Temperature-Dependent Changes of Chloride Transport Kinetics in Human Red Cells J. B R A H M F r o m the D e p a r t m e n t o f Biophysics, University o f C o p e n h a g e n , P a n u m Institute, DK-2200 C o p e n h a g e n N, D e n m a r k

A B $ T R A C T Chloride self-exchange in h u m a n red cells was studied between 0°C and 38°C. At higher t e m p e r a t u r e s the flow-tube method was used. Although the general features of chloride transport at 0°C and 38°C are similar, the following differences were found: (a) the maximum p H of chloride self-exchange flux was lowered 0.6 p H unit from 7.8 to 7.2 when t e m p e r a t u r e was increased from 0°C to 38°C; (b) the a p p a r e n t half-saturation constant increased from 28 mM at 0°C to 65 mM at 38°C; (c) chloride transport at body t e m p e r a t u r e is slower than predicted by other investigators by extrapolation from low-temperature results. Chloride transport increased only 200 times when t e m p e r a t u r e was raised from 0°C to 38°C, because the a p p a r e n t activation energy decreased from 30 kcal mol -~ to 20 kcal tool -~ above a t e m p e r a t u r e o f 15°C; (d) a study o f t e m p e r a t u r e d e p e n d e n c e o f the slower b r o m i d e self-exchange showed that a similar change o f activation energy occurred a r o u n d 25°C. Both in the case o f CI- (15°C) and in the case o f B r - (25°C), critical t e m p e r a t u r e was reached when the anion self-exchange had a turnover n u m b e r o f about 4" 109 ions cell -1 s-l; (e) inhibition of chloride transport by DIDS (4,4'-diisothiocyano-stilbene-2,2'-disulfonate) revealed that the deflection persisted at 15°C at partial inhibition (66%) presumably because DIDS inactivated 66% of the transport sites, It is suggested that a less t e m p e r a t u r e - d e p e n d e n t step o f anion exchange becomes rate limiting at the t e m p e r a t u r e where a critical turnover n u m b e r is reached. INTRODUCTION

It is t h e p u r p o s e o f t h e p r e s e n t w o r k to p r e s e n t a s t u d y o f c h l o r i d e s e l f - e x c h a n g e at 38°C a n d to p r o v i d e a l i n k to p r e v i o u s s t u d i e s o f c h l o r i d e t r a n s p o r t p e r f o r m e d at low t e m p e r a t u r e s ( G u n n et a l . , 1973a; D a l m a r k , 1975). It has been an open question whether the low-temperature data can be e x t r a p o l a t e d to t h e p h y s i o l o g i c a l t e m p e r a t u r e r a n g e . F o r i n s t a n c e , it h a s r e c e n t l y b e e n q u e s t i o n e d ( C h o w et a l . , 1976) w h e t h e r t h e p r o n o u n c e d t e m p e r a t u r e d e p e n d e n c e o f c h l o r i d e e x c h a n g e f o u n d b e t w e e n 0°C a n d 10°C also a p p l i e s to the physiological temperature range. From studies of bicarbonate transport, C h o w et al. (1976) c o n c l u d e d t h a t Q10 o f i n o r g a n i c a n i o n e x c h a n g e b e t w e e n 25°C a n d 37°C was as low as 1.7 (EA ~ 8.8 k c a l . m o l - t ) . I n t h e p r e s e n t w o r k c h l o r i d e s e l f - e x c h a n g e h a s b e e n s t u d i e d in t h e w h o l e r a n g e b e t w e e n 0°C a n d 38°C. It was THE jOURNAL OF GENERAL PHYSIOLOGY " VOLUME 70, 1977 " pages 283-306

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f o u n d that the t r a n s p o r t process could not he described by a single value o f the A r r h e n i u s activation e n e r g y . T h e activation e n e r g y decreases f r o m 30 kcal" mo1-1 in the low t e m p e r a t u r e r a n g e (0-15°C) to 20 kcal. tool -1 between 15°C and 38°C. T h e s e values c o r r e s p o n d to a decrease o f Q10 f r o m 7 in the low to 3 in the high t e m p e r a t u r e r a n g e . A study o f p H d e p e n d e n c e a n d o f the concentration d e p e n d e n c e o f chloride e x c h a n g e at 38°C also suggests that the exact p r o p e r t i e s o f the t r a n s p o r t process c a n n o t be predicted f r o m data obtained at low t e m p e r a t u r e s , a l t h o u g h the general features o f the t r a n s p o r t process are similar. B r o m i d e self-exchange was also studied at 0-38°C. A similar c h a n g e o f activation e n e r g y was f o u n d , but at a h i g h e r t e m p e r a t u r e (25°C). T h e fact that the deflection a p p e a r e d at d i f f e r e n t t e m p e r a t u r e s d e p e n d i n g on the anion studied does not s u p p o r t the hypothesis that a t e m p e r a t u r e - i n d u c e d c h a n g e o f the lipids in s o m e way may influence the function o f the t r a n s p o r t proteins. It is suggested that the activation e n e r g y o f anion t r a n s p o r t changes w h e n a critical t u r n o v e r n u m b e r o f a b o u t 4 . 1 0 ~ ions cell -1 s -1 is e x c e e d e d . It has been shown (Cabantchik a n d Rothstein, 1974) that anion e x c h a n g e is selectively inhibited by the p o t e n t a m i n o r e a g e n t 4,4'-diisothiocyano-stilbene2,2'-disulfonate (DIDS) which binds to integral m e m b r a n e proteins involved in anion t r a n s p o r t . In the p r e s e n t study e x p e r i m e n t s were p e r f o r m e d a f t e r partial inhibition o f chloride t r a n s p o r t with DIDS. T h e results show that 66% inhibition o f chloride t r a n s p o r t did not r e m o v e the deflection at 15°C w h e r e the t r a n s f e r rate was also f o u n d to be r e d u c e d to o n e - t h i r d o f the uninhibited t r a n s p o r t n u m b e r . It is t h e r e f o r e likely that partial inhibition o f chloride t r a n s p o r t with D I D S inactivated two-thirds o f the t r a n s p o r t sites, whereas the t u r n o v e r rate at the uninhibited t r a n s p o r t sites was u n c h a n g e d a f t e r D I D S t r e a t m e n t o f the cells. MATERIALS,

METHODS,

AND

CALCULATIONS

Electrolyte Media In the experiments performed with erythrocytes the following three media were used (mM). MEDIUM A 145 NaC1, 1.5 CaCI~, 1.0 MgCI2, 5 D-glUcose, and 27 glycyl-glycine (total CI-: 150). MEDIUM B 141 Na-acetate, 1.5 Ca(acetate)~, 1.0 Mg(acetate)~, and 27 glycyl-glycine. By adding ammonium chloride to medium B the chloride concentration was varied from 2 to 600 mM. MEDIUM C 150 NaBr, 5 D-glucose, and 27 glycyl-glycine. Three media with KCI concentrations of 165,320, and 600 mM, all buffered with 2 mM Tris, were used in the experiments performed with ghosts (viz. Tables I and II and Fig. 10).

Labeling and Packing of Cells and Ghosts 20-30 ml of freshly drawn, heparinized human blood was centrifuged at room temperature and the plasma and buffy coat were removed. The cells were washed once in medium A or three times in medium B or C, incubated at the temperature of the subsequent experiment, and titrated to the desired pH with either CO~ or 150 mM NaHCO~. The cells were treated gently by this titration and the following washes

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removed the bicarbonate. At p H values below 6.0 and above 8.5 small amounts o f 150 mM HCI or N a O H , respectively, were a d d e d . After titration, the cells were washed three times in the m e d i u m o f a p p r o p r i a t e ionic strength and p H , and r e s u s p e n d e d to a hematocrit o f 60-80% before addition o f either 610/~Ci ~CI (obtained as the sodium salt, sp act 0.3-0.5 mCi mmo1-1, concentration range 150-225 mM NaCI, Radiochemical Centre, A m e r s h a m , England) or 70-80 ~Ci S~Br (obtained as the sodium salt, sp act 4-6 mCi mmo1-1, concentration range 120-140 mM NaBr, AEK, Ris~, Denmark). T h e cells were equilibrated with the tracer anions at room t e m p e r a t u r e for at least 3 rain which by far exceeds six half-times o f the anion exchange at this temperature. T h e suspension was divided into a major portion centrifuged in 10-ml glass tubes and a minor portion centrifuged in 0.7-ml nylon tubes (ID 3 mm; F u n d e r and Wieth, 1967) for the determination o f chloride or bromide concentration, cell water, and radioactivity in cells and medium. Both portions were spun at 50,000 g for 15 min (Sorvall RC-5 centrifuge, Du Pont Instruments, Sorvall Operations, Newtown, Conn.). T h e supernate and the u p p e r 2-ram layer o f cells in the main portion were drawn off and the remaining cells were stored in a 5-mi syringe at 4°C until use. In another set o f experiments ghost cells were p r e p a r e d with intracellular KCI concentrations o f 165, 320, and 600 mM as described by Schwoch and Passow (1973). T h e procedures o f tracer equilibration and the subsequent separation o f ghosts and extracellular medium were carried out as described above. Experiments were also p e r f o r m e d with ghosts treated with DIDS which is a potent and selective inhibitor o f anion exchange (Cabantchik and Rothstein, 1974). T h e DIDS was synthesized by Dr. M. Hancock, Chemical Lab. II, University o f Copenhagen. Two batches o f ghost cells with a chloride concentration o f 165 mM were incubated with DIDS (5 ~1 or 50 ~1 1 mM DIDS solution per milliliter o f cell suspension with a hematocrit o f 50%) for 45 min at 38°C before the packing procedure. At these DIDS concentrations the anion exchange was inhibited by 66% and 99.6%, respectively, in the two batches o f cells used for experiments between 0°C, and 38°C. T h e extracellular medium t r a p p e d between intact red cells and between ghosts determined as the [3H]inulin space was 1.6% (wt/wt) and 8.2% (wt/wt), respectively, as determined by F u n d e r and Wieth (1976). T h e volume o f the ghosts was calculated from the ghost counting (Coulter C o u n t e r model DN, Counter, Electronics, Inc., Hialeah, Fla.) and the cytocrit after correcting the latter for the extracellular medium t r a p p e d between the packed ghosts.

