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Ba2+-sensitive K+ channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule
Volume 243, number 2, 173-176
FEB 06719
January 1989
Ba 2 +-sensitive K + channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule Christian Jacobsen, Hans Roigaard-Petersen and M.I. Sheikh Institute o f Medical Biochemistry, University o f Aarhus, DK-8000 Aarhus C, Denmark Received 16 N o v e m b e r 1988 This paper describes properties of 8ORb+ fluxes through a novel K + channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate > SO~4- > CI-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+> Rb+> Li+> Na+= choline+ . K + channel; Ba"+ sensitivity; Membrane vesicle; (Pars convoluta)
1. I N T R O D U C T I O N
2. M A T E R I A L S
Ionic channels constitute substantial routes for t h e m o v e m e n t o f i o n s a c r o s s e p i t h e l i a l cell m e m b r a n e s . T h e l u m i n a l m e m b r a n e is e s s e n t i a l l y c o n d u c t i v e f o r N a +, w h i l e t h e b a s o l a t e r a l m e m b r a n e is g e n e r a l l y c o n d u c t i v e f o r K +. H o w e v e r , in c e r t a i n e p i t h e l i a s u c h as m a m m a l i a n c o l o n [ 1 - 3 ] a n d p r o x i m a l t u b u l e [ 4 - 7 ] , t h e r e is c o n s i d e r a b l e e v i d e n c e in t h e l i t e r a t u r e f o r t h e e x i s t e n c e o f K + c h a n n e l s b o t h in l u m i n a l a n d b a s o l a t e r a l m e m b r a n e s . B y t h e use o f h i g h l y p u r i f i e d l u m i n a l m e m b r a n e vesicles f r o m t w o d i s t i n c t s e g m e n t s o f nephron we have recently been able to demonstrate the presence of an amiloride-sensitive Na + channel in p a r s r e c t a o f r a b b i t p r o x i m a l t u b u l e [8]. U s i n g t h e s a m e e x p e r i m e n t a l p r o c e d u r e as d e s c r i b e d in [8,9], we n o w d e s c r i b e s o m e c h a r a c t e r i s t i c s o f a n o v e l B a 2 + - s e n s i t i v e K + c h a n n e l in l u m i n a l - m e m b r a n e vesicles i s o l a t e d f r o m p a r s c o n v o l u t a o f rabbit proximal tubule.
2.1. Preparation of luminal-membrane vesicles Luminal-membrane vesicles were isolated from pars convoluta (outer cortex) of the proximal tubule of rabbit kidney according to [10] and the method is only briefly described here. Outer cortical tissue was obtained by taking slices <0.3 mm thick from the surface of the kidney containing pars convoluta. The tissue was homogenized and vesicles were prepared by differential centrifugation and by Mg 2+ precipitation analogous with Ca 2+ precipitation as described in [10]. The final pellet was washed for 20 min by a solution consisting of 3.75 mM EDTA, 298 mM sucrose and 27 mM Hepes/Tris buffer, pH 7.4, and resuspended in the same medium minus EDTA. The purity of the membrane vesicle preparations was examined by electron microscopy [11] and by measuring specific activities of various enzyme markers as in [12]. The amount of protein was determined as described by Lowry et al. [13] and as modified by Peterson [14] and with serum albumin as a standard. All solutions used in this study were sterilized before use.
