee plots exhibited systematic deviations f r o m linearity which suggest a m o r e c o m p l e x p a t t e r n o f c o n c e n t r a t i o n response. The d e v i a t i o n s o b s e r v e d at high chloride c o n c e n t r a t i o n s are n o t caused by the s u b s t i t u t i o n o f chloride for a second a n i o n species. T h e y are o b s e r v e d

90

Pft~gers Arch. 375 (1978) CITRATE ( 0 ~ pH 7,2 )

'%,

%, minxg ceils

xlO4

moles

/

/

[Cllin

m

/

I

minxg cells moles

/

: 300 mM

Ks(ex } = 50 mM

50

x10 4

e/

100M-1

.f

I

100 M-1

1/[Ci]in

SULFATE (O~ xlO 4

I

50

1/[Cl ]ex

~

[Cllex = 300 mM Ks{in) - 21 mM

o/e/e

mihxgceUs moles

7,1 ) 1%1 x104

min

/

xg cells

/

2

2

Fig. 2

/

/

/ [CI ]in = 300 mM

[Cl]ex = 300 rnM

Ks(ex ) = 160 mM ,

50 1/[Ci]ex

o

,

100 M-1

/

//

Ks(in) = 59 mM i

1

50

100 M-1

Lineweaver-Burk plots. The plots were made from the flux/concentration curves shown in Figure 1. The reciprocal chloride flux l/Jcl is plotted vs. the reciprocal extracellular chloride concentration 1/[C1]ex (upper and lower left panel) and vs. the reciprocal intracellular chloride concentration 1/[C1]i. (upper and lower right panel), respectively, pH and temperature as indicated in the Figure

1/ [ CI ]in

in a similar fashion in sucrose solutions (not shown in Fig. 3). These deviations probably reflect a self-inhibition of the unidirectional chloride flux which cannot be directly recognized from the flux/concentration curves shown in Figure 1. In sulfate media, an inflection in the curves is additionally observed at low chloride concentrations which may be caused by the inhibitory effect of sulfate on the chloride transport system. The apparent chloride half-saturation constants K~(cl) and the apparent maximal chloride flux Jmax(Cl) obtained from the Lineweaver-Burk plots are compiled in Table 1. If the chloride concentrations were raised at both membrane surfaces using K-citrate as a substitute, an apparent half-saturation constant of 28 m M (0 ~ C; pH7.2) was observed. For red blood cells under comparable conditions, chloride half-saturation concentrations ranging from 25 - 30 m M (0 ~ C; p H 7.2) in the absence of citrate were reported [6, 8,14, 29]. This confirms that citrate has little effect upon the concen-

tration dependence of the chloride self-exchange. In contrast, the apparent half-saturation constants for chloride in sulfate solutions were conspicuously higher. The apparent chloride half-saturation constants for the inner and for the outer membrane surface exhibit considerable differences. Under asymmetric conditions in both citrate and sulfate media, the chloride halfsaturation constants for the outer membrane surface were approximately 2.5 times higher than the halfsaturation constants for the inner membrane surface. In sucrose solutions the chloride half-saturation constants for the outer membrane surface were slightly lower than in citrate solutions. Theoretically, sucrose should be the best substitute for our purposes since it should not react with the chloride transport sites. For technical reasons, however, it is impossible to reseal red cell ghosts in all sucrose solutions. Hence it is impossible to determine the chloride half-saturation constant for the inner membrane surface by using sucrose as a substitute.