Determination of the Rate of Efflux All experiments were p e r f o r m e d u n d e r steady-state conditions which means that the efflux o f tracer from the labeled cells is counterbalanced by an equal influx o f nonradioactive ions. In the present study two methods were used to d e t e r m i n e the rate o f tracer wash-out from the packed, labeled cells. In experiments where the half-time exceeded 1-2 s, successive cellfree filtrates were collected from a well-stirred, thermostated cell suspension by the Millipore-Swinnex filtering technique described by Dalmark and Wieth (1972). More rapid reactions were d e t e r m i n e d by means o f the flow-tube technique, the principle o f which is shown in Fig. 1. T h e internal dimensions o f the mixing c h a m b e r and the flowtube were the same as those r e p o r t e d by Piiper (1964). Six filtering units were connected by exchangeable lengths o f constant bore tubing. Cellfree samples o f extracellular medium were collected beneath the ports by inserting either Millipore filters (pore diameter 1 ~ m , Millipore Corp., Bedford, Mass.) or Nucleopore filters (pore diameter

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0.2 /~m, Shandon Nucleopore Corp., Pleasanton, Calif.). T h e flow-tube and its accessories were thermostated with a precision of - 0 . I°C. T h e constancy of the flow-rate (-+ 1%) d u r i n g the experiments was monitored with a strain gauge which measured the buoyancy of a perspex rod placed in the effluent cylinder (8, Fig. 1). T h e linear velocity always exceeded 110 cm s -x which ensured a turbulent flow as d e t e r m i n e d by the flow-pressure relationship described by Sidel and Solomon (1957). A turbulent flow was obtained at a lower linear velocity in the present study although the internal diameter was almost equal in the two flow-tubes (0.20-0.22 cm) because: (a) the filters in the flow-tube used in the present experiments bulged slightly into the bloodstream, resulting in a Bernouilli effect stimulating turbulence; (b) the suspension leaving the mixing chamber had a low viscosity because the hematocrit was only 0.3-1.0% d e p e n d i n g on the relative amounts o f packed erythrocytes o r ghosts and m e d i u m used. Problems o f hemolysis are greatly r e d u c e d and sufficient amounts of filtrates are easily obtained (ca. 0.5 cm 3 at each port) when the hematocrit is low, offering an advantage over the concentrated cell suspensions used by other authors (Tosteson, 1959; Piiper, 1964; Hemingway et al., 1970). T h e total volume of filtrates collected constituted a maximum of 1-1.5% o f the total volume. In experiments p e r f o r m e d at p H 7.4 and 9.0, the degree of hemolysis in the six filtrates and the equilibrium sample was examined spectrophotometrically at the Soret band (411 nm). At p H 7.4 the lysis expressed as the percentage o f the total hemoglobin was 0.7% (range: 0.6-0.8%). At p H 9.0 where the cells are considerably more fragile the lysis was enhanced to 7.1% (range: 4.7-8.6%). Determination o f Radioactivity and Cell W a t e r Content Cell water, chloride, and activity of 3eCI- or S2Br- in plasma, cells, whole blood, and filtrates were measured as described by Dalmark and Weith (1972). CALCULATIONS T h e experiments were p e r f o r m e d u n d e r steady-state conditions and the results showed that the kinetics o f the exchange was well described by a closed two-compartment system with the following relation between specific activity in the medium and time: at = a® (1 -- e -bt) + aoe bt,

(1 a)

where ao, at, and a® are the specific activities (cpm mmol -l) at zero time, at the time t, and at isotopic equilibrium, respectively. T h e equation can be rewritten as follows: ln(i-

a)

= -bt+ln(1-

a,).

(lb)

Since the exchange followed first-order kinetics, the relation between ln[(1 - at~a®)] and time was linear in a semilogarithmic plot. T h e slope of the line, - b , is d e t e r m i n e d by linear regression analysis and is equal to the efflux rate coefficient k(s-1), because the extracellular volume is at least two orders o f magnitude larger than the cellular volume (hematocrit below 1%). T h e term ln[(1 - ao/a®)] expresses the interception with the ordinate at t = 0 and d e p e n d s ideally on the fraction o f extracellular tracer t r a p p e d in the medium between the packed cells. T h e unidirectional chloride flux at steady state is defined as V J = k ' ~ ' C l = P'CI (mol'cm-2"s-1),

(2a)

where V is the intracellular solvent volume (cmZ), A is the m e m b r a n e area available for the exchange (cm2), Cl is the cellular anion concentration (mol'cm-3), and P is the a p p a r e n t

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Chloride Transport in Human Red Cells

'

'I

FIGURE 1. Schematic diagram of the flow tube. 1-2 cm a of packed, labeled red cells in the syringe (1) m o u n t e d in a continuous infusion p u m p (Perfusor IV, B. Braun Melsungen, Melsungen) were injected into the mixing chamber (2) and mixed with 350 ml of medium from the container (3). Air pressure (0.2-2.0 atm) was used as the driving force of the medium and was controlled by a precision pressure valve (4). T h e suspension flowed past six filtration units (6) connected by exchangeable lengths of constant bore tubing (ID 2 mm). Filters with pore diameters of 0.21.0/xm supported by glass fiber filters bulged slightly into the bloodstream, thereby avoiding adsorption of cells to the filters. T h e dimensions of the filtration slit were 10 x 2 mm. A valve (7) at the outlet directed the effluent to either a waste bucket (9) or a graduated cylinder (8) where the effluent was collected d u r i n g the steady-state filtration period. T h e duration of this period was registered by an electrical stopclock controlled by the valve (7). Pairs of test tubes (10), one for waste filtrate, the other for the steady-state filtrate, were placed beneath each port. T h e equipment was kept in a box thermostated by circulation of thermostated air (-+0.2°C). The temperature was measured by three thermocouples (5). permeability (cm" s-!). Cell water and cell volume are d e p e n d e n t on pH. In the present study: d38o{liter cell water. (kg cell solids) -1} = 3.61(SD -+0.12) - 0.26(SD -+0.02). pH. It is convenient to relate the anion transport to the cell solid content in order to refer to a given n u m b e r of cells and thereby to a fixed m e m b r a n e area. Therefore, Eq. (2a) was used in the following form

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M = k ' d ' C l (retool.(3.1- 10lz cells" min)-l),

(2b)

where d ensures the constant area because 3.1 • 10la normal erythrocytes with a mean area of 142/~m 2 contain 1 kg cell solids and have a membrane area of 4.4.107 cm 2 (Wieth et al., 1974). Correspondingly, in the experiments with ammonium chloride-loaded cells, d was corrected for the increased content of solids. In order to be able to compare fluxes in red cells and ghosts, chloride transport in ghosts was expressed in mmol. (3.1" 10la cells-min) -~ (cf. Funder and Wieth, 1976). The Arrhenius activation energy of anion exchange was calculated by linear regression analysis of the relation between the natural logarithm of the flux and the reciprocal of the absolute temperature according to the equation: In M = - E__~A.I+ const, R

T

(3)

where EA is the apparent activation energy (cal'mol-~), R is the gas constant (1.99 cal.(mol" K) -:) and T is the absolute temperature (K). In the experiments with cells treated with ammonium chloride-acetate, the activation energies were calculated according to Exner (1964):

R" TI"~22

EA = ~ --

(ln MI - In M2).

