Gen Physiol Biophys (1997), 16 , 273- 284 273

Potassium Channels of the Lamprey Erythrocyte Membrane Exhibit a High Selectivity to K+ over Rb+: a Comparative Study of 8 6Rb and 41K Transport

G P G U S E V , D G F L E I S H M A N , V A N I K I F O R O V , \ N D A O S H E R S T O B I T O V

Sechenou Institute of Evolutionary Physiology and Biochemistry

Russian Academy of Sciences

St Petersburg Russia

Abstract. In older to assess the specificity of potassium channels, we examined the K+ transpoit pathways in the lampiey red blood cells using 8RRb and stable isotope 41K as tiaceis measured by the mass spectrometiic method Upon replacing 4 mmol/1 K+ with 4 mmol/1 Rb+ in the incubation medium, the íate coefficient foi unidirectional 86Rb influx (A"m) was significantly reduced from 1 48 ± 0 10 h _ 1 to 0 82 ± 0 05 h - 1 (P < 0 001) Addition of 1 mmol/1 Ba2 + to the incubation medium significantly decieased 86Rb influx m K+- (by 53%) and Rb+-media (by 26%) Thus, the i eduction in the late coefficient foi 80Rb influx in Rb+-medium as compaied to K+-modium was mainly due to the Ba2+-sensitive component (0 23 ± 0 04 vs 0 78 ± 0 10 h - 1 , P < 0 001) The ouabain-sensitive component of 8eRb influx was also highei in K+-(Am = 0 62 ± 0 05 h"1) than in Rb+-medmm (A'ln = 0 54 ± 0 05 h - 1 , P < 0 05) In the piesence of 4 mmol/1 41KC1, the aveiage value of the íate coefficient foi the total 41K influx was 8 1 ± 0 5 h"1 The íate coefficient foi 41K influx was íeduced to 0 82 ± 0 13 h _ 1 in the piesence of 1 mmol/1 Ba2 + Under the conditions of our assays, the rate of 8eRb uptake via potassium channels of the lampiey led cells was only about 11% in K+-medium and only about 3% m Rb+-medium compaied to 41K influx These data cleaily demonstiate a high selectivity of potassium channels for K+ ovei Rb+ , as already reported foi some potassium channels in a vanety of cells and tissues

Key words: K+ channels — Na+ , K+-pump — K + /Rb+ selectivity — Lamprey erythiocytes

Correspondence to G P Gusev, Sechenov Ins t i tu te of Evolut ionary Physiology and Biochemistry Russian Academy of Sciences, 194223, Thore7 pr 44 St Pe tersburg , Russia Tel (812) 552-32-91, Fax (812)552-30-12 E-mail gusev Sief spb su

274 Gusev ct al

In troduct ion

There has been a large number of studies that suggested the ability of R b + to substitute sa t i s fac tory for K + m the investigation of ion transport mechanisms in a variety of cells and tissues (Castronovo et al. 1968; Beauge and Ortiz 1970; Bernstein and Israel 1970; Cheval et al. 1991; Ikehara et al. 1993). Therefore, K+ transport through cell membranes is often assessed by measuring 8eRb because of the cost and the short half-life of potassium radionuclides. However, some potas­sium channels have long been known to be able to discriminate between these two ions (Adrian 1964; Hille 1973, Lattore and Miller 1983; Gallacher et al 1984: Van Driessche and Zeiskc 1985; Silver et al. 1994). This selectivity foi K+ over Rb+ is a property shared by many of the numerous well-defined potassium channels, although the degree of selectivity differs among classes of the channels

The lamprey red blood cells arc unique among erythrocytes of many othei species studied so far. The lamprey erythrocyte membrane shows a low chloride permeability (Nikinmaa and Railo 1987; Gusev and Sherstobitov 1993). On the other hand, several recent studies (Kirk 1991; Gusev et al. 1992) have shown that the lamprey red cells contain potassium channels which can be inhibited by B a 2 + , quinine, tetraethylammoiimm and amiloride. The channels provide a major con­tribution to total K + influx in the led cells under physiological conditions, but theii properties lemain unexplored. In previous investigations the pathways of K +

movement through the lamprey erythrocyte membrane have been examined using 8eRb as a tracer. Kirk (1991) has observed that the transport of 86Rb in the lamprey erythrocytes was definitely smaller than that of 43K when the incubation medium contained 4 mmol/1 K + However, in this study no detailed experiments have been done with replacing external K + with R b + to ascertain the t iue K + / R b + selectivity for the potassium channels in the lamprey erythrocytes. Therefore, it is of interest to define more clearly the discrimination between K + and R b + t ransport via the potassium channels in the lamprey erythrocyte membrane which is a convenient model for investigating ion transport mechanisms.

