Pflfigers Arch (1985) 405 (Suppl 1):S 143-S 146 Pflfigers Archly European Journal of i~ysio~y

�9 Springer-Verlag 1985

Cellular electrophysiology of potassium transport in the mammalian cortical collecting tubule*,** Bruce Koeppen i and Gerhard Giebiseh z

1 Departments of Medicine and Physiology, University of Connecticut Health Center, Farmington, CT 06032, USA 2 Department of Physiology, Yale University School of Medicine, New Haven, CT 06510, USA

Abstract . Electrophysiological studies were carried out on single perfused cortical and medullary collecting ducts to define the potassium and sodium transport properties of their apical and basolateral cell membranes. In addition, the effects of chronic mineralocorticoid hormone treatment on the mechanism of transport of potassium ions were evaluated. Studies included the measurement of transepithe- lial and cell potentials, and the resistance of individual cell membranes.

The apical cell membrane of principal cells of the cortical collecting duct is characterized by separate potassium and sodium conductances. The basolateral cell membrane has also a potassium conductance, whereas the intercellular shunt pathway is largely permeable to chloride ions. Stimu- lation of potassium secretion by mineralocorticoids is as- sociated with the following events: 1) Increased cell potassium uptake across the basolateral cell membrane due to stimulation of Na-K ATPase and a more favorable electri- cal driving force for passive entry, 2) facilitated exit of potassium from the cell to the tubule lumen by a more favorable electrochemical gradient (apical cell membrane depolarization) and 3) enhanced potassium secretion by in increase of the potassium conductance of the apical cell membrane.

Some properties of single potassium channels in the apical membrane of rabbit cortical collecting tubules are also described.

Key words: Collecting duct - Potassium transport - Minera locort i co id hormone

Since the introduction of the method of isolation and in vitro microperfusion of the cortical collecting tubule this nephron segment has been recognized to play a key role in the regulation of sodium, potasgium and hydrogen ion transport. Such studies have also identified the cortical collecting tubule as a major site where mineralocorticoids augment the reabsorption of sodium and stimulate potassium secretion [3, 4, 16, 17, 20, 24].

* This work was supported by NIH grants 32489 and 17433 ** Portions of this work have been published previously in Am J

Physiol 244: F35- F47 (1983) and in "Regulation and develop- ment of membrane transport processes". Graves JS (ed) John Wiley and Sons, Inc, 1985, pp 89--104

Offprint requests to: B. Koeppen at the above address

The successful application of cell potential measure- ments to various portions of the collecting duct [7, 8, 9 - 11, 18], when combined with transepithelial potential and resistance measurements, allows one to examine directly the electrical properties of the apical and basolateral cell membranes, and thereby assess the electrochemical driving forces and membrane conductances for transcellular and paracellular electrolyte movement. In addition, the cell sites where mineralocorticoids regulate sodium and potassium transport can be evaluated [9, 10, 19].

Studies of the transport properties of various portions of the cortical and medullary collecting duct have demon- strated considerable axial heterogeneity. In particular, the cortical collecting duct is a site of avid sodium reabsorption and potassium secretion [3, 17, 20, 24], and of either net hydrogen ion or bicarbonate secretion [13, 14]. The secretory capacity for potassium diminishes sharply with the transi- tion to the outer medulla [24]. It is, however, along the collecting duct in both the outer and inner stripe of the outer medulla that net hydrogen ion secretion is most pronounced [13, 141.

Recently, considerable heterogeneity in the electro- physiological characteristics of the different portions of the collecting duct has been described [8]. Table 1 summarizes the transepithelial and cellular electrical properties of the cortical and outer medullary portions of the collecting duct.

The transepithelial voltage (liT) is oriented lumen negative in the cortex and outer stripe, but becomes lumen positive within the inner stripe of the outer medulla. A progressive decline in the magnitude of the basolateral mem- brane voltage (Vbl) along the length of the collecting duct is also observed.