Correspondence address: M.I. Sheikh, Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
AND METHODS
2.2. Assay o f Rb + transport Influx of SeRb+ was measured in K + (55 mM) loaded vesicles analogous to flux measurement procedures reported previously [8,9]. Dowex AG50W-X8 columns were prepared in 1 ml tuberculin syringes. The resin was washed with 1 M Tris to bring it into the Tris form, followed by 5-6 vols of 298 mM sucrose, 27 mM Hepes/Tris, pH 7.4, containing 1 mg/ml bovine serum albumin. In the standard loading procedure, vesicles, 500/~1 (approx. 10 mg/ml), were suspended in a buffer of 185 mM sucrose, 3.75 mM EDTA, 1 mM ouabain, 55 mM KCI (or
Published by Elsevier Science Publishers B.V. (Biomedical Division) 00145793/89/$3.50 © 1989 Federation of European Biochemical Societies
I73
FEBS LETTERS
Volume 243, number 2
55 mM RbCI, NaCI, LiCI, choline chloride, K-gluconate or 27.5 mM K:SO4), 27 mM Hepes/Tris, pH 7.4, for 1.5 h on ice. The vesicles were collected by centrifugation (25000 x g for 30 min at 2°C) and resuspended in the same buffer, to a protein concentration of approx. 15 mg/ml. Portions of 300/~l were applied on separate columns. The individual eluates were diluted to 1.0 ml with 298 mM sucrose, 27 mM Hepes/Tris, pH 7.4, buffer, and 75/zl were withdrawn for protein determination. In references BaC12 and amiloride were added in final concentrations of 10 mM and 350/~M, respectively. The experiment was initiated by adding 86Rb+ (210 kBq; 25 nmol) to the incubation medium. Samples of 140/zl were taken out at various time intervals (1, 3, 5, 7, 10, 15 and 60 rain) and uptake was stopped by passing the samples through the Dowex columns by centrifugation. The eluates were diluted to 500/d and used for radioactivity determinations.
3. RESULTS AND DISCUSSION
3.1. Effects o f external Ba 2+ and ouabain Fig.1 presents the intravesicular 86Rb+ content as a function o f time in the absence and presence o f 10 mM Ba 2+ in KCI (55 mM) loaded luminalmembrane vesicles from pars convoluta. With or without external Ba + the vesicles continued to accumulate 86Rb+ throughout the 15 min of
January 1989
measurements. It is seen that the external Ba 2+ reduced the magnitude of the 86Rb+ uptake throughout the entire period o f measurement, suggesting that part of the influx proceeded through aqueous channels. Barium is widely used as a blocker of K + channels in epithelial cells (review [15]). It is likely that part of the Ba2+-insensitive S6Rb+ uptake reflects passive Rb+/K + exchange through the Na+,K+-exchange pump [16,17]. In a separate experiment, addition of 1 mM ouabain slightly reduced (approx. 10°70) the Ba2+-insensi tive uptake. In the present communication, we have been concerned specifically with transport through aqueous channels. Therefore, in order to reduce the level o f the background Ba2+-insensitive S6Rb+ flux, 1 mM ouabain has been included in the incubation medium in all of the experiments described below. We have also examined the effect of amiloride (350/~M) on the uptake of S6Rb+. No significant inhibitory effect of this diuretic on the uptake of isotope was observed (not shown), suggesting that influx of S6Rb+ occurred via a K + channel. Incidentally, these findings rule out the possibility of appreciable contamination of pars convoluta vesicles with the membrane fragments from pars recta, in which Na + channels are shown to be present [8].
12o
_
20 L 9/~/~
/
e
~
~ + lo mM BaCI2
.L
I
I
I
5
10
15
Time (min) Fig. 1. Time course of S6Rb+ uptake into luminal-membrane vesicles from pars convoluta of rabbit proximal tubules. One ml of vesicles (protein conc. 5 mg/ml), Ki+ = 55 mM, K+ut = 0 raM, was incubated at room temperature with 86Rb+ (24/~M) in the absence (curve 1, 0), and presence of 10 mM BaCI2 (curve 2, o). Means _+ SE of four independent preparations are shown.
174
3.2. Cation selectivity Whether or not the 86Rb+ transport is conductive is further explored by examining the effect of loading vesicle with various alkali cations on the influx of 86Rb+. In these experiments, the vesicles were loaded with different cations and the ability of opposing gradients of these cations to sustain Ba2+-sensitive 86Rb+ accumulation was examined. Fig.2A shows that all the cations tested in this paper reduced the uptake o f S6Rb+ to various extents as compared to the influx o f S6Rb+ by K+-loaded vesicles (control). Thus, these measurements indicate that the radioactive tracer movement in the presence o f ouabain proceeds largely through a conductive pathway. The observed ranking order K + > Rb + > Li + > Na + = choline + is a reflection of the decreasing permeability of the membrane vesicle of interest to these cations. The results could mean that the K + channels themselves show this order of selectivities, but it is more probable that K + channels show a higher selectivity to K + than to Na +, and in addition the membrane
Volume 243, number 2
FEBS LETTERS
vesicles are s o m e w h a t selective for all c a t i o n s c o m p a r e d to a n i o n s .