The present study was undertaken to examine the extent to which R b + would substitute foi K + in the transport pathways through the lamprey erythrocyte1 mem­brane. We combined two approaches to study K + and R b + transfer across the cell membrane. First, the rates of Ba'2+- and ouabain-sensitive 8eRb uptake were com­pared at the presence of 4 mmol/1 KC1 or 4 mmol/1 RbCl in the media. Secondly, the rate of Ba2+-.sensitivc influx of the stable isotope 41K into the red cells was stud­ied in media containing 4 inmol/1 41KC1. The data presented in this paper indicate that the potassium channels are highly selective for K + over Rb+. In contiast there is a relatively small discrimination ability (~20%) between K + and R b + t ransport via the Na-K-pump.

K and Rb Transport m Lamprey Ervthrocvtes 2 7 5

Mater ia l s a n d M e t h o d s

Animals

Experiments weie earned out on nvei lampreys (Lampetra fluviatihs) weighing 50 80 g The animals weie kept foi 4 months (November Febiuary) m a q u a n a with tap watei at 2-4°C undei continuous aeration The water m the a q u a n a was periodically íeplaced with fiesh dechloimated tap water

Preparation of alls

Blood was diawn aftei íapid decapitation in a tube containing hepaiinized saline Red cells weie sepaiated by centiifugation (2700x g for 5 mm) Aftei aspnat ion of saline and the buffy coat the eiythrocytes weie washed thiee times in a s tandaid saline containing 145 mmol/1 NaCl and 10 mmol/1 Tns-HCl (pH 7 4 at 20 °C) Cell suspensions in saline (hematocrit 30 40%) were stoied at loom tempera ture foi no moie than 10 mm befoie expenments

Miasnument of *6Rb and 41A uptake

K + and R b + influxes were measuied using íachoactive 86Rb and stable isotope 11K as t iaceis \11 expeiiments weie peifoimed at loom tempera tu ie (18 20°C) Incubation media weie piepaied b} adding to the s tandaid salmc of 10 mmol/1 glucose and 140 mmol/1 KC1, RbCl oi 4 1 kCl solutions to a final concentiation of 4 mmol/1 Flux media also contained 1 mmol/1 BaCb oi 0 1 mmol/1 ouabain as lequiicd The cell suspension was added to the incubation media to a final hematociit of 2 3% Then, 86Rb (0 01 mCi/ml) was added to the media containing KC1 and RbCl at zeio time At \ a n o u s time m t e n a l s 1 ml ahquots of well mixed suspension weu íeinoved and injected into 10 ml ice-cold s tandaid saline Aftei centiifugation (2700x g foi 1 nun at 4°C) a small amount of the supernatant was taken foi the d e t a initiation of íadioac tivity The led cells weie washed twice with the same ice cold saline Cell pellets weie lysed in distilled watei the radioactivity of the media and lysates was deteimined using a gamma-countei As shown m this study 8 eRb is an inaccurate tracei foi the measuiement of K + t ranspoi t through the lampre\ e i>thioc}te membiane Theiefoie, 8 eRb uptake was calculated as follows Ju/ ~ ^RBC / 4 \ / whcie \RBC I S the íadioactivitj of 1 ml packed cells and AM IS the íadioactivitj of 1 ml medium As 8 eRb uptake was linear up to 60 mm (Fig 1) the la te coefficients foi 8eRb influx (h" 1 ) weie calculated from 60 mm 89Rb uptake At the same concentiation of K + and R b + in media, the la te coefficients íeflect ion influxes (mmol/1 cells/h)

In the expeiiments with 41KC1, the 4 1K/3 qK isotope íat io was measuied in washed eiythiocytes using a mass spectrometei with the triple filament ion souice (Fleishman et al 1992) In brief, 20 30 ft\ of washed eiythrocytes weie lysed in 5 ml distilled watei and about 3 //l of lysates was deposited on one evapoiator of the ion