Heterogeneity in the conductive properties of these segments is further apparent from the values of transepithe- lial resistance (RT) and fractional membrane resistance (fR~)- Ra- increases progressively along the collecting duct, as does fractional resistance. This latter parameter relates the resis- tance of the apical cell membrane to the total transcellular resistance, and indicates a marked reduction of the apical membrane conductance in the medullary segments.

The nature of the changes in apical membrane con- ductance can be analysed by observing the effects of adding amiloride and BaC12 to the luminal fluid. In the cortical segment, the apical membrane contains both sodium and potassium conductive pathways, as represented by the effects of amiloride and BaC12, respectively. As indicated, this membrane is predominantly potassium selective. In the outer stripe, the potassium conductance is markedly re- duced, while the apical membrane in the inner stripe does

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Tablel, Transport and eleetrophysioloNc heterogeneity of the rabbit collecting duct

ark ~ VT Vbl RT (pEq/mm/min) (mV) (mV) (kO cm)

f.~ f .2

Amiloride BaC12

CCD (n = 34) -17.8 -16.2_+2.0 -85.2-t-3.4 14.7_+1.4 0.40-t-0.03 0.49+0.04 ~ 0.76+__0.06 c OMCDo(n=8) - 3.1 - 2.1___4.2 --55.6-+3.7 22.1-+3.6 0.80_+0.04 0.98_+0.01 ~ 0.84_+0.03 OMCDi (n = 23) + 0.1 +16.5_+1.9 -29.2+2,1 56,6+6.4 0.99+0.01 0.99___0.01 0.99+_0.01

CCD = cortical collecting duct; OMCDo = outer stripe of outer medullary collecting duct; OMCDi = inner stripe of outer medullary collecting duct

From reference 24, negative values indicate secretion b Added to the lumen; amiloride (10 -s M), BaC12 (2 x 10 -3 M) ~ P < 0.05 when compared to control values

not contain significant conductive pathways to either ion species.

Taken together with the known rates of net K + secretion by each of these collecting duct segments, also summarized in Table 1, it is clear that the cortical collecting duct is the major site for potassium secretion. We shall now consider in more detail the cellular mechanism of potassium transport in this segment, and in particular the role of mineralocorticoid hormones in regulating this process [9, 10, 19].

The transport of potassium across the collecting duct can be analysed by reference to a two-membrane model (see Fig. 4) that represents the properties of the principal cell 1. Potassium ions are secreted across the tubule against a trans- epithelial electrochemical potential difference, and can therefore be termed active transport [3]. The energy source for this process is the Na-K-ATPase located in the baso- lateral cell membrane and it is this active pump-driven uptake of potassium that raises the intracellular potassium activity. Potassium in turn enters the tubule lumen passively down its electrochemical gradient via a barium-inhibitable conductive pathway in the apical cell membrane, and net potassium secretion results.

Mineralocorticoid hormones such as DOCA or aldos- terone stimulate net potassium secreton [4, 17, 20, 24]. In principle, this could occur by one or more of three general mechanisms: 1) an increase in the activity of the Na-K- ATPase, 2) an increase in the conductance of the apical cell membrane to potassium, or 3) by an increase of the electrochemical gradient for potassium across one or both cell borders.

One well-known action of mineralocorticoid hormones is to increase Na-K-ATPase activity in the cortical collecting duct [2, 16]. In addition, we now present direct evidence that changes also occur in the apical membrane potassium conductance, and the transcellular electrochemical driving forces for potassium.

Figure 1 summarizes the electrophysiologic properties of cortical collecting ducts obtained from control rabbits (C), and from rabbits treated with deoxycorticosterone acetate or DOCA (D) [9, 10]. In all the studies to be reviewed here rabbits received daily injections of 5 mg of DOCA for 1 0 - 1 4 days.