(4)

This equation was also used to calculate the decrease of activation energy due to different degrees of saturation of the transport system. RESULTS

1. Temperature Dependence of Chloride Exchange at [Cl]o = 150 mM By e m p l o y i n g two m e t h o d s (cf. Materials, Methods, and Calculations) it was possible to measure the chloride e x c h a n g e in the whole t e m p e r a t u r e r a n g e f r o m 0°C to body t e m p e r a t u r e , and to d e t e r m i n e w h e t h e r the p r o n o u n c e d t e m p e r a ture d e p e n d e n c e at 0-10°C (Dalmark and Wieth, 1972) extrapolates into the physiological t e m p e r a t u r e range. Identical results were obtained at the same t e m p e r a t u r e (10°C) with the two m e t h o d s , thereby checking the reliability o f both techniques at their limits o f p e r f o r m a n c e . In Fig. 2 the relation between unidirectional chloride flux a n d the reciprocal o f the absolute t e m p e r a t u r e is shown in an A r r h e n i u s diagram. At 0-10°C the results were obtained by means o f the Millipore filtering technique. Between 10°C a n d 38°C the e x c h a n g e was d e t e r m i n e d by means o f the flow-tube m e t h o d . Each point on the g r a p h represents the average o f the calculated chloride t r a n s p o r t o f 2-10 experiments, all p e r f o r m e d at p H 7.2. T h e a p p a r e n t activation energies o f the t r a n s p o r t at 0-15°C, at 15-38°C, and at 0-38°C were calculated by regression analyses. In the low t e m p e r a t u r e r a n g e an activation e n e r g y o f 30 kcal. tool -1 (SD ---0.6; r = 0.999) was f o u n d to be contrasting with an activation e n e r g y o f 20 kcal. mo1-1 (SD +0.5; r = 0.999) at 15-38°C. An activation e n e r g y o f 24 kcal-mo1-1 (SD -+1.1; r = 0.993) was f o u n d between 0°C and 38°C if the deflection o c c u r r i n g a r o u n d 15°C was neglected in the regression analysis.

2. pH Dependence of Chloride Transport at 38°C All the experiments o f this section were p e r f o r m e d with the flow-tube m e t h o d . Fig. 3 shows the rate o f tracer e x c h a n g e in f o u r e x p e r i m e n t s c o n d u c t e d at p H

BnAHM Chloride Transport in Human Red Cells

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b e t w e e n 6.0 a n d 9.0. W h e n p H was i n c r e a s e d f r o m 6.0 to 9.0 a p p r o a c h o f 36C1- to i s o t o p i c e q u i l i b r i u m d e c r e a s e d f r o m 0.2 r a t e c o e f f i c i e n t i n c r e a s e d l i n e a r l y w i t h p H as s h o w n in Fig. t h e e x c h a n g e at p H 7.2 t h a t g a v e a m a x i m u m f l u x (viz. Fig.

the half-time of the s to 0.025 s, a n d t h e 4. T h e h a l f - t i m e o f 5) was 0.05 s (k 13.3

S-l).

Flow-tube technique

12~ • t "~MiUipom-Swlnnex technique ,~ !1

~9;7.tO_

~8 t,,a7 -

~mol

302Ikcollmol

c

-e,.e" :12

L 2~o ,, i~" 1,e s. o. 33 3A 3~5 3.6

FIGURE 2. T e m p e r a t u r e d e p e n d e n c e o f chloride exchange at 0-38°C. T h e logarithm of chloride transport (In M) is depicted vs. the reciprocal of the absolute t e m p e r a t u r e (l/T). T h e results were obtained by the Millipore-Swinnex filtering technique at 0-10°C and by the flow-tube method at 10-38°C. All experiments were p e r f o r m e d at p H 7.2. Each point on the g r a p h is an average o f chloride transport calculated from more than two experiments. T h e ranges are shown when they exceeded the size o f the point. A high a p p a r e n t activation energy o f 30 kcal. mol -~ in the low-temperature interval decreases to 20 kcal. mol -~ above 15°C. T h e extrapolated Q,0 o f 5 in the high t e m p e r a t u r e range predicted from the data obtained at 0-10°C (Q10 = 7) is thus too high, as the present results give a value of 3.

0,5

/2=52m s

pH g0,Ti/2=25m s 0.1i

[~'

pH~O,Tv2:38 ms

~ '~

....

~0

ms

FXGUKE 3. T h e p H d e p e n d e n c e o f the rate o f chloride exchange u n d e r steadystate conditions at 38°C. T h e rate of 3"C1 exchange was measured as the tracer washout from labeled, packed cells into an electrolyte medium identical to the incubation medium. T h e fiow-tube method was used in all experiments. T h e logarithmic ordinate is the fraction o f the tracer r e m a i n i n g in the cells at a given time and the abscissa is time in milliseconds. T h e negative slopes o f the graphs are equal to the efflux rate coefficients u n d e r the present conditions o f a closed two-compartment system with an extracellular volume o f more than 99% o f the total volume. T h e r a t e c o e f f i c i e n t s w e r e u s e d to c a l c u l a t e t h e f l u x e s as d e s c r i b e d in M a t e r i als, M e t h o d s , a n d C a l c u l a t i o n s . T h e c a l c u l a t e d u n i d i r e c t i o n a l f l u x e s a r e s h o w n in Fig. 5 w h e r e c h l o r i d e t r a n s p o r t is d e p i c t e d as a f u n c t i o n o f e x t r a c e l l u l a r p H . A b e l l - s h a p e d g r a p h was f o u n d , as was t h e case at 0°C ( G u n n et a l . , 1973a), b u t t h e m a x i m a w e r e f o u n d at d i f f e r e n t p H v a l u e s . A t 38°C t h e f l u x h a d a p e a k e d

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m a x i m u m at p H 7.2 and decreased steeply at both m o r e acid and more alkaline p H values, whereas a b r o a d m a x i m u m was f o u n d between p H 7.0 and 8.0 at 0°C. T h e p H d e p e n d e n c e o f chloride t r a n s p o r t was e x a m i n e d in resealed erythrocyte ghosts at 38°C in o r d e r to eliminate the effect o f p H on intracellular chloride concentration and m e m b r a n e potential. T h e results o f Table I show that the increase o f chloride self-exchange between p H 5.6 and 7.2 was similar to that f o u n d in intact cells. However, in ghosts the flux decreased m u c h more g r a d u 30

/2

'

20 k: 7195 -39.70/95 pH- 39.702

10 os, D

s.o

,,,~•

7.0 ao pH

9b

~o

FIGURE 4. The rate coefficients k(s -~) as a function of extracellular pH at 38°C. The coefficients were calculated as described in Materials, Methods, and Calculations. The rate coefficients increased linearly with increasing pH according to the equation: k = 7.20 (SD -+ 0.21)'pH - 39.70 (SD +- 1.58).

"2 xlO"s •[~ ).s

= 1.0 ~_ 0.5 E x

3 la,.

o~0

~0

7'0

pH.

8.0

~o

,d.0

FIGURE 5. The pH 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 in h u m a n red cells at 38°C. The points are average values o f the calculated f l u x o f c h l o r i d e f r o m 2 to 10 experiments, e.g., at pH 7.2: M = 149.5 mo1.(3.1.10 TM cells' min) -a (SD --+ 8.8, n = 10). The maximum flux at pH 7.2 equals a transport rate of 4.8- 10TM ions. (cell. s)-~. ally above p H 7.2 than in erythrocytes. T h e decrease was about 15% per p H unit. At p H 9, where the flux in intact red cells had decreased by 60%, the flux had decreased only by 25% in the ghosts. It may be seen that the t r a n s p o r t capacity f o u n d in the ghosts in these e x p e r i m e n t s ( - 1 5 0 mol (3.1.10 TM cells, rain) -~) was the same as that f o u n d in intact cells (viz. Fig. 5).

3. Concentration Dependence of Chloride Transport at 0-38°C T o evaluate the concentration d e p e n d e n c e o f chloride t r a n s p o r t the cellular chloride concentration was varied by loading cells with a m m o n i u m chloride in

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BRAHM Chloride Transport in Human Red Cells

an acetate m e d i u m as described by G u n n et al. (1973a). T h e e x p e r i m e n t s were p e r f o r m e d at 0 °, I0 °, 25 °, a n d 38°C. Chloride t r a n s p o r t was m e a s u r e d at p H 7.2 w h e r e the t r a n s p o r t h a d a m a x i m u m at physiological ionic s t r e n g t h in the whole t e m p e r a t u r e interval, because the b r o a d m a x i m u m f o u n d at 0°C b e c a m e p e a k e d by a shift only o f the alkaline limb when t e m p e r a t u r e was raised. In Fig. 6 the fluxes are shown as a function o f the cellular chloride concentration. T h e g r a p h s d e m o n s t r a t e that the e x c h a n g e is a saturable process at all t e m p e r a t u r e s . At 0°C the flux decreased at cellular chloride concentrations above 200 m M . This is the p h e n o m e n o n t e r m e d "self-inhibition" by D a l m a r k (1976). This self-inhibition is not a p p a r e n t at h i g h e r t e m p e r a t u r e s in the concentration r a n g e studied. T h e a p p a r e n t half-saturation constant, i.e. the cellular chloride concentration TABLE

I

pH DEPENDENCE OF CHLORIDE SELF-EXCHANGE IN GHOSTS AT 38°C ( [ C I ] , = [C1]o : 165 mM) Chloride flux pH

Experimental values

Mean

tool (3.l .IO ta edls .rain) -I

5.6 6.0 6.5 7.0 7.2 7.6 8.0 9.0 10.0

7.2-7.5 71--74 91--108 145--151 144 141-141 125-132 107-123 77-87

7.4 73 100 148 144 141 129 115 82

T h e rate o f ~CI efflux was m e a s u r e d by m e a n s o f t h e flow-tube t e c h n i q u e . T h e increase o f flux with increasing p H f r o m 5.6 to 7.0 at 38°C is similar in both ghosts a n d erythrocytes. T h e steep decrease o f chloride flux in erythrocytes at p H values above 7.2 (viz. Fig. 5) contrasts with a g r a d u a l decrease o f 50% over 3 p H units in ghosts.

at which h a l f - m a x i m a l flux was f o u n d , increased f r o m 28 m M at 0°C to 65 m M at 38°C. T h e lower right c u r v e in Fig. 6 shows that the t r a n s p o r t system is unsaturated at physiological ionic strength a n d p H at 38°C as a result o f the t e m p e r a ture effect on the half-saturation constant. This finding raises the question o f w h e t h e r the c h a n g e in a p p a r e n t activation e n e r g y is caused solely by a decrease in saturation s e c o n d a r y to a decrease in a p p a r e n t affinity at the h i g h e r t e m p e r a tures.