A
A
I00
e" O U
80
I
60 + .g=
~g
_m
40
i
20
e,0~ m o e=
g om
z
140
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o¢ J
January 1989
120 I00
3.3. Electrical nature F i g . 2 B p r o v i d e s direct evidence t h a t the Ba2+-inhibited S6Rb+ a c c u m u l a t i o n is d r i v e n b y a n electrical p o t e n t i a l . I n these e x p e r i m e n t s t h e l u m i n a l - m e m b r a n e vesicles f r o m p a r s c o n v o l u t a were l o a d e d with either 55 m M KC1, 27.5 m M K2SO4 o r 55 m M K + - g l u c o n a t e , a n d t h e u p t a k e o f S6Rb+ was s t u d i e d in the a b s e n c e a n d presence o f Ba 2+. It is a p p a r e n t f r o m fig.2B t h a t the rate o f i s o t o p e u p t a k e was highest for g l u c o n a t e , int e r m e d i a t e with sulfate m e d i u m a n d lowest with c h l o r i d e . T h e results c a n be e x p l a i n e d as follows. It is k n o w n t h a t t h e passive p e r m e a b i l i t y o f the vesicle m e m b r a n e for these a n i o n s is as follows: g l u c o n a t e < SO42- < C l - [18,19]. T h e c a t i o n d i f f u sion p o t e n t i a l s h o u l d t h e r e f o r e be p r o g r e s s i v e l y s h o r t - c i r c u i t e d b y a n i o n s o f increasing p e r m e a b i l i ty a n d the r a t e o f the S6Rb+ u p t a k e s h o u l d f o l l o w t h e o b s e r v e d o r d e r g l u c o n a t e > SO 2- > C l - . I n c o n c l u s i o n , the results p r e s e n t e d in this c o m m u n i c a t i o n r e v e a l e d the presence o f a novel Ba2+-sensitive K + c h a n n e l in l u m i n a l - m e m b r a n e vesicles p r e p a r e d f r o m p a r s c o n v o l u t a o f r a b b i t p r o x i m a l tubule. F u r t h e r m o r e , o u r p r e l i m i n a r y o b s e r v a t i o n indicates t h a t the K + c h a n n e l activity is r e g u l a t e d b y C a 2+. T h e s e e x p e r i m e n t s a r e in p r o gress in o u r l a b o r a t o r y .
"6
Acknowledgements: This study was supported in part by the
8O --,
60
+
G
Danish Medical Research Council, The Danish Biotechnology Programme, Aarhus Universitets Forskningsfond, P. Carl Petersens Fond, Fonden til La~gevidenskabens Fremme, Kong Christian Den Tiendes Fond, Fogh-Nielsens Legat and NOVO Fond.
t~
4O
_¢ t~ ¢-
20
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Fig.2. Time course of S6Rb+ uptake into luminal-membrane vesicles from pars convoluta. (A) Vesicles loaded with either 55 mM KCi, 55 mM RbC1, 55 mM LiCI, 55 mM NaCI or 55 mM choline chloride. (B) Vesicles loaded with either 55 mM KCI, 27.5 mM KzSO4 or 55 mM K-gluconate and measured in absence and presence of 10 mM BaCI2. The experimental protocol was as described in legend to fig. 1.