1 Structure-function studies strongly suggest that it is the principal cell that is involved in the reabsorption of sodium and the secre- tion of potassium unlike the intercalated cell which is thought to be responsible for potassium reabsorption and urinary acidifica- tion [6, 15, 21-23]

VT Vbl Vo fR o (rnV) (mY) (mY)

P<.OOl

oo I oo N oo o F -80 -80 80 P<.05 0.8

-60 P<.O01 -60 60 0.6 NS

-20 -20 20 0.2

0 O 0 0.0 C O C D C D C D

Fig. 1. Electrical properties of cortical collecting ducts from control (C) and DOCA-treated rabbits (D). VT: transepithelial potential difference, Vbj: basolateral membrane potential, Va: apical mem- brane potential, fRa : fractional membrane resistance [R,/(R~ + Rbl)] (from [9] with permission)

As is apparent, DOCA administration resulted in marked changes in the transcellular electrical profile. Both the transepithelial voltage (Vr) and the basolateral mem- brane voltage (Vbl) hyperpolarized. In contrast, the apical membrane voltage (Va) depolarized. Although the fractional resistance of the apical membrane 0rR,) was unchanged by DOCA treatment, it could be shown that significant changes in resistance of both the apical and basolateral cell membranes had in fact occured. Accordingly, the specific resistances of the individual membranes were calculated by equivalent circuit analysis [1, 9]. Figure 2 provides informa- tion on the cellular resistance values normalized to luminal surface area. With DOCA treatment, the resistance of both cell membranes decreased in parallel; the apical membrane (Ra) from 57 to 27 s �9 cm 2, and the basolateral membrane (Rb0 from 80 to 42 O" cm 2. The resistance of the shunt pathway (Rs), which represents all conductive pathways in parallel to the cell impaled with the microelectrode, in- creased markedly from 230 to 710 O. cm 2. Thus, with DOCA treatment, the conductive properties of the tubular epithelium reflect to a larger degree the properties of the cellular pathway. Similar results have recently been reported by Sansom and O'Neil [19]).

Since the apical membrane contains conductive pathways for both sodium and potassium, additional experi- ments were carried out in an effort to determine whether the DOCA-induced decrease in apical membrane resistance could be attributed to a change in either one or both of

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O

E c-

O

100 1 0 0 0 Ra Rb I Re

80 ~ 8 0 0

//H //// 60 ~///~ 600

40 ~ ~ ~9"//~//�89 t 400

20 ~ 200

C D C D C D Fig. 2. Effect of DOCA on membrane resistances. All values are normalized to luminal area. Ra: apical membrane resistance; Rbh basolateral membrane resistance; Rs: resistance ofparcellular shunt pathway. (Data from [9])

f R a

t.O

0.8

0.6 N8

0 , 2

0 . 0

Control

NS

T

BaCI 2

NS

T

BaCl 2 §

Arnllorlde Fig. 3. Effect of DOCA on fractional membrane resistance. Effect of t0- 5 M amiloride and 2 • 10- 3 M barium chloride on fRa. Note thatboth drugs increased fRa but that the change of fRa was not different in control (open bars) and DOCA-treated (hatched bar) tubules. (Data from [9])

these ion pathways. Figure 3 summarizes the results of these experiments. BaC12 was added to the tubule lumen, and its effect on the fractional membrane resistance determined. Since BaC12 blocks the potassium pathway in the apical membrane [10, 18], its effect on the fractional resistance can be used to assess the relative sodium and potassium conductances of this membrane. As can be seen, BaCI2 in- creased the fractional resistance to the same extent, in both control and DOCA-treated tubules, indicating that the rela- tive sodium and potassium conductances of this membrane were unaltered between the two groups. Thus, The DOCA- induced fall in apical membrane resistance resulted from an increase in the conductance of both the sodium and potassium pathways. In addition, the observation that the fractional resistance approached unity in the presence of amiloride and BaC12 indicates that sodium and potassium are the major if not only conductive ion species, across the apical membrane and that DOCA treatment does not induce an additional conductance pathway.