4. Temperature Dependence of Chloride Exchange at High Chloride Concentrations T h e n a t u r e o f the c h a n g e in activation e n e r g y is f u r t h e r e x a m i n e d in T a b l e I I a and b, T h e activation energies in the two t e m p e r a t u r e ranges c o n s i d e r e d are shown in T a b l e I I b . T h e y were calculated b o t h f r o m the results o b t a i n e d with a m m o n i u m chloride-loaded cells a n d f r o m results o b t a i n e d with resealed erythrocyte ghosts with intracellular chloride concentrations o f 165,320, a n d 600 m M .

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T h e table c o m p a r e s these results with the results o b t a i n e d with intact e r y t h r o cytes. In all cases the activation e n e r g y d e c r e a s e d f r o m 28-32 kcal mol -~ (0-15°C) to 19-21 kcal mol -~ (15-38°C). It is shown in the Discussion that u n s a t u r a t i o n o f a b o u t 35% o f the t r a n s p o r t system at 38°C only accounts for a decrease o f the e x p e r i m e n t a l l y d e t e r m i n e d activation e n e r g y by 2-3 kcal mo1-1 a n d it is therefore not surprising that the s a m e values are f o u n d in all t h r e e sets o f experiments. T a b l e I I a also shows the large r a n g e o f half-times at each t e m p e r a t u r e , which excludes the idea that the decrease o f activation e n e r g y is an artifact o f the m e t h o d , b e c o m i n g manifest when chloride t r a n s p o r t is rapid.

5. Temperature Dependence of Bromide Self-Exchange T h e e x p e r i m e n t s were c o n d u c t e d like the e x p e r i m e n t s o f chloride self-exchange

5

-~"

31(

-*

*C k~2zs(.,.)

. . . . . .

lo'c" kl/235(n'tM)

31U;: i

xlO-'

51

i

xlO-s

1.5[

---.

3 as',c o.s 38"c 1 ~ / , kl1247(mM ! ~¢ , , , k?lZ~(mM) 100 300 500 100 300 500 CELLULAR CHLORIDECONCENTRATION(mM)

FIGURE 6, The chloride exchange as a function of the cellular chloride concentration. The cells were loaded with ammonium chloride in acetate media as described by Gunn et al. (1973). The experiments were performed at pH 7.2. At all temperatures the transport shows saturation kinetics. The half-saturation constant (Kv2), defined as the cellular chloride concentration at which the flux is half maximal, increases with temperature from 28 to 65 mM. At 38°C and a cellular chloride concentration of 110 raM, the transport system is therefore operating at 60-70% of its maximum capacity. r e p o r t e d in Section 1. T h e Millipore-Swinnex filtration technique was used in e x p e r i m e n t s at t e m p e r a t u r e s u p to 25°C because the rate o f bro~aide e x c h a n g e is slower. In T a b l e I I I the half-times o f e x c h a n g e , the self-exchange fluxes, and the a p p a r e n t activation energies are shown. It can be seen that the t e m p e r a t u r e d e p e n d e n c e o f b r o m i d e self-exchange is not well described by a single value o f activation energy. T h e kink in the vicinity o f 25°C c o r r e s p o n d s to a decrease o f activation e n e r g y f r o m 32 kcal mo1-1 to 22 kcal tool -1.

6. Temperature Dependence of Inhibited Chloride Transport T h e following inhibition study elucidates w h e t h e r the c h a n g e o f activation e n e r g y persists when chloride t r a n s p o r t is inhibited by DIDS, which is a p o t e n t a n d selective inhibitor o f anion t r a n s p o r t (Cabantchik a n d Rothstein, 1974; L e p k e et al., 1976). T w o sets o f e x p e r i m e n t s were p e r f o r m e d , one w h e r e chloride t r a n s p o r t was inhibited 99.6%, a n d a n o t h e r w h e r e the inhibition was 66%. Both batches o f

BRAHM

Chloride Transport in Human Red Cells TABLE

293 I It2

H A L F - T I M E S ( T t, s) A N D C H L O R I D E T R A N S P O R T (M, mol" [3.1" 1013 cells, m i n ] -1) IN H U M A N E R Y T H R O C Y T E S A N D T H E I R G H O S T S A T 0-38°C ( p H 7.2) Erythrocytes [C|]o (mM)

...

N H.Cl-loaded erythrocytes

Ghosts

150

165

320

600

Tt M

17.2 0.75

26.7 0.77

63.0 0.52

103.6 0.44

5 Tt M

6.52 1.71

9.23 2.21

20.3 1.62

33.6 1.36

10 Tt M

2.32 4.93

3.63 5.63

8.13 4.03

12.6 3.63

15 Tt M

0.89 11.6

1.87 11.0

3.29 9.97

5.20 8.82

20 Tt M

0.454 23.8

1.04 19.7

1.88 17.5

2.87 16.0

25 Tt M

0.230 39.2

0.523 39.1

0.968 33.9

1.58 28.9

30 Tt M

0.098* 83.3

0.293 69.7

150-350

~C

0

38 Tt M

0.052 149.5

0.162§ 126.4

_

0.324§ 101.3

0.551~ 83.2 0.469§ 97.8

27.7 0.49

3.30 3.81 _

0.636 33.2 _

0.221 126.3

The half-times and the chloride transport between 0°C and 38°C in: (a) erythrocytes washed in medium A having a constant extracellular chloride concentration of 150 raM; (b) ghosts prepared with three cellular KCI concentrations; and (c) erythrocytes washed in medium B with NH4CI concentrations that gave maximal fluxes at the temperatures concerned. *, ~, and § denote that the experiments were performed at 32°C, 35°C, and 37.5°C, respectively. TABLE

APPARENT

ACTIVATION

Erythrocytes [Clio (mM)

0-15°C 15-38°C 0~38°C

. ..

I I b

E N E R G I E S (kcal mo1-1) O F C H L O R I D E I N R E D C E L L S A T 0-38°C

EXCHANGE N H4Cl-loaded erythrocytes

Ghosts

150

165

320

600

150-350

30.2+-0.6 (0.999) 19.7+-0.5 (0.999) 23.5+-1.3 (0.993)

28.1---1.6 (0.997) 19.8-+0.9 (0.997) 22.8+1.0 (0.994)

30.6~-0.9 (0.999) 18.3-+0.7 (0.998) 23.6-*-1.6 (0.989)

31.2-+0.9 (0.999) 19.4-+0.4 (0.999) 23.7-+1.4 (0.992)

31.6" 19.0" 24.6+-1.8 (0.995)

The activation energies were calculated according to Eq. (3) and (4) (see Materials, Methods, and Calculations). T h e correlation coefficients are stated in brackets. * Indicates that Eq. (4) was used.

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ghosts and the efflux m e d i u m were titrated to p H 7.2 at 38°C which e n s u r e d that chloride transport was m e a s u r e d at p H values in the whole t e m p e r a t u r e interval where noninhibited chloride t r a n s p o r t was maximal. Fig. 7 displays the temperature d e p e n d e n c e o f chloride t r a n s p o r t that was 66% inhibited. It can be seen TABLE

I 1 I

HALF-TIMES, UNIDIRECTIONAL FLUXES, AND APPARENT ACTIVATION ENERGIES OF BROMIDE SELF-EXCHANGE IN HUMAN ERYTHROCYTES AT 0-38°C ([Br]o = 150 rnM, pH 7.20) T t (s) Range M tool (3.1.10 la cells, rain) -1

E^ (kcal tool -~)

0°C

5°C

10°C

15°C

20°C

25°C

30"C

35°C

38°C

160.3 158.2-162.4

46.8 46.5-47.1

15.0 15.0-15.0

5.0 4.6-5.4

2.0 1.9-2.0

0.97 0.94-0.99

0.400 0.398-0.401

0.227 0.225-0.228

0.174 0.171-0.177

0.08

0.26

0.82

2.01

5.14

10.5

20.5

36.2

47.2

0-25°C

25-38°C

0-38°C

31.5-1.8 (0.998)

21.5--. 1.4 (0.998)

2 9 . 0+- 1.9 (0.994)

T h e rate coefficients of bromide self-exchange were determined as described in Materials, Methods, and Calculations. T h e half-times'(Ti = ! - ~ ) in the table are average values o f one to two double runs. T h e ranges o f the half-times are also shown. T h e apparent activation energies (kcal tool -t) o f bromide transport in the two temperature intervals as well as the whole temperature range are calculated according to Eq. (3) in Materials, Methods, and Calculations. T h e correlation coefficients of the linear regression analyses are stated in brackets.