276 Guse\ et al

souice The isotope composition of endogenous (natuial) potassium corresponds to a u k / i 9 K ratio of 0 072 The mtiacellulai K"1" concentiation m the washed led cells was deteimmed using a Flapho-40 flame photometei (Jena, Geimanv) As could be shown m oui expeiiments the concentiation of mtiacellulai K + lemained constant at am incubation penod The K + concentiation, measuied by flame pho tometn (C'K) IS a sum ot d i K and 0 > K OI CR = C U R 4- C i % / i , wheie / is the Ci^/Cw^

íatio Thus the concentiation of 4 I K in the led cells can be calculated as follows CM K = Ck ' / ( I + i ^ At / e io time the concentiation of endogenous 41K was equal to C'K 0 072/(1 + 0 072) U K uptake In the lampiey eivthiocytes was calculated as the1 diffeienrt between 41K concentiation at time t and the endogenous content of UK The "K uptake was expiessed as iiimol pel liter packed cells The íate coefficient foi l lK uptake was calculated fiom the initial lmeai n K uptake divided In n I \ concentiation in the medium and the incubation time

Reagents

All chemicals used weie of analytical giade Ouabain was obtained fiom Sigma (St

Louis MO USA) Ouabain was dissolved in a flux medium to make a 10 mmol/1

stock solution MiRb and UKC1 (96% 41K. 4% vyK) weie obtained fiom ISOTOP

(St Pete isbmg, Russia)

Statistics

All icsults aie gnen as the mean value ± standaid e n o i (S E ) Statistical signifi­

cance was deteimined by the Student 's f-test foi paned obseivations

R e s u l t s

Timt covis< of*bRb uptake in K+- and Rb+ -media

The fust seiies of expeiiments was undeitaken to compare 8eRb uptake b> the

lampiey led cells incubated m media containing 4 mmol/1 K + oi 4 mmol/1 R b +

As can be seen m Fig 1.4, the íate of 8 eRb uptake was significantly gieatei in

K + medium than in Rb+- i i iedium In both media the 8 eRb uptake by lampiey

eiythiocvtes was appoximately hneai up to 60 mm of incubation The calculated

ía te coefficients foi 86Rb uptake weie 1 66 ± 0 16 h" 1 in K+- and 0 86 ± 0 06

h " 1 m Rb + -media (P < 0 001) Addition of potassium channel blockei B a 2 + (1

mmol/1) to the medium caused a íeduction in the ía te of 8 eRb uptake by the

lampiey led cells (Fig IB) The mhibitoiy effect of B a 2 + was two times gieatei in

K+-mediuni as compaied to Rb + -medmm In the piesence of 1 mmol/1 B a 2 + , the

uptake of 8(iRb was lineal ovei 60 mm. and the íate coefficients weie 0 7 2 ± 0 04 h _ 1

and 0 60 ± 0 03 h " 1 in K + - and Rb + -media , respectively The diffeience in B a 2 + -

msensitive components of 8 eRb uptake between both media was lelatively small but

K and T^b Transport in Lamprey Erythrocytes 277

20 -i

E = 15

•o E

E Q. O

ffl U

E Q.

© •X s Q. 3

QĹ CO

•- 4 mmol/l KCI in medium

O- 4 mmol/l RbCl in medium

*

Incubation time (min)

Figure 1. Time course of 8 6Rb ujptake by lamprey erythrocytes incubated in media con­taining 4 mmol/1 K + or 4 mmol/l R b + without (.4) and with 1 mmol/1 B a 2 + (B) Red cells were premcubated for 5 mm in the respective media and 8 6Rb was added at time zero T{b uptake was measured as described in Materials and Methods 8 6Rb uptake is expressed ab the ratio of radioactivity (cpm) of 1 ml packed cells to radioactivity (cpm) of 1 nil medium The straight lines were fitted using linear regression analysis Data are means ± S E for 6 separate experiments * P < 0 05 as compared with R b + medium (paired f-test)

statistically significant (P < 0 01, paned r-test) Thus, replacement of R+ with Rb+ in the incubation medium resulted in considerable decrease m 8 eRb uptake by the lamprey erythrocytes This deciement in 86Rb uptake m Rb+-medmm was mainly accounted for by inhibition of the Ba2+-sensitive component of 8 8Rb transport across potassium channels in the lamprey erythrocyte membrane (Kirk 1991, Gusev et al 1992)