K. F. Schnell et al. : Chloride Transport in Red Cell Ghosts

91 CITRATE (0 ~C: pH Z2)

JCI

0,

moles min xg cells

x10 -z,

moles rain xg cells

xlO -/~ e \e

\ =300 mM

~

e

[Cllex = 300ram

\ \ ml minxg cells 10

i 5

ml rain xg cells

;

JcI /[CI)ex

,'0

~Cl /[Cllin

SULFATE (O*C;pH 7,2 ) JCI

moles rain xg cells

rain

xlO-/'

moles xg cells

x 10-z'

2

.\

Fig. 3 Hofstee plots. The plots were made from the flux/concentration curves s h o w n in Figure 1 by plotting the chloride flux Jc~ vs. the ratio fo/[Cl]o, (upper and lower left panel) and Jc]/[CIJi~ (upper and lower right panel), respectively. The straight lines were drawn in order to demonstrate the deviations from linearity

[Ci]in =300 mM

,t

[CI]ex = 300raM

\ m{ mmxg cells

ml

mlnxg cells

i

5 ~CI / [CI]ex

1LO

0

i

i

5 JCl / ICI]in

.

10

Table 1. Apparent half-saturation constants Ks and apparent maximal chloride flux fm,x" The headings of the columns specify the experimental conditions (0~C; pH 7.2). The constants for the outer membrane surface (subscript ex), for the inner membrane surface (subscript in) and for symmetric conditions (subscript in = ex) were determined by using Lineweaver-Burk plots as shown in Figure2 Medium

K-Citrate

[Cl]e x 10 - 300 mM [ClJi n 300 mM

[Cl]ex 300 mM [C1]~n 10 - 300 mM

[C1]ex 10 - 300 mM [C1]i~ 10 - 300 mM

mM

moles x rain- t x g cells - 1

mM

mM

2.97 • 10 -4

21

2.39 x 10 .4

3.33xi0

59

227x10

53

K2SO 4

160

Sucrose

25

4

3.03 x 10 -4

.

Figure 4 displays the inhibition of the unidirectional chloride flux by intracellular and extracelMar sulfate. The dose-response curves and the corresponding Dixon p l o t s a r e s h o w n . T h e c h l o r i d e c o n c e n t r a t i o n s a t t h e cism e m b r a n e side w e r e 10 m M a n d 100 m M , r e s p e c t i v e l y .

moles x min- 1 x g cells 1

.

28

4 .

250

moles x rain- t x g cells- 1 3.18 x 10 -4 4.34xi0

~

.

T h e s u l f a t e c o n c e n t r a t i o n s a t t h e c i s - m e m b r a n e side w e r e r a i s e d to 1 5 0 r a M b y s u b s t i t u t i n g K - s u l f a t e f o r isoosmotic amounts of K-citrate. The chloride concent r a t i o n a t t h e t r a n s - m e m b r a n e side w a s 300 m M w i t h a b a c k g r o u n d c o n c e n t r a t i o n o f 10 m M K - c i t r a t e b u f f e r .

92

Pflfigers Arch. 375 (1978)

SULFATE/ CITRATE ~'CI

1/~CI

min~ils

x 10-4i~

min•

cells

moles

x 104

%,[CI]ex

[Cl]ex

~

9

100mM [Cllin=300ram

9

3

Ki(ex ) :70raM

/

10mM [

I

50

.oI

100

I

mM

0

I

50

[S04]ex

•C[

I

100

mM

[ S04 ] ex

minxg cells moles

moles

min xg cells

xlO-4

x10 l'

[CHin

[Cllin

21

,

s

,OOm.

4

t ~ ~

~

[Cl'ex =300ram

1

3

lOmM

=125mM 10raM

Ki(in)

~ e / e

2

/

100 mM

--

/

0

I

50

I

I00 [ S04 ]in

9

~

i

I

mM

--I

0

[

50

I

I

100

Fig. 4 Inhibition of the unidirectional chloride flux by sulfate. The unidirectional chloride flux fcl is plotted vs. extracellular sulfate concentration [SO4]~x and vs. the intracellular sulfate concentration [SO4]~, (upper and lower left panel). To the right, the corresponding Dixon plots are shown. The chloride concentrations at the cismembrane side were 10mM KCI and 100mM KC1 as labeled at the respective curves. The sulfate concentrations were varied by substituting 244 mM K-citrate solutions with 264mM K2SO 4 solutions. The solutions at the trans-membrane side contained 300mM KC1 and 10 mM K-citrate. The experiments were executed at 0~ C, pH 7.2

mM

[S04lin

Table 2. Inhibition constants for sulfate and for citrate. The apparent inhibition constants were determined by using Dixon plots as shown in Figure 4 Anion