~ 8.11

30.0'k¢~l *'2 mol'l~

| E

3.2

34

I031T

~6

FIGURE 7. The Arrhenius plot of partially inhibited chloride transport shows that the deflection persisted at 15°C although the transport was 66% reduced with DIDS. There is a good agreement between results obtained with the two methods employed (cf. flux at 25°C). It also appears that the change of activation energy could be determined with the slow filtration method, when the rate of chloride exchange is reduced by inhibition. that also u n d e r these conditions the a p p a r e n t activation e n e r g y decreased f r o m 30 to 18 kcal mo1-1 above ca. 15°C as in the case o f noninhibited chloride transport (viz. Fig. 2). I f chloride transport, however, were almost completely inhibited (99.6%), the relation between t r a n s p o r t and t e m p e r a t u r e could be described by the linear equation: In M = -10.26(SD ---0.25)" (10s T -1) +39.21(SD

BRAHM

Chloride Transport in Human Red Cells

295

- 0 . 8 5 ) , having a constant activation e n e r g y o f 20.4(SD - 1 ) kcal mo1-1 between 0°C and 38°C. DISCUSSION

The pH Dependence of Chloride Transport In Fig. 8 the p H d e p e n d e n c e o f chloride self-exchange at 38°C is c o m p a r e d with results obtained at 0°C (Gunn 1972). At each p H value chloride fluxes have been depicted as a fraction o f the maximal flux at the a p p r o p r i a t e t e m p e r a t u r e in o r d e r to facilitate comparison. It a p p e a r s that both g r a p h s exhibit a m a x i m u m and that the acidic branches have similar positions. In contrast, the alkaline limb was displaced in the acidic direction when t e m p e r a t u r e was raised. T h e r e f o r e , x 1.0

3

U.. -J ~t

O5 I.-

75

0H

FIGURE 8. The fractional flux as a function of extracellular pH at 0°C (©) and 38°C (0). The values at 0°C are calculated from Gunn (1972). The chloride flux at each pH value is depicted as a fraction of the maximal flux at the two temperatures, respectively. The broad maximum between pH 7 and pH 8 obtained at 0°C became more peaked at pH 7.2 when temperature was raised to 38°C. If the shape of the acid limb is due to titration of a group in the membrane, the pK must be about 6 (Dixon and Webb, 1964). The fractional flux ( M t r a e t ) below 7.2 at 38°C is well described as a function of pH, of pK, and of the maximal chloride transport by the equation: [H+]'~_,

Mfraet = (1 + 1--0-2E3/ .

The alkaline limb is discussed in the text.

effects o f low and high p H values on the fractional fluxes will be discussed separately. CHLORIDE TRANSPORT BELOW PH 7 Chloride t r a n s p o r t decreases to the same d e g r e e at 0°C and 38°C when p H is lowered f r o m 7 to 5.7. It has been concluded f r o m studies at 0°C that the transport system is titrated into a f o r m that does not mediate chloride e x c h a n g e when one or m o r e h y d r o g e n ions are b o u n d to a p r o t o n acceptor in the m e m b r a n e (Gunn 1972, 1973). G u n n (1973) based his analysis o f the acid p H effect on the assumption that the inhibition o f chloride t r a n s p o r t was caused by the p r o t o n a t i o n o f a single g r o u p . Later, Dalmark (1975) p r o p o s e d that the decrease o f the flux was a function o f the second p o w e r o f the h y d r o g e n ion concentration, a n d suggested that the h y d r o gen ion inhibition might involve at least two p r o t o n b i n d i n g groups. This second

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power d e p e n d e n c e o f inhibition on H + concentration has not yet been confirmed by o t h e r investigators. In resealed ghosts with an intact anion t r a n s p o r t system F u n d e r and Wieth (1976) f o u n d it sufficient to assume that one H + ion reacts with the transport system to explain the effect o f p H on chloride t r a n s p o r t at 0°C. T h e present results at 38°C also agree with the assumption that the binding o f one p r o t o n is sufficient to block the transport. It may also be noted that chloride t r a n s p o r t is only inhibited by 90% at p H 5 (Gunn et al., 1975), although the inhibition should have been 99% if H + reacts with two p r o t o n acceptors with a p p a r e n t pK values o f 6 or more. It must be noted, however, that chloride permeability increases spontaneously with time when red cells are incubated at p H values below 6 (Gunn et al., 1975) and this p h e n o m e n o n makes it difficult to analyze the true n a t u r e o f the h y d r o g e n ion effect at low p H values. T h e i m p o r t a n t thing to note in the present study is that the a p p a r e n t pK o f the h y d r o g e n ion inhibition is very insensitive to variations o f t e m p e r a t u r e between 0°C and 38°C. T h e ionization enthalpy o f a possible p r o t o n acceptor g r o u p must t h e r e f o r e be very low. CHLORIDE TRANSPORT ABOVE PH 7 T h e m a x i m u m o f chloride transport is peaked at 38°C c o m p a r e d to the r o u n d e d shape f o u n d at low t e m p e r a t u r e s . This is exclusively d u e to a displacement o f the alkaline limb in the acidic direction. It is worth noting that the m a x i m u m at both low and high t e m p e r a t u r e s is f o u n d at that p H where the chloride distribution ratio (Clcell water/Clmedlum) is about 0.75, meaning that in both cases intracellular chloride concentration is 110 mM when the extracellular concentration is held constant at 150 mM. Fig. 9 shows both the chloride distribution ratio (left ordinate) and the cellular chloride concentration (right ordinate) as a function o f p H . T h e dashed line represents the chloride distribution at 0°C ( G u n n e t al., 1973a). At 38°C a parallel line was obtained, but it was shifted ~0.6 p H unit in the acidic direction, in a m a n n e r similar to the displacement o f the transport m a x i m u m . G u n n (1972, 1973) suggested that the decreasing flux with increasing p H was caused by the d e p r o t o n a t i o n o f a g r o u p in the t r a n s p o r t system which would function only when the g r o u p was p r o t o n a t e d . Dalmark (1975) showed that the decrease o f flux with increasing p H disappears when chloride transport at 0°C is studied in red cells loaded with high concentrations o f potassium chloride. It might be a r g u e d that this finding is expected if O H - and CI- c o m p e t e for the transporting g r o u p , so that C1- would not be displaced by O H - when the chloride concentration was increased to 600 mM as in the studies o f Dalmark (1975). T h e studies o f F u n d e r and Wieth (1976) showed that this explanation cannot be correct. In their study o f ghosts with a constant intracellular chloride concentration o f 165 mM, an increase o f p H f r o m 7 to 10.5 had no effect on chloride e x c h a n g e flux, and they stressed that the similarity between the ghosts and the KCl-enriched cells o f Dalmark was that the effect o f p H on intracellular chloride concentration (and thereby also on m e m b r a n e potential) had been efficiently abolished in both cell preparations. It is obvious that the effect o f alkaline p H on chloride transport in ghosts at 38°C differs f r o m the low t e m p e r a t u r e results cited above. Table I shows that the flux decreases with increasing p H . This observation raises the question o f whether the d i f f e r e n c e between the 0°C and the 38°C results could be caused by a

297

BR^HM Chloride Transport in Human Red Cells

t e m p e r a t u r e - d e p e n d e n t c h a n g e o f p K o f a functional g r o u p in the t r a n s p o r t system. A s s u m e , for instance, that the t r a n s p o r t system possesses a critical functional g r o u p with a p K o f 12 at 0°C. This g r o u p will not be d e p r o t o n a t e d to any m e a s u r a b l e d e g r e e at 0°C in the accessible p H r a n g e , explaining why F u n d e r a n d Wieth (1976)found a constant e x c h a n g e flux u p to p H 10.5. I f the p K o f the g r o u p c h a n g e s to 10 w h e n t e m p e r a t u r e is increased f r o m 0°C to 38°C, o n e would expect to find a decrease o f the flux by 50% at p H 10 as was actually the case (Table I). H o w e v e r , the table also shows that t h e r e was an almost linear decrease o f the flux, o v e r t h r e e p H units, o f - 1 5 % p e r p H unit. T h i s g r a d u a l decrease has no r e s e m b l a n c e to that caused by the d e p r o t o n a t i o n o f a single functional g r o u p in the t r a n s p o r t system. O n the o t h e r h a n d it c a n n o t be e x c l u d e d that the reduction o f chloride e x c h a n g e is related to a n u m b e r o f g r o u p s in the m e m b r a n e with a p K distribution which would " s m e a r " the titration curve o v e r