REFERENCES [1] Wills, N.K., Zeiske, W. and Van Driessche, W. (1982) J. Membrane Biol. 69, 187-197. [2] Wills, N.K., Alles, W.P., Sandier, G.I. and Binder, H.J. (1984) Am. J. Physiol. 247, 749-757. [3] Dawson, D.C., Van Driessche, W. and Helman, S.I. (1985) Fed. Proc. 44, 1745. [4] G6gelein, H. and Greger, R. (1984) Pflfigers Arch. 401, 424-426. [5] Parent, L., Cardinal, J. and Sauve, R. (1986) Biophys. J. 49, 159a. [6] G6gelein, H. and Greger, R. (1987) Pfliigers Arch. 410, 288-295. 175
Volume 243, number 2
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[7] Parent, L., Cardinal, J. and Sauve, R. (1988) Am. J. Physiol. 254, F105-F113. [8] Jacobsen, C., R~igaard-Petersen, H. and Sheikh, M.I. (1988) FEBS Lett. 236, 95-99. [9] Garty, H., Rudy, B. and Karlish, J.D. (1983) J. Biol. Chem. 258, 13094-13099. [10] Sheikh, M.I. and Moller, J.V. (1987) in: Biochemical Toxicology: A Practical Approach (Snell, K. and Mullock, B. eds) Ch.7, pp.153-182, IRL Press, Oxford, England. [11] Kragh-Hansen, U., R~igaard-Petersen, H. and Sheikh, M.I. (1985) Am. J. Physiol. 249, F704-F712. [12] Sheikh, M.I., Kragh-Hansen, U., Jorgensen, K.E. and Roigaard-Pctersen, H. (1982) Biochem. J. 208, 377-382.
176
January 1989
[13] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [14] Peterson, G.L. (1977) Anal. Biochem. 83, 346-356. [15] Wills, N.K. and Zweifach, A. (1987) Biochim. Biophys. Acta 906, 1-31. [16] Karlish, S.J.D. and Stein, W.D. (1982) J. Physiol. (London) 328, 295-316. [17] Kenney, L.J. and Kaplan, J.H. (1985) in: The Sodium Pump (Glynn, I.H. and Ellory, J.C. eds) pp.535-539, The Company of Biologists Ltd, Cambridge. [18] Fr6mter, E., Muller, C.W. and Wick, T. (1971) in: Electrophysiology of Epithelial Cells (Giebisch, G. ed.) pp.119-146, F.K. Schattauer, Stuttgart. [19] Schneider, E.G. and Sacktor, B. (1980) J. Biol. Chem. 255, 7645-7649.
FEB 06719
January 1989
Ba 2 +-sensitive K + channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule Christian Jacobsen, Hans Roigaard-Petersen and M.I. Sheikh Institute o f Medical Biochemistry, University o f Aarhus, DK-8000 Aarhus C, Denmark Received 16 N o v e m b e r 1988 This paper describes properties of 8ORb+ fluxes through a novel K + channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate > SO~4- > CI-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+> Rb+> Li+> Na+= choline+ . K + channel; Ba"+ sensitivity; Membrane vesicle; (Pars convoluta)
1. I N T R O D U C T I O N
2. M A T E R I A L S
Ionic channels constitute substantial routes for t h e m o v e m e n t o f i o n s a c r o s s e p i t h e l i a l cell m e m b r a n e s . T h e l u m i n a l m e m b r a n e is e s s e n t i a l l y c o n d u c t i v e f o r N a +, w h i l e t h e b a s o l a t e r a l m e m b r a n e is g e n e r a l l y c o n d u c t i v e f o r K +. H o w e v e r , in c e r t a i n e p i t h e l i a s u c h as m a m m a l i a n c o l o n [ 1 - 3 ] a n d p r o x i m a l t u b u l e [ 4 - 7 ] , t h e r e is c o n s i d e r a b l e e v i d e n c e in t h e l i t e r a t u r e f o r t h e e x i s t e n c e o f K + c h a n n e l s b o t h in l u m i n a l a n d b a s o l a t e r a l m e m b r a n e s . B y t h e use o f h i g h l y p u r i f i e d l u m i n a l m e m b r a n e vesicles f r o m t w o d i s t i n c t s e g m e n t s o f nephron we have recently been able to demonstrate the presence of an amiloride-sensitive Na + channel in p a r s r e c t a o f r a b b i t p r o x i m a l t u b u l e [8]. U s i n g t h e s a m e e x p e r i m e n t a l p r o c e d u r e as d e s c r i b e d in [8,9], we n o w d e s c r i b e s o m e c h a r a c t e r i s t i c s o f a n o v e l B a 2 + - s e n s i t i v e K + c h a n n e l in l u m i n a l - m e m b r a n e vesicles i s o l a t e d f r o m p a r s c o n v o l u t a o f rabbit proximal tubule.