278 Gusev et al.

Total, Ba"+- and ouabain-sensitive mRb uptake

In the second set of experiments, 8 eRb uptake was measured in the lamprey ery­throcytes incubated for 60 min in s tandard saline containing 4 mmol/1 KC1 or 4 mmol/1 RbCl. Effects of both B a 2 + and ouabain on ion transport were exam­ined in the experiments. The data summarized in Table 1 illustrate that the total and Ba2 •"-insensitive 80Rb uptake in the presence of K + were significantly greater than those in the presence of R b + . A marked decrease in the Ba2 f-sensitive 8bRb influx was observed when K + in incubation medium was replaced with R b + (Ta­ble 1). These data are m good agreement with the results of a pievious series of experiments (Fig. 1). Addition of 0.1 mmol/1 ouabain to the incubation medium in the presence of 1 mmol/1 Ba 2 + led to a significant reduction in 86Rb uptake. The ouabain-sensitive influx of 80Rb was only slightly higher in K + -medium than m Rb + -mediuni , the difference however being significant using paired statistical anal­ysis. There was no statistical difference between the residual, (Ba 2 + plus ouabain)-resistant, components of 86Rb uptake by the lamprey red cells in both media (Ta­ble 1). These data clearly indicate that Ba2 +-sensit ive potassium channels markedly discriminate between K + ions and R b + ions.

Table 1. Rb influx into lamprey erythrocytes incubated in K - and lib -medium

Rate coefficient of 80Rb influx (h _ 1) Medium

Control Ba2+ Ouabain + Ba~+ Ba~+-sensitive Ouabain-sensitive

KC'l 148 ± 0 10 0.70 ±0.05 0.082 ±0.012 0 78 ± 0.10 0.62 ± 0.05 (n) (13) (13) (6) (13) (6)

RbCl 0.82 ± 0.05** 0.59 ± 0 02** 0.068 ± 0.006 0.23 ± 0 04** 0 54 ± 0.05* (") (13) (13) (6) (13) (6)

The erythrocytes were premcubated for 30 mm in media containing (mmol/1): 140 NaCl; 1 CaCl2; 10 Tris-HOl; 10 glucose and 4 KC1 or RbCl (pH 7.1 at 20°C) without or with 1 mmol/1 Ba and Ba"+ plus ouabain. Then, Tib was added and its uptake was measured over 60 min The data represent mean ± S E for 6-13 independent experiments. * P < 0.05; ** P < 0.01 as compared with medium containing 4 mmol/1 KC'l (paired r-test).

41A" uptake by lamprey erythrocytes

In order to assess the true transport rate of K + ions across the lamprey erythrocyte

membrane, the uptake of stable isotope 41K by the red cells was studied. Lamprey

erythrocytes were incubated in a medium containing 4 mmol/1 41KC1, and 41K ac­

cumulation in the red cells was determined using mass spectrometry (see Materials

K and ^ ib Transport in Lamprey Erythrocytes 279

15-i

Ô10 E E

S Q. 3

5 -

0-*

B

10 20 30 40 50 60 70 Time (min)

8 16 Time (min)

24

Figure 2. Time dependence of 41R uptake by lamprey erythrocytes Red cells were prem cubated for 5 mm in a medium containing (mmol/l) 144 NaCl 1 CaC'b 10 Tris-HCl 10 glucose (pH 7 4 at 20°C) Then at time zero 140 mmol/1 41RC1 solution was added to a final concentration of 4 mmol/1 Samples of the suspension were taken at various time intervals over 65 mm (A) and a 24 mm period (B) Data points were fitted to single exponential function by a computer program [A) The straight line (B) has been drawn according to linear regression Values are mean ± S F of 3 (A) and 6 (B) separate experiments

and Methods) In the fiist series of experiments, the uptake of 41K was measuied m cells incubated foi 25, 45, and 65 mm As can be seen in Fig 2A, 41K rapidly accumulated m the lamprey red cells within the first 25 mm and the rate of 41K uptake decreased during the following 40 mm of cell incubation Thus, a non lineal time dependency was observed during the 65 mm period of cell incubation There­fore additional experiments were performed to assess the initial rate of 41K uptake by measuring 41K accumulation in cells incubated for 8, 16, and 24 mm Fig 2B cleaily lllustiates that 41K uptake was linear over the 24 mm period The rate co efficient of 41K uptake m lamprey eiythrocytes was 8 1 ± 0 5 h""1 This value did not differ from the rate coefficient of 41K uptake (7 0 ± 1 1 h"1) calculated from the initial slope of the cuive m the first series of experiments (Fig 2A) Blockade of potassium channels by 1 mmol/1 Ba2+ was associated with a significant reduc­tion in the rate of 41K uptake by lampiey erythrocytes The uptake of 41K in the piesence of Ba2 + was lineal ovei 120 mm of cell incubation (1 7 ± 0 4 for 65 mm