Medium

Osmolarity mosm/1

pH

Ki(e~) mM

Ki(i,i mM

Citrate

Sucrose/Citrate

660

7.2

125

-

Sulfate

Sucrose/Sulfate Sucrose/Sulfate

330 660

7.0 7.2

9 16

-

Sulfate

Citrate/Sulfate Citrate/Sulfate

330 660

7.2 7.2

34 70

113 125

T h e g r a p h i c a l l y d e t e r m i n e d i n h i b i t i o n c o n s t a n t s are listed in T a b l e 2. T h e D i x o n p l o t s in all cases e x h i b i t e d a competitive type of inhibition. In sucrose solutions, a citrate i n h i b i t i o n c o n s t a n t o f 1 2 5 m M a n d a sulfate i n h i b i t i o n c o n s t a n t o f 16 m M f o r the o u t e r m e m b r a n e s u r f a c e w e r e o b s e r v e d . T h i s i n d i c a t e s t h a t citrate to a c e r t a i n e x t e n t i n t e r a c t s w i t h the c h l o r i d e t r a n s p o r t sites o n the o u t e r m e m b r a n e surface. H o w e v e r , its a f f i n i t y to these t r a n s p o r t sites is m u c h l o w e r t h a n the a f f i n i t y o f

sulfate. In c i t r a t e s o l u t i o n s , the sulfate i n h i b i t i o n c o n s t a n t s for the o u t e r m e m b r a n e s u r f a c e are c o n s i d e r a b l y h i g h e r t h a n in s u c r o s e s o l u t i o n s . T h i s is due to the f a c t t h a t in these e x p e r i m e n t s , an i n h i b i t o r w i t h a l o w affinity to the c h l o r i d e t r a n s p o r t sites (citrate) h a d to be r e p l a c e d by an i n h i b i t o r w i t h a h i g h a f f i n i t y to the c h l o r i d e t r a n s p o r t sites (sulfate). C o n s e q u e n t l y a n o v e r e s t i m a t i o n o f the sulfate i n h i b i t i o n c o n s t a n t is to be expected.

K. F. Schnell etal. : Chloride Transport in Red Cell Ghosts The sulfate inhibition constants for the outer membrane surface are considerably lower than the sulfate inhibition constants for the inner membrane surface (Table 2). This means that sulfate has a lower relative affinity to the chloride transport sites at the inner membrane surface than to the chloride transport sites at the outer membrane surface. The differences between the intracellular and the extracellular inhibition constants of sulfate cannot be attributed to a high affinity of citrate to the chloride transport sites on the inner membrane surface. The concentration responsiveness of the unidirectional chloride flux in citrate media yielded lower chloride half-saturation constants for the inner membrane surface than for the outer membrane surface, indicating that the affinity of citrate to the chloride transport sites at the inner membrane surface is lower than its affinity to the chloride transport sites at the outer membrane surface. Hence the differences between the intracellular and the extracellular sulfate inhibition constants primarily reflect differences of the relative affinities of chloride and sulfate for a common transport site. Furthermore, the sulfate inhibition constants increase with increasing tonicity and/or ionic strength and the differences between the inhibition constants for the outer and the inner membrane surface reduce as the ionic strength of the incubation solution is increased (Table2). This phenomenon is not quite understood. It possibly reflects the contribution of electrostatic forces to the adsorption of anions to the membrane surfaces.