'•1.2 ts0"~

~J 10 .~O.8

xxxx •

~0.21

120

xx 6O

r~.~=2.887-0.300pH(,) ~ \-. ~ ' c ---3.18-0.3t2 pH ~--)¢x~ ~ ~ , ~ 50

60

~0

oH

80

SO

~o ~

FIGURE 9. Chloride distribution ratio and internal chloride concentration (mM) as a function of extracellular pH at 0°C and 38°C. At both temperatures the external chloride concentration is 150 mM. The dashed line shows the distribution at 0°C according to the equation: r o = 3.18(SD -+0.05) - 0.312(SD -+0.007)" pH (Gunn et al., 1973 a). At 38°C the distribution is also a linear function of extracellular pH (rcl = 2.89 (SD -+0.09) - 0.300(SD -+0.01). pH) parallel to the curve at 0°C but shifted 40.6 pH unit in the acid direction. several p H units. In that case the effect on chloride t r a n s p o r t could be indirect, namely by r e d u c i n g the local chloride concentration t h r o u g h an effect on the surface potential o f the m e m b r a n e . Such an effect would be seen if d e p r o t o n a tion o f q u a r t e r n a r y a m m o n i u m g r o u p s a n d o f p h o s p h a t e g r o u p s in the p o l a r heads o f the p h o s p h o l i p i d s a r e critical for the local chloride c o n c e n t r a t i o n in an electrical double layer " f e e d i n g " the e x c h a n g e t r a n s p o r t system (Hall a n d Latorre, 1976). Fig. 5 shows that the flux d e c r e a s e d m u c h m o r e steeply with increasing p H in erythrocytes t h a n in ghosts. T h i s is qualitatively similar to the cited findings at 0°C. D a l m a r k (1975) showed that the effect o f p H on chloride t r a n s p o r t in erythrocytes at 0°C could be grossly a c c o u n t e d for by a s s u m i n g that the r e d u c t i o n o f flux is d u e to a r e d u c e d f o r m a t i o n o f chloride c o m p l e x e s with a t r a n s p o r t i n g g r o u p . T h e n u m b e r o f these c o m p l e x e s was a s s u m e d to be determ i n e d by equilibrium reactions, as suggested by G u n n (1972), a n d the ratios between u n c o m p l e x e d sites at the two surfaces (S~" a n d S+ ) were a s s u m e d to vary a c c o r d i n g to the relation Cl~'/Clo- = S+/S~-. It m u s t be n o t e d that the r e p o r t e d a g r e e m e n t between e x p e r i m e n t a l findings a n d the m o d e l was only qualitative. For instance, the m o d e l (Dalmark 1975, Fig. 10) predicts a 20% decrease o f flux

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at p H 9.5, whereas reductions o f 30% were f o u n d e x p e r i m e n t a l l y at the s a m e p H value (Dalmark, 1975, Fig. 5) a n d a reduction by 70% at the same p H value has been r e p o r t e d by others ( G u n n et al., 1973a). Qualitative a g r e e m e n t similar to that r e p o r t e d by D a l m a r k (1975) applies to the p r e s e n t results obtained at 38°C with the single addition that it is necessary to take into consideration the effect, f o u n d in ghosts, o f p H on chloride e x c h a n g e at constant intracellular chloride concentration (Table I). T h e conclusion, t h e r e f o r e , is that the effect o f alkaline p H on chloride t r a n s p o r t in red cells can also be i n t e r p r e t e d at 38°C in t e r m s o f the effects o f p H on intracellular chloride concentration and t h e r e b y on m e m b r a n e potential, which may in t u r n d e t e r m i n e the steady-state distribution o f t r a n s p o r t sites between the two surfaces o f the m e m b r a n e as originally suggested by D a l m a r k (1975).

The Temperature Dependence of Chloride and Bromide Transport Self-exchange o f chloride in red cells has not previously been m e a s u r e d at 38°C. Various a u t h o r s have a s s u m e d that the rate o f e x c h a n g e at body t e m p e r a t u r e can be e x t r a p o l a t e d f r o m results obtained at 0-10°C if one assumes that the activation e n e r g y o f chloride t r a n s p o r t r e m a i n s constant o v e r the whole t e m p e r ature interval. H o w e v e r , the p r e s e n t study (Fig. 2) shows that the activation e n e r g y d e c r e a s e d f r o m a b o u t 30 to a b o u t 20 kcal mol -I when the t e m p e r a t u r e was increased. T h e value o b t a i n e d by extrapolation o f l o w - t e m p e r a t u r e results exceeds the actual chloride flux at 38°C by a factor o f f o u r (600 vs. 150 mol [3.1.10 x3 cells, mini-l). Chloride self-exchange above 10°C has previously been d e t e r m i n e d in a single investigation at 25°C by T o s t e s o n (1959) who f o u n d a halftime o f 220 ms at p H 7.4. T h e c o r r e s p o n d i n g flux was 1.4.10 -8 tool cm -2 s -1 to be c o m p a r e d with a value o f 1.5.10 -8 calculated f r o m the p r e s e n t results. POSSIBLE "ROLE OF UNSTIRRED LAYER" T h e decrease o f activation e n e r g y is m u c h smaller than that r e p o r t e d f o r c h l o r i d e - b i c a r b o n a t e e x c h a n g e by Chow et al. (1976). Nevertheless, it is necessary to consider w h e t h e r the decrease r e p o r t e d h e r e might be an e x p e r i m e n t a l artifact. O n e could ask w h e t h e r diffusion o f chloride t h r o u g h an u n s t i r r e d layer might c o n t r i b u t e significantly to chloride permeability when m e m b r a n e t r a n s p o r t is rapid. T h e unstirred layer has been estimated to c o r r e s p o n d to an a q u e o u s layer o f 5 - 6 / ~ m in a stop-flow a p p a r a t u s (Sha'afi et al., 1967). This value m a y be taken as an u p p e r limit u n d e r the p r e s e n t conditions, w h e r e flow is t u r b u l e n t . With a diffusion coefficient for chloride o f 2" 10 -5 cm 2" s -1 the permeability coefficient (P = D/Ax) o f the unstirred layer is 4.10 -2 c m . s -x, almost 100 times larger t h a n the permeability m e a s u r e d at 38°C, p H 7.2 (Pel: 5' 10 -4 c m " s - l ) . A n o t h e r p r o o f that the deflection o f the activation e n e r g y is not directly related to the rate coefficient o f chloride e x c h a n g e is f o u n d in the e x p e r i m e n t s with raised cellular chloride concentrations. It can be seen f r o m the results in T a b l e I I b that activation e n e r g y also d e c r e a s e d in highchloride ghosts a n d in a m m o n i u m chloride-loaded cells where the half-time o f e x c h a n g e is u p to 10 times longer than in ceils at physiological ionic strength. THE POSSIBLE ROLE OF PARTIAL SATURATION It was shown in Fig. 6 that the chloride t r a n s p o r t system o f h u m a n red cells with an intracellular chloride concentration o f 110 m M is not s a t u r a t e d at 38°C. T h e t r a n s p o r t system is o p e r a t i n g at only 60-70% o f its m a x i m u m capacity, a n d o n e must consider how

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much this could decrease the a p p a r e n t activation energy. Calculations show that the activation e n e r g y would only increase by 2-3 kcal mol -* between 15°C and 38°C if the chloride transport system was saturated in the whole range between 15°C and 38°C. In this context it is worth noting that the activation e n e r g y was in fact not significantly different in cells with an intracellular chloride concentration o f 110 mM and in chloride-loaded cells or ghosts, where saturation o f transport was achieved in the whole t e m p e r a t u r e interval. CRITICAL TEMPERATURE OR CRITICAL RATE T h e evidence thus shows that the change o f activation e n e r g y with t e m p e r a t u r e is in fact real. It cannot be excluded that the change is m o r e gradual than indicated by the straight lines o f the regression analyses (viz. Fig. 2). H o w e v e r , it simplifies the evaluation if one assumes that the curves can be resolved into two straight lines with a distinct point o f intersection. T h e r e a p p e a r to be at least two possibilities: (a) that the energy barrier o f chloride t r a n s p o r t decreases at a critical t e m p e r a t u r e (e.g., due to a phase transition o f some m e m b r a n e c o m p o n e n t ) ; or (b) that chloride exchange is rate limited by one step in the transport process when the rate is slow (below 150C), whereas a n o t h e r step with a somewhat lower activation e n e r g y becomes rate limiting at high t e m p e r a t u r e s . Critical Temperature

It cannot be excluded that the state o f the m e m b r a n e lipids indirectly may affect the transport rates o f ions and hydrophilic molecules by modifying the function o f m e m b r a n e lipoproteins as suggested by W a r r e n et al. (1975). Studies o f anion transport in red cells f r o m various animals show a correlation between membrane lipid composition and rate o f transport (Deuticke and G r u b e r 1970; Wieth et al., 1974). Changes o f the activation e n e r g y o f sugar transport in Escherichia coli strains correlate with the t e m p e r a t u r e s at which m e m b r a n e lipids u n d e r g o a phase transition (Linden and Fox 1973). It is not clear w h e t h e r a phase transition o f erythrocyte m e m b r a n e lipids actually occurs between 0°C and 38°C. Aloni et al. (1974) d e t e r m i n e d the microviscosity and fusion e n e r g y o f lipids in intact erythrocytes, in ghost m e m b r a n e s , and in liposomes o f the lipid extracts without finding a phase transition between 0°C and 40°C. Several results reviewed by Old field and C h a p m a n (1972) s u p p o r t this observation. H o w e v e r , o t h e r investigators have f o u n d evidence o f a phase transition occurring a r o u n d 10-20°C (Johnson, 1975; Z i m m e r and Schirmer, 1975; Bieri and Wallach, 1976). I f it is assumed that the change o f activation e n e r g y is d u e to a phase transition, it is a necessary consequence that the critical t e m p e r a t u r e is influenced by the anions o f the m e d i u m . T h e deflection in the A r r h e n i u s diagram o c c u r r e d at 15°C in the case o f chloride transport, at 25°C for b r o m i d e transport, and it does not a p p e a r to be present for anions that are t r a n s p o r t e d m o r e slowly than b r o m i d e (Dalmark and Wieth, 1972). T h e effects o f anions on the t e m p e r a t u r e o f phase transitions in lipids recently described by C h a p m a n et al. (1977) a p p e a r to be too small to account for the present effects. Critical Rate