280 Gusev et al

and 3 2 ± 0 5 mmol/1 cells foi 120 min) and the rate coefficient foi K + up take was 0 39 ± 0 11 h l (Fíg 2 4) The mean intiatelluiar concentiation of K + measuied bv flame pho tomenv was 56 9 ± 6 1 and 75 7 ± 4 5 mmol / l cells foi the h is t and tht second sc nes of expeiiments lespectively

D i s c u s s i o n

The lesults of the pieseut study aie consistent with the eailiei íepoi t of Knk (1991) and piovide iurthc i evidence foi a high selectivity of the potassium c hannels foi K + ovei R b + in the lampiev eiythrocyte membrane Our comparative data on the rate of 8 6Rb and UK influxes into the lamprey led cells via the B a 2 +

sensitive potassium channels are summanzed in Table 2 Theic is good quant i ta t ive agicement between the results obtained heie with 41K and those obtained pieviously with J i K (Knk 1991) \t external K + concentiation of 4 mmol/1 the channel has a 9 fold preference foi 11K ovei 86Rb and an 8 fold pieference for 4 3K ovei 8eRb at external K + concentration of 5 mmol/1 In addition we could show tha t 8 eRb mflux into the lampiey led cells via potassium channels was significantly dee leased aftei leplacement of external K + with R b + suggesting that R b + mteiae ts with the channel and behaves as a paitial blockei It is evident that at the same K +

and R b + concentiations m the medium the tiacer permeability of the potassium channels to Rb+ must have only been about 3% of the permeability to I \ + The possibility cannot be niled out that the effect of R b + on the potassium channels may be clue to changes m membrane potential In pieccdmg studies ( K n k 1991 Guse\ et al 1992) K + movement across the lamprey erythrocyte membiane was examined using 8 fRb as a tracei and in the piesence of 1 4 mmol/1 KC1 m the media Undei these conditions the B a 2 + mhibitable uptake of 8BRb only leflects about 10% of the t ine peimeabihty of the potassium channels to K + Recently an mwaidly rectifying potassium curient has been identified in lamprey erythrocytes using whole cell patch clamp technique (Virkki and Nikinmaa 1995) The authois

T a b l e 2 . R a t e coefficients for B a 2 + sensitive influxes of 41K and 86Rb into l amprey ery th ro ty te s ( h _ 1 )

Medium

86Rb influx

RbCl RC1

0 23 ± 0 04 0 78 ± 0 10*

41K influx

41KG1

7 28 ± 0 5**

The red cells were incubated in s tandard media containing 4 mmol/1 RbCl KC1 or 41KC1 The values are mean ± S E of 13 (86R,b influx) and 6 (41K influx) determinat ions * P < 0 001 as compared with RbCl medium, ** P < 0 001 as compared with 8 6Rb influx

K and Tib Transport in Lamprey Erythrocytes 2 8 1

have found the K + channels to exhibit a much greater permeability to K + over R b + as indicated by the reversal potential for channel currents.

The use of potassium radionuclides (42K and i3K) is limited due to their cost and a short half-life (about l l h and 23h, respectively). There have been numerous studies to demonstrate the possibility of using 8eRb as an accurate quantitative marker of K + transport in a variety of cells and tissues (Beauge and Ortiz 1970: Cheval et al. 1991; Zhou and Wingo 1992). Therefore. 86Rb is routinely employed as a substitute for K + to assay ion transport mechanisms in many types of cells for reasons of convenience (half-life of about 18 days) and cost. However, there is some evidence that R b T may not be a perfect substitute for K + m all cases Numerous patch-clamp and tracer studies have shown that some types of potassium channels do not discriminate between K + and R b + ions, but other potassium channels have a considerably lower conductance to R b + (for review see Hille 1973; Lattore and Miller 1983; Van Driessche and Zeiske 1985: Nichols and Lederer 1991). It has been known for a long time that the inward rectifier is slightly permeable to R b + in a number of tissues such as frog skeletal muscle (Adrian 1964; Spalding et al. 1982). macrophages (Gallin and Livengood 1981), bovine artery endothelium (Silver et al. 1994) and guinea-pig ventricular cells (Matsuda 1996). The conductance of the inward rectifier channel for Rb+ was extremely low. with a R b + / K + ratio of 0.03-0.04. while the íelative Rb+ permeability was only 0.3-0.5. These studies have suggested that R b + inteiferes with the movement of K + through the channels and alteis its kinetics. Similarly, potassium channels of some epithelial tissues have also been shown to exhibit a low conductance to R b + but their permeability for R b + is nearly the same as for K + (Gallacher et al. 1984; Gogelein et al. 1987; Bleich et al. 1990). Ca 2 +-act ivated potassium channels of lymphocvt.es (Gnssmer et al. 1993) and human eiythrocytes (Christophersen 1991) display relatively small discrimination with respect to both the R b + / K + permeability (0.55-0.96) and R b + / K + conductance (0.36-0.60).