Discussion

The chloride transport across the red blood cell membrane is a tightly coupled exchange reaction which can be saturated [2, 3, 6 - 8,11,14, 29]. Recent biochemical studies have shown that the band-3-protein which spans the erythrocyte membrane plays a crucial role in anion transport [4, 5,18 - 22, 30]. However, the mechanism of the chloride transport across the red cell membrane is still controversial. The chloride transport could either be mediated by a mobile carrier [12-15] or by a dielectric pore the access to which is regulated by superficial anion adsorption sites [23,28]. Studies with the non-penetrating inhibitors phlorizin [24] and DAS (2-[4'-aminophenyl]-6-methylbenzene-3,T-disulfonic acid) [16] have shown that the inhibition of the chloride transport system is asymmetric. The inhibition of the chloride transport by phlorizin and by DAS is noncompetitive suggesting that these inhibitors do not directly interact with the anion transport sites (Gerhardt and Schnell, unpublished results). The characterization of the anion transport sites at the inner and at the outer membrane surface therefore requires the

93 study of the concentration responsiveness of the unidirectional chloride flux under conditions where the intracellular and the extracellular chloride concentrations were independently varied. Furthermore, it seemed interesting to study the action of a nonpenetrating competitive inhibitor on the chloride transport (for review cf.: Dalmark [8] and Deuticke [10]). Irrespective of the particular model used for the description of the transport processes, the transport of an anion across the red cell membrane can be subdivided into three consecutive steps: (1) The adsorption of the anion to the cis-membrane surface, (2) the translocation of the anion across the membrane, and (3) the desorption of the anion at the trans-membrane surface. Both the carrier and the pore concept assume the translocation of anions to be the rate-determining step for the hnion transport across the red cell membrane. In contrast to an enzymatic reaction, the chloride transport across the red blood cell membrane is not only a function of the concentrations of chloride and of other anions at the cis-membrane side but it additionally depends upon the anion concentrations at the transmembrane side. If the chloride concentrations at the trans-membrane side are kept constant and if the rate of translocation itself is not affected by variations of the chloride concentrations, then the unidirectional chloride flux from the cis- to the trans-compartment should be determined by the concentration of adsorbed chloride anions at the cis-membrane surface. The adsorption of anions to the membrane surfaces adheres to the law of mass action. It is determined by the bulk concentrations of the anions in the adjacent aqueous phase and by their affinities to the membrane sites. Hence plotting procedures such as the LineweaverBurk plot, the Hofstee plot, and the Dixon plot may serve as diagnostic tools which permit a more precise analysis of the concentration dependence and of the mode of inhibition of the chloride flux. The concentration dependence of the unidirectional chloride flux is asymmetric. For the inner membrane surface, lower apparent half-saturation constants were obtained than for the outer membrane surface (Table 1). The concentration dependency of the chloride flux at the inner membrane surface follows a simple Michaelis-Menten kinetics. For the outer membrane surface a more complex pattern of concentration dependence was observed. Particularly the Hofstee plots (Fig. 3) exhibited strong deviations from linearity which indicate that the unidirectional flux does not follow a simple saturation kinetics. The deviations at high chloride concentrations seem to reflect a selfinhibition of the chloride flux that cannot be directly perceived from the flux/concentration curves as shown in Figure 1. Since the self-inhibition for chloride [3, 6 8] is much less pronounced than the self-inhibition for