2.1. Preparation of luminal-membrane vesicles Luminal-membrane vesicles were isolated from pars convoluta (outer cortex) of the proximal tubule of rabbit kidney according to [10] and the method is only briefly described here. Outer cortical tissue was obtained by taking slices <0.3 mm thick from the surface of the kidney containing pars convoluta. The tissue was homogenized and vesicles were prepared by differential centrifugation and by Mg 2+ precipitation analogous with Ca 2+ precipitation as described in [10]. The final pellet was washed for 20 min by a solution consisting of 3.75 mM EDTA, 298 mM sucrose and 27 mM Hepes/Tris buffer, pH 7.4, and resuspended in the same medium minus EDTA. The purity of the membrane vesicle preparations was examined by electron microscopy [11] and by measuring specific activities of various enzyme markers as in [12]. The amount of protein was determined as described by Lowry et al. [13] and as modified by Peterson [14] and with serum albumin as a standard. All solutions used in this study were sterilized before use.
Correspondence address: M.I. Sheikh, Institute of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark
AND METHODS
2.2. Assay o f Rb + transport Influx of SeRb+ was measured in K + (55 mM) loaded vesicles analogous to flux measurement procedures reported previously [8,9]. Dowex AG50W-X8 columns were prepared in 1 ml tuberculin syringes. The resin was washed with 1 M Tris to bring it into the Tris form, followed by 5-6 vols of 298 mM sucrose, 27 mM Hepes/Tris, pH 7.4, containing 1 mg/ml bovine serum albumin. In the standard loading procedure, vesicles, 500/~1 (approx. 10 mg/ml), were suspended in a buffer of 185 mM sucrose, 3.75 mM EDTA, 1 mM ouabain, 55 mM KCI (or
Published by Elsevier Science Publishers B.V. (Biomedical Division) 00145793/89/$3.50 © 1989 Federation of European Biochemical Societies
I73
FEBS LETTERS
Volume 243, number 2
55 mM RbCI, NaCI, LiCI, choline chloride, K-gluconate or 27.5 mM K:SO4), 27 mM Hepes/Tris, pH 7.4, for 1.5 h on ice. The vesicles were collected by centrifugation (25000 x g for 30 min at 2°C) and resuspended in the same buffer, to a protein concentration of approx. 15 mg/ml. Portions of 300/~l were applied on separate columns. The individual eluates were diluted to 1.0 ml with 298 mM sucrose, 27 mM Hepes/Tris, pH 7.4, buffer, and 75/zl were withdrawn for protein determination. In references BaC12 and amiloride were added in final concentrations of 10 mM and 350/~M, respectively. The experiment was initiated by adding 86Rb+ (210 kBq; 25 nmol) to the incubation medium. Samples of 140/zl were taken out at various time intervals (1, 3, 5, 7, 10, 15 and 60 rain) and uptake was stopped by passing the samples through the Dowex columns by centrifugation. The eluates were diluted to 500/d and used for radioactivity determinations.
3. RESULTS AND DISCUSSION
3.1. Effects o f external Ba 2+ and ouabain Fig.1 presents the intravesicular 86Rb+ content as a function o f time in the absence and presence o f 10 mM Ba 2+ in KCI (55 mM) loaded luminalmembrane vesicles from pars convoluta. With or without external Ba + the vesicles continued to accumulate 86Rb+ throughout the 15 min of
January 1989
measurements. It is seen that the external Ba 2+ reduced the magnitude of the 86Rb+ uptake throughout the entire period o f measurement, suggesting that part of the influx proceeded through aqueous channels. Barium is widely used as a blocker of K + channels in epithelial cells (review [15]). It is likely that part of the Ba2+-insensitive S6Rb+ uptake reflects passive Rb+/K + exchange through the Na+,K+-exchange pump [16,17]. In a separate experiment, addition of 1 mM ouabain slightly reduced (approx. 10°70) the Ba2+-insensi tive uptake. In the present communication, we have been concerned specifically with transport through aqueous channels. Therefore, in order to reduce the level o f the background Ba2+-insensitive S6Rb+ flux, 1 mM ouabain has been included in the incubation medium in all of the experiments described below. We have also examined the effect of amiloride (350/~M) on the uptake of S6Rb+. No significant inhibitory effect of this diuretic on the uptake of isotope was observed (not shown), suggesting that influx of S6Rb+ occurred via a K + channel. Incidentally, these findings rule out the possibility of appreciable contamination of pars convoluta vesicles with the membrane fragments from pars recta, in which Na + channels are shown to be present [8].