It is generally accepted that membrane transport carriers do not markedly discriminate between K + and R b + ions. It is commonly admitted that R b + can substi tute for K + in Na-K-ATPase studies. In a variety of cells and tissues 42K and 86Rb yielded similar results when used as tracers to assay the activities of the Na-K-pump and cotransporters (Castronovo et al. 1968; Bernstein and Israel 1970; Schafer and Troutman 1986). However, the da ta of the present study indi­cate that the ouabain-sensitive 8 eRb influx in the lamprey red cells somewhat (by ~ 16%i) decreases after replacing 4 mmol/1 KC1 in the media with 4 mmol/1 RbCl (Table 1). The results are consistent with the study of Warden et al. (1989) who have demonstrated a reduced activity of the Na,K-ATPase in several segments of rabbit nephron in the presence of 5 mmol/1 R b + (~ 20%i) compared with 5 mmol/1 K + . Cheval and Doucet (1990) used the same approach to study the selectivity of ouabain-sensitive 86Rb uptake in single segments of rat nephron by incubating

282 Gusev et al

tubule piepaiations with 5 mmol/ l R b + 01 K + They have found that the active t ranspoi t ía te elicited by K + was 12 70% higher than that m the presence of R b +

In anothci study (Soltoff and Mandel 1984), the ouabain-sensitive ía te of icspira-tion m a suspension of lenal pioximal tubules was íeduced to 83%) in the piesence of 5 mmol/1 R b + as compaied with 5 mmol/1 K + Thus the magnitude of the active t ianspoi t will be nuclei estimated when 86Rb is used as a tracei and R b + as a substi tute foi k + m external medium When the incubation medium contains K+ ions, 8<3Rb can be consideied to be an accuiatc maikei foi measuiing the true activity of the Na.K-pump m the lampiey eiytlnocytes and othei cell types The K + and R b + influxes m the lampiey led cells measuied using 8GRb as a t iacei m the piesence of B a 2 + plus ouabain weie indistinguishable (Table 1), implying that the (Ba 2 + + ouabain)-iesistant pathway foi the tiacei movement cloes not discnminate between K+ and R b +

In summaiy the pieseut studv cleaily demonstiates that the potassium chan­nels in the lampiey e n t h i o c y t e membiane have a stiong pieference foi K + ovei R b + as shown by the compaiison of the Ba 2 +-sensi t i \e influxes between 41K and 8bRb The 8eRb influx via the channel decieased when all external K + was ieplaced with R b + , suggesting that R b + may paitiallv inhibit the channel conductance In its selectivity piopeities the potassium channel m the lamprey erythrocyte mem­biane lesembles a numbei of potassium channels so fai studied m vanous cell types Although theie weie quan t i t a tne diffeiences between K + and R b + t i anspoi t m the lampiey eiythiocvtes we found no evidence suggesting that these ions aie trans­p o s e d by diffeient mechanisms It should be emphasized that when 8 eRb is used as a maikei foi studv of the K + t i anspoi t pathways, it is pieferable to avoid substi tu­tion of R b + foi K + m incubation media The piesent study shows the possibility of using stable UK isotope to investigate ion transpoit mechanisms acioss biological membianes

Acknowledgements. The present study was made possible m part bv giants No NVO000 fiom the International Science Foundation No NVO 500 from the Inter­national Science Foundation and the Russian Government

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K and T ib Transport in Lampiey Erythrocytes 283

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Final version accepted September 16, 1997