94 sulfate [23,25,28] a n d for p h o s p h a t e (yon der Mosel a n d S c h n e l l , u n p u b l i s h e d results), it might easily escape detection if suitable p l o t t i n g p r o c e d u r e s are n o t applied. The deviations f r o m linearity could either reflect the c o n t r i b u t i o n o f positively c h a r g e d a m m o n i u m g r o u p s to the a d s o r p t i o n o f anions to the outer m e m b r a n e surface [28] or it c o u l d indicate the interaction o f chloride with a m o d i f i e r site which inhibits the t r a n s l o c a t i o n o f the l o a d e d a n i o n - c a r r i e r across the red cell m e m b r a n e [ 6 - 8 ] . D u e to the self-inhibition o f the chloride t r a n s p o r t , the c o n d i t i o n s for a m a x i m a l activation o f the a n i o n t r a n s p o r t system are difficult to specify. Trials runs with isotonic KC1/K-citrate solutions ( 3 3 0 m o s m / l ; p H 7 . 2 ; 0 ~ under asymmetric c o n d i t i o n s yielded the same m a x i m a l chloride fluxes. However, it was difficult to perceive f r o m the f l u x / c o n c e n t r a t i o n curves p a r t i c u l a r l y for the o u t e r m e m b r a n e surface w h e t h e r or n o t a s a t u r a t i o n o f the unidirectional chloride flux was reached. Therefore, the experiments were c o n d u c t e d in d o u b l e isotonic solutions, where the chloride c o n c e n t r a t i o n s could be varied over a greater c o n c e n t r a t i o n range. The D i x o n plots (Fig.4) d i s p l a y e d a competitive inhibition o f the u n i d i r e c t i o n a l chloride flux b y intracellular a n d by extracellular sulfate. This indicates that chloride a n d sulfate interact with the same anion a d s o r p t i o n sites at the inner a n d at the o u t e r m e m b r a n e surface a l t h o u g h sulfate at 0~C is n o t t r a n s p o r t e d across the red cell m e m b r a n e . The inhibition o f the chloride flux by sulfate is asymmetric. F o r the outer m e m b r a n e surface a lower sulfate inhibition c o n s t a n t was o b t a i n e d t h a n for the inner m e m b r a n e surface. Conversely, the chloride h a l f - s a t u r a t i o n c o n s t a n t s for the o u t e r m e m b r a n e surface were higher t h a n those for the inner m e m b r a n e surface. The fact t h a t the chloride flux is c o m p e t i t i v e l y inhibited by sulfate u n d e r conditions where sulfate is n o t t r a n s p o r t e d suggests t h a t b o t h anions interact with c o m m o n a n i o n t r a n s p o r t sites at the inner a n d the o u t e r m e m b r a n e surface which are p o s i t i o n e d in front o f the m a i n diffusion b a r r i e r o f the red b l o o d cell m e m b r a n e . T h e results o f o u r studies d o n o t p e r m i t to distinguish between a carrier a n d a p o r e t r a n s p o r t mechanism. The s a t u r a t i o n o f the chloride t r a n s p o r t is consistent with either a p o r e or a carrier t r a n s p o r t system. Either there are fixed a n i o n a d s o r p t i o n sites at the inner a n d at the o u t e r m e m b r a n e surface or there is a carrier which has access to b o t h m e m b r a n e surfaces. The a s y m m e t r y o f the c o n c e n t r a t i o n response o f the chloride flux a n d o f the i n h i b i t i o n o f the chloride flux by sulfate p o i n t s to differences in the affinities o f the anion a d s o r p t i o n sites for the various a n i o n species. Alternatively, the a s y m m e t r y could indicate an asymmetric d i s t r i b u t i o n o f the a n i o n carrier across the red cell m e m b r a n e . The self-inhibition o f the chloride flux,

Pflfigers Arch. 375 (1978) which has been o b s e r v e d for the outer m e m b r a n e surface at high chloride concentrations, can be accomplished either by the b l o c k i n g o f a p o r e [28] or by the i n t e r a c t i o n o f chloride with an inhibitor site [ 6 - 8 ] . A c c o r d i n g to o u r results the i n h i b i t o r site s h o u l d be p o s i t i o n e d on the o u t e r m e m b r a n e surface.

Acknowledgements. This paper was supported by the Deutsche Forschungsgemeinschaft. We wish to thank cand. reed. R. yon der Mosel and Dr. R. Vangala for reading the paper and for helpful comments. The support by Prof. C. Albers is greatly acknowledged.

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Received December 6, 1977