12o
_
20 L 9/~/~
/
e
~
~ + lo mM BaCI2
.L
I
I
I
5
10
15
Time (min) Fig. 1. Time course of S6Rb+ uptake into luminal-membrane vesicles from pars convoluta of rabbit proximal tubules. One ml of vesicles (protein conc. 5 mg/ml), Ki+ = 55 mM, K+ut = 0 raM, was incubated at room temperature with 86Rb+ (24/~M) in the absence (curve 1, 0), and presence of 10 mM BaCI2 (curve 2, o). Means _+ SE of four independent preparations are shown.
174
3.2. Cation selectivity Whether or not the 86Rb+ transport is conductive is further explored by examining the effect of loading vesicle with various alkali cations on the influx of 86Rb+. In these experiments, the vesicles were loaded with different cations and the ability of opposing gradients of these cations to sustain Ba2+-sensitive 86Rb+ accumulation was examined. Fig.2A shows that all the cations tested in this paper reduced the uptake o f S6Rb+ to various extents as compared to the influx o f S6Rb+ by K+-loaded vesicles (control). Thus, these measurements indicate that the radioactive tracer movement in the presence o f ouabain proceeds largely through a conductive pathway. The observed ranking order K + > Rb + > Li + > Na + = choline + is a reflection of the decreasing permeability of the membrane vesicle of interest to these cations. The results could mean that the K + channels themselves show this order of selectivities, but it is more probable that K + channels show a higher selectivity to K + than to Na +, and in addition the membrane
Volume 243, number 2
FEBS LETTERS
vesicles are s o m e w h a t selective for all c a t i o n s c o m p a r e d to a n i o n s .
A
A
I00
e" O U
80
I
60 + .g=
~g
_m
40
i
20
e,0~ m o e=
g om
z
140
A
o¢ J
January 1989
120 I00
3.3. Electrical nature F i g . 2 B p r o v i d e s direct evidence t h a t the Ba2+-inhibited S6Rb+ a c c u m u l a t i o n is d r i v e n b y a n electrical p o t e n t i a l . I n these e x p e r i m e n t s t h e l u m i n a l - m e m b r a n e vesicles f r o m p a r s c o n v o l u t a were l o a d e d with either 55 m M KC1, 27.5 m M K2SO4 o r 55 m M K + - g l u c o n a t e , a n d t h e u p t a k e o f S6Rb+ was s t u d i e d in the a b s e n c e a n d presence o f Ba 2+. It is a p p a r e n t f r o m fig.2B t h a t the rate o f i s o t o p e u p t a k e was highest for g l u c o n a t e , int e r m e d i a t e with sulfate m e d i u m a n d lowest with c h l o r i d e . T h e results c a n be e x p l a i n e d as follows. It is k n o w n t h a t t h e passive p e r m e a b i l i t y o f the vesicle m e m b r a n e for these a n i o n s is as follows: g l u c o n a t e < SO42- < C l - [18,19]. T h e c a t i o n d i f f u sion p o t e n t i a l s h o u l d t h e r e f o r e be p r o g r e s s i v e l y s h o r t - c i r c u i t e d b y a n i o n s o f increasing p e r m e a b i l i ty a n d the r a t e o f the S6Rb+ u p t a k e s h o u l d f o l l o w t h e o b s e r v e d o r d e r g l u c o n a t e > SO 2- > C l - . I n c o n c l u s i o n , the results p r e s e n t e d in this c o m m u n i c a t i o n r e v e a l e d the presence o f a novel Ba2+-sensitive K + c h a n n e l in l u m i n a l - m e m b r a n e vesicles p r e p a r e d f r o m p a r s c o n v o l u t a o f r a b b i t p r o x i m a l tubule. F u r t h e r m o r e , o u r p r e l i m i n a r y o b s e r v a t i o n indicates t h a t the K + c h a n n e l activity is r e g u l a t e d b y C a 2+. T h e s e e x p e r i m e n t s a r e in p r o gress in o u r l a b o r a t o r y .