A second possibility is that chloride exchange is rate limited by two d i f f e r e n t rate coefficients. T h e A r r h e n i u s diagram in Fig. 10 shows that the change o f activa-

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tion e n e r g y o f chloride a n d o f b r o m i d e t r a n s p o r t did not occur at the s a m e t e m p e r a t u r e . It can be seen that the kink a p p e a r e d at the two t e m p e r a t u r e s where chloride a n d b r o m i d e fluxes were identical: i.e. at the s a m e t r a n s p o r t rate. For chloride t r a n s p o r t the deflection was f o u n d at 15°C w h e r e chloride t r a n s p o r t was 11.5 mol (3.1.1013 cells, min) -1, equalling 3.7" 109 ions (cell. s) -~. At 25°C, w h e r e the deflection was f o u n d in case o f b r o m i d e , the t r a n s p o r t a m o u n t e d to 3.5" 109 ions (cell. s) -x. I f it is accepted as a working hypothesis that the activation e n e r g y changes when a critical t u r n o v e r n u m b e r is r e a c h e d , the simplest explanation of this p h e n o m e n o n would be that the overall t r a n s p o r t rate is d e t e r m i n e d by two "reactions" with d i f f e r e n t activation energies, in such a way that one step with a high EA is rate limiting at low t e m p e r a t u r e s , whereas a n o t h e r step with a lower xlO 9

50 20

tt

2

~.

II!:

~6

• Chloride ~lf-e*~t'm*~e ftux o ~ iHdf-jpte.J~ulge flux ( m e a l (3V 1013Cells.rain) -1 )

r.

--4

3e

3o

3.2

3~3

"C

20

3J*

%

,o

3'.5

o

36

FIGURE 10. Arrhenius plot of the temperature dependence of chloride and of bromide self-exchange at 0-38°C. The logarithmic righthand ordinate shows the turnover number of ions per cell per second. The data of chloride self-exchange are repeated from Fig. 2. The results of bromide self-exchange were obtained by means of the Millipore-Swinnex filtering technique at 0-25°C and by means of the flow-tube method above 25°C. The pH of the experiments was 7.2. The extracellular halide concentration was 150 mM in all experiments. EA b e c o m e s limiting at h i g h e r t e m p e r a t u r e s . A l t h o u g h n o t h i n g is k n o w n a b o u t the molecular n a t u r e o f chloride e x c h a n g e it is likely that it involves m o r e t h a n one rate process. A classical carrier m o d e l includes at least t h r e e rate coefficients: (a) the f o r m a t i o n o f the a n i o n - c a r r i e r complex; (b) translocation o f the complex; a n d (c) liberation o f the anion f r o m a site on the carrier. T h e deflection o f EA could t h e n be the result o f a highly t e m p e r a t u r e - d e p e n d e n t step that is rate limiting at low t e m p e r a t u r e s (EA ~ 30 kcal tool-l), whereas a less t e m p e r a t u r e - d e p e n d e n t process b e c o m e s rate limiting at the t e m p e r a t u r e w h e r e the critical t u r n o v e r rate o f the t r a n s p o r t system is e x c e e d e d . At this point it m u s t be i n t e r p o l a t e d that the a m o u n t o f evidence showing that a 10S dalton m e m b r a n e p r o t e i n is involved in anion t r a n s p o r t m a k e s it unlikely that anion t r a n s p o r t in red cells is m e d i a t e d by a simple mobile carrier (Rothstein et al., 1976). H o w e v e r , the general considerations above may as well be applied to a m o d e l w h e r e (a) the b i n d i n g o f an anion to a site in a m e m b r a n e protein

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leads to (b) a conformational change in the protein which (c) allows the f u r t h e r passage o f the anion t h r o u g h the m e m b r a n e . It is implied by the present interpretation that the t r a n s p o r t system functions at a slower rate when it is t r a n s p o r t i n g b r o m i d e instead o f chloride. It appears f r o m Fig. 10 that the exchange flux o f b r o m i d e at any t e m p e r a t u r e is 3-10 times smaller than that o f chloride. According to the present interpretation the smaller flux o f b r o m i d e at any given t e m p e r a t u r e c a n n o t be due to a decreased n u m b e r o f t r a n s p o r t sites all functioning with the rates chracteristic for chloride transport. T h e differing rates o f halide t r a n s p o r t have been observed by Tosteson (1959) and by Dalmark and Wieth (1972). Determinations o f the halfsaturation constants for chloride, b r o m i d e , and iodide ( G u n n et al., 1973b) clearly indicate that these rates are not d u e to differing affinities o f the anions for the t r a n s p o r t system. T h e r e f o r e , it is reasonable to assume that the rate o f translocation is modified by the chemical n a t u r e o f the halide. ACTIVATION

ENERGIES FOR T R A N S P O R T

OF A N I O N S O T H E R

THAN

CHLORIDE

AND BROMIDE It is t h e r e f o r e concluded that the activation e n e r g y o f anion transport remains constant and high until a critical t r a n s p o r t rate is e x c e e d e d , and that it decreases f r o m 30 to 20 kcal.mo1-1 when the t u r n o v e r o f anions exceeds 4.109 ions (cell. s) -1. This hypothesis would also explain why the activation energies o f m o r e slowly t r a n s p o r t e d anions have been f o u n d to r e m a i n constant even at h i g h e r t e m p e r a t u r e s . Dalmark and Wieth (1972) f o u n d that self-exchange o f iodide has a constant activation e n e r g y o f 36 kcal" mol -~ between 0°C and 38°C, and studies o f sulfate t r a n s p o r t have also d e m o n s t r a t e d constant activation energies o f 33-38 kcal. mo1-1 between 0°C and 38°C (Lepke and Passow, 1971; Wieth, 1970a). T h e s e findings a g r e e with the concept o f a critical t u r n o v e r n u m b e r that will not be reached by I - or by S O ¥ - , which are t r a n s p o r t e d 102-104 times slower than chloride. THE

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CELLS T h e anion t r a n s p o r t system is not the only t r a n s p o r t mechanism o f the red cell m e m b r a n e that shows a decrease o f activation e n e r g y between 0°C and 38°C. Sen and Widdas (1962) f o u n d an activation e n e r g y o f glucose transfer o f 20 kcal.mol -~ a r o u n d 20°C, decreasing to 7-10 kcal-tool -~ a r o u n d 37°C. This is interesting because glucose t r a n s p o r t , according to T a v e r n a and L a n g d o n (1973), is ascribed to that g r o u p o f integral m e m b r a n e proteins which are believed to be involved in anion transport. Recent studies d o not exclude this possibility (Kasahara and Hinkle, 1976; Zala and Kahlenberg, 1976). H o a r e (1972) studied the activation e n e r g y o f the facilitated diffusion o f L-leucine and f o u n d a decrease f r o m 47 to 17 k c a l ' m o l -~ above 20°C. T h e s e changes in activation e n e r g y have been f o u n d in NaCl media in t r a n s p o r t systems which presumably function considerably m o r e slowly than the chloride-transporting system. I f the activation energies o f sugar and amino acid t r a n s p o r t b e c o m e constant in, e.g., iodide media, one might hypothesize that the t h e r m o d y n a m i c properties o f the t r a n s p o r t systems d e p e n d on the n a t u r e o f the anions present. In this context it may be noted that facilitated diffusion o f erythritol t h r o u g h the hexose t r a n s p o r t system is inhibited when chloride is replaced by the foreign anions thiocyanate or salicylate (Wieth, 1971) and that very complex changes o f