"6
Acknowledgements: This study was supported in part by the
8O --,
60
+
G
Danish Medical Research Council, The Danish Biotechnology Programme, Aarhus Universitets Forskningsfond, P. Carl Petersens Fond, Fonden til La~gevidenskabens Fremme, Kong Christian Den Tiendes Fond, Fogh-Nielsens Legat and NOVO Fond.
t~
4O
_¢ t~ ¢-
20
C
Fig.2. Time course of S6Rb+ uptake into luminal-membrane vesicles from pars convoluta. (A) Vesicles loaded with either 55 mM KCi, 55 mM RbC1, 55 mM LiCI, 55 mM NaCI or 55 mM choline chloride. (B) Vesicles loaded with either 55 mM KCI, 27.5 mM KzSO4 or 55 mM K-gluconate and measured in absence and presence of 10 mM BaCI2. The experimental protocol was as described in legend to fig. 1.
REFERENCES [1] Wills, N.K., Zeiske, W. and Van Driessche, W. (1982) J. Membrane Biol. 69, 187-197. [2] Wills, N.K., Alles, W.P., Sandier, G.I. and Binder, H.J. (1984) Am. J. Physiol. 247, 749-757. [3] Dawson, D.C., Van Driessche, W. and Helman, S.I. (1985) Fed. Proc. 44, 1745. [4] G6gelein, H. and Greger, R. (1984) Pflfigers Arch. 401, 424-426. [5] Parent, L., Cardinal, J. and Sauve, R. (1986) Biophys. J. 49, 159a. [6] G6gelein, H. and Greger, R. (1987) Pfliigers Arch. 410, 288-295. 175
Volume 243, number 2
FEBS LETTERS
[7] Parent, L., Cardinal, J. and Sauve, R. (1988) Am. J. Physiol. 254, F105-F113. [8] Jacobsen, C., R~igaard-Petersen, H. and Sheikh, M.I. (1988) FEBS Lett. 236, 95-99. [9] Garty, H., Rudy, B. and Karlish, J.D. (1983) J. Biol. Chem. 258, 13094-13099. [10] Sheikh, M.I. and Moller, J.V. (1987) in: Biochemical Toxicology: A Practical Approach (Snell, K. and Mullock, B. eds) Ch.7, pp.153-182, IRL Press, Oxford, England. [11] Kragh-Hansen, U., R~igaard-Petersen, H. and Sheikh, M.I. (1985) Am. J. Physiol. 249, F704-F712. [12] Sheikh, M.I., Kragh-Hansen, U., Jorgensen, K.E. and Roigaard-Pctersen, H. (1982) Biochem. J. 208, 377-382.
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[13] Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. [14] Peterson, G.L. (1977) Anal. Biochem. 83, 346-356. [15] Wills, N.K. and Zweifach, A. (1987) Biochim. Biophys. Acta 906, 1-31. [16] Karlish, S.J.D. and Stein, W.D. (1982) J. Physiol. (London) 328, 295-316. [17] Kenney, L.J. and Kaplan, J.H. (1985) in: The Sodium Pump (Glynn, I.H. and Ellory, J.C. eds) pp.535-539, The Company of Biologists Ltd, Cambridge. [18] Fr6mter, E., Muller, C.W. and Wick, T. (1971) in: Electrophysiology of Epithelial Cells (Giebisch, G. ed.) pp.119-146, F.K. Schattauer, Stuttgart. [19] Schneider, E.G. and Sacktor, B. (1980) J. Biol. Chem. 255, 7645-7649.