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the activation e n e r g y o f passive s o d i u m a n d potassium t r a n s f e r have been f o u n d w h e n chloride in the cells a n d m e d i u m has been replaced by the same anions (Wieth, 1970b). ACTIVATION ENERGY OF INHIBITED CHLORIDE TRANSPORT I f the above int e r p r e t a t i o n is correct it follows that the c h a n g e o f activation e n e r g y should persist if chloride t r a n s p o r t is inhibited by an inhibitor which reduces the m a x i m u m flux by r e m o v i n g a fraction o f the t r a n s p o r t sites without affecting the t r a n s p o r t rate o f the r e m a i n i n g sites. A t t e m p t s have b e e n m a d e to characterize the inhibition caused by the a m i n o r e a g e n t D I D S which is an irreversible inhibitor o f anion t r a n s p o r t (Cabantchik and Rothstein, 1974; L e p k e et al., 1976). It is shown in Fig. 7 that a similar t e m p e r a t u r e d e p e n d e n c e o f chloride t r a n s p o r t was f o u n d when the e x c h a n g e was r e d u c e d by two-thirds with DIDS. T h e results t h e r e f o r e strongly suggest that D I D S inactivated two-thirds o f the a n i o n - t r a n s p o r t i n g sites, whereas the r e m a i n i n g fraction o f sites o p e r a t e d with a t u r n o v e r rate as high as in the case o f noninhibited chloride t r a n s p o r t . I f the chloride t r a n s p o r t was r e d u c e d by m o r e t h a n 99%, h o w e v e r , a constant activation e n e r g y o f 20 kcal mo1-1 was f o u n d in the whole t e m p e r a t u r e interval between 0°C a n d 38°C. Previous studies have shown that the almost c o m p l e t e inhibition o f chloride t r a n s p o r t with salicylate (Dalmark a n d Wieth, 1972) and with trinitrocresolate ( D a l m a r k et al., 1972) also caused a constant activation e n e r g y . This is s u r p r i s i n g if o n e believes that the a m i n o r e a g e n t a n d the a r o m a t i c anions are " n o n - c o m p e t i t i v e inhibitors" in the sense that they act by r e d u c i n g the rate o f t r a n s p o r t o f the sites. H o w e v e r , it m u s t be stressed that these inhibition studies were all p e r f o r m e d u n d e r conditions w h e r e chloride e x c h a n g e was inhibited by m o r e t h a n 99%. It c a n n o t be e x c l u d e d that t h e r e exist m i n o r classes o f t r a n s p o r t sites which always function at a rate lower t h a n the critical t u r n o v e r rate a n d which are m o r e resistant to inhibitors t h a n the vast majority o f t r a n s p o r t sites. In that case the n a t u r e o f inhibition may be o b s c u r e d w h e n almost c o m p l e t e inhibition is achieved.

The Turnover Rate of Chloride Ions It is shown in T a b l e I I that chloride t r a n s p o r t at 0°C was a b o u t 0.7 m o l . (3.1" 1013 cells" min) -1 which equals 2.3. l0 s ions. (cell. s) -1. At 38°C the chloride t r a n s p o r t o f 150 m o l . ( 3 . 1 . 1 0 TM cells.min) -1 c o r r e s p o n d s to a value o f 4.9"10 l° ions. (cell. s) -1. T h e t r a n s p o r t rate thus increased a b o u t 200 times when t e m p e r a t u r e was raised f r o m 0°C to 38°C. It has previously b e e n m e n t i o n e d that t h e r e is s o m e evidence that an integral m e m b r a n e protein with a molecular weight o f a b o u t 105 daltons is involved in anion t r a n s p o r t . Steck (1974) estimated that t h e r e are a b o u t 106 copies o f these protein molecules p e r cell. T h e n u m b e r o f t r a n s p o r t sites may be smaller if anions are t r a n s p o r t e d t h r o u g h a d i m e r or a t e t r a m e r o f the m e m b r a n e protein. It has b e e n suggested (Cabantchik a n d Rothstein, 1974; Rothstein et al., 1976) that each cell m e m b r a n e carries a b o u t 3-5" 105 inhibitor b i n d i n g sites. T h e same o r d e r o f m a g n i t u d e was f o u n d by H o a n d Guidotti (1975) using the sulfanilate anion or the isothiocyanate derivative o f the sulfanilate anion as inhibitor.

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H o w e v e r , it has been s u g g e s t e d by Zaki et al. (1975) that the actual n u m b e r m a y be higher because their studies o f the c o m p e t i t i o n between b i n d i n g o f a stilbene derivative a n d d i n i t r o f l u o r o b e n z e n e revealed a n u m b e r o f 0.8-1.2" 106 sites cell-l.,A recent study o f L e p k e et al. (1976) shows that the b a n d 3 proteins bind as m a n y as 1.2.106 H~DIDS molecules p e r cell. It m a y be o f interest to c o m p a r e the t u r n o v e r rate for chloride t r a n s p o r t in red cell m e m b r a n e s with the t u r n o v e r rates o f ion t r a n s p o r t t h r o u g h m o d i f i e d bimolecular lipid m e m b r a n e s . I f o n e assumes that each red cell m e m b r a n e carries 106 t r a n s p o r t sites, the t u r n o v e r n u m b e r p e r site is 5.104 ions s -1 at 38°C. For bimolecular lipid m e m b r a n e s data are available both for p o t a s s i u m t r a n s p o r t m e d i a t e d by valinomycin, which is believed to be a cycling carrier, a n d for potassium t r a n s p o r t m e d i a t e d by gramicidin, which is believed to f o r m pores or channels t h r o u g h the m e m b r a n e . T h e c a r r i e r t r a n s p o r t o f potassium by valinomycin had a t u r n o v e r n u m b e r o f 104 ions s -1 at r o o m t e m p e r a t u r e (L/iuger, 1972). When an activation e n e r g y o f 14-19 k c a l - m o l -~ is used (Benz et al., 1973) the t u r n o v e r rate at 38°C is a b o u t 3" 104 s -1. In contrast, the p o r e - m e d i a t e d potassium t r a n s p o r t in g r a m i c i d i n - t r e a t e d m e m b r a n e s a m o u n t s to 2.107 ions- s -1 at 25°C (Hladky, 1974). A c c o r d i n g to G i n s b u r g a n d Noble (1974) the net activation e n e r g y o f p o r e f o r m a t i o n a n d cation t r a n s f e r is 9.3 kcal. mo1-1, resulting in a t u r n o v e r rate o f 4. l0 T s -1 at 38°C. It is obvious that a c o m p a r i s o n o f the t u r n o v e r rate o f chloride t r a n s p o r t with the rates f o u n d with i o n o p h o r e s in b i m o l e c u l a r lipid m e m b r a n e s does not lend i n f o r m a t i o n a b o u t the m o l e c u l a r n a t u r e o f chloride t r a n s p o r t . Still it is o f interest to note that if o n e assumes the n u m b e r o f t r a n s p o r t sites to be o f the o r d e r o f 1 million, the t u r n o v e r rate o f the biological t r a n s p o r t system is two to three o r d e r s o f m a g n i t u d e smaller than that f o u n d in the n a r r o w gramicidin p o r e , a n d a p p e a r s to be within one o r d e r o f m a g n i t u d e f r o m the t u r n o v e r rate o f the valinomycin carrier. Chloride t r a n s p o r t is t h e r e f o r e not so r a p i d that it is necessary to postulate a r a p i d t r a n s f e r o f chloride t h r o u g h m e m b r a n e pores. O n the c o n t r a r y the t r a n s p o r t process a p p e a r s to be slow e n o u g h to a c c o m m o d a t e several successive steps, as e.g., the b i n d i n g to a site followed by a m i n o r c o n f o r m a t i o n a l c h a n g e in a protein molecule which, as suggested by Rothstein et al. (1976), may p r e c e d e the actual p e n e t r a t i o n o f the m e m b r a n e barrier.

The Hetero-Exchange of Chloride and Bicarbonate It must be e m p h a s i z e d that the activation energies o f chloride a n d b r o m i d e transport are high in the whole t e m p e r a t u r e r a n g e o f 0-38°C. T h e lower activation e n e r g y o f 20 kcal mol -~ above the critical t u r n o v e r rate m e a n s that Qa0 is 3-4 and not 5 as p r e d i c t e d f r o m results at low t e m p e r a t u r e s . A Q~0 o f 1.2-1.7 (EA 4-9 kcal. tool -1) between 24°C a n d 38°C, as r e p o r t e d by L u c k n e r (1948) a n d by Chow et al." (1976) for the h e t e r o - e x c h a n g e o f chloride for bicarbonate, is significantly lower than the p r e s e n t values. In both these studies anion m o v e m e n t s were registered electrometrically by an electrode system: silver-silver chloride electrode (Luckner, 1948), a n d pH-glass electrode (Chow et al., 1976). T h e low activation energies are very close to those f o u n d for free diffusion o f electrolytes in water. It is obvious that the effect o f diffusion on the r e s p o n s e time m i g h t

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have b e e n a real p r o b l e m in t h e e x p e r i m e n t s of L u c k n e r , which were c a r r i e d o u t with a layer o f u n s t i r r e d r e d cells. A similar e x p l a n a t i o n does not a p p e a r to a p p l y to the e x p e r i m e n t s o f Chow et al. (1976), b e c a u s e the r e s p o n s e time o f t h e i r p H stop-flow t e c h n i q u e has b e e n r e p o r t e d to be below 5 ms ( C r a n d a l l et al., 1971). As stated a b o v e , it has not yet b e e n e x c l u d e d t h a t i n d i v i d u a l a n i o n species can e x e r t specific effects on the activation e n e r g y o f a n i o n t r a n s p o r t at physiological t e m p e r a t u r e s . I f t h e r e is such an effect, the results o f Chow et al. m a y be d u e to a specific effect o f b i c a r b o n a t e on t h e t h e r m o d y n a m i c s o f the t r a n s p o r t process at high t e m p e r a t u r e s . I wish to express my gratitude to Dr. J. O. Wieth for his advice during all phases of the work. The valued technical assistance of Mrs. Annie Jcrgensen and Mrs. Birgitte Dolherg Olsen is gratefully acknowledged. This work was carried out during the term of a research scholarship from the University of Copenhagen. Received for publication 9 April 1976. REFERENCES

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