Figure 4 summarizes the cell schemes for sodium and potassium transport in control and DOCA-stimulated tubules. The transcellular voltage profiles are also indicated.

C o n t r o l D O C A

: 84o 1 0-~ -44.8 0-

t I 61.0J ~ 105,8 Va Vbl

Va %1

Fig. 4. Potential profile of cells and model of regulation of Na + and K + transport by mineralocorticoid hormones. Both sodium and potassium transport are stimulated. Elements of stimulation of potassium transport include increased cell uptake across the basolateral cell membrane by increased ATPase activity and by a more favorable electrical gradient for passive potassium entry. Translocation of potassium ions from cell to lumen is also facilitated, both by a more favorable electrical gradient (depolariza- tion of the apical cellmembrane) and by an increase in the potassium conductance of this cell membrane. (From [9] with permission)

In control tubules, the potassium that is brought into the cell via the Na-K-ATPase exits across both cell borders. Because of the transcellular voltage profile, exit across the luminal membrane is favored, although some recycling of potassium across the basolateral cell membrane occurs.

With DOCA treatment, cellular uptake of potassium by the Na-K-ATPase is increased. The cellular effiux of potassium into the lumen is facilitated by an increase in the potassium conductance of the apical cell membrane, and by a depolarization of the apical membrane voltage from 72 to 61 mV. The recycling of potassium across the basolateral cell membrane is reduced, secondary to the marked hyperpotarization of the membrane voltage from 84 to 106 mV. Indeed, the magnitude of this voltage likely exceeds the equilibrium potential for potassium, and could therefore lead to thepassive uptake of potasium into the cell.

Taken~together, these DOCA-induced changes in the properties of the principal cell allow for maximally efficient potassium secretion. Moreover, this provides a mechanism by which the ratio of net sodium reabsorption to net potassium secretion can be varied over a wide range, even though the coupling ratio of the Na-K-ATPase may be fixed.

Two independent studies have recently been carried out to define further the properties of the apical potassium conductance [5, 12]. In both series of experiments, re- cordings of single potassium channel activity in the apical membrane of rabbit cortical collecting tubules were obtained by patch-clamp techniques. The properties of the apical potassium channel included a selectivity ratio of potassium/ sodium 9:1, channel activation by calcium ions in the physiological concentration range and inhibition by barium and by lowering pH [5]. Finally, it was also observed that depolarization of the apical membrane increased the open- channel probability (Hunter M, Giebisch G, unpublished observations). This is of interest in view of the evidence

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cited above that D O C A treatment depolarizes the apical cell membrane and increases the potassium conductance. The precise relationship of these two events needs further study.

References

1. Fr6mter E, Gebler B (1977) Electrical properties of amphibian urinary bladder epithelia. III. The cell membrane resistances and the effect of amiloride. Pfltigers Arch 371:99-108

2. Garg LC, Knepper MA, Burg MB (1981) Mineralocorticoid effects on Na-K-ATPase in individual nephron segments. Am J Physiol 240: F536- F544

3. Grantham JJ, Burg MB, Orloff J (1970) The nature of transtu- bular Na and K transport in isolated renal collecting tubules. J Clin Invest 49:1815-1826

4. Helman SI, O'Neil RG (1977) Model of active transepithelial Na and K transport of renal collecting tubules. Am J Physiol 233: F559- F571

5. Hunter M, Lopes AG, Boulpeap EL, Giebisch G (1984) Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc Nat1 Acad Sci USA 81:4237-4239

6. Kaissling B, Kriz, W (1979) Structural analysis of the rabbit kidney. Adv Anat Embryol Cell Biol 56:1 -- 123

7. Koeppen BM (1985a) Conductive properties of the rabbit outer medullary collecting duct: inner stripe. Am J Physiol 248 : F 500 -- F 506

8. Koeppen BM (1985b) Electrophysiologic heterogeneity of the rabbit collecting duct. Kidney Int 27:313

9. Koeppen BM, Giebisch G (1985) Mineralocorticoid regulation of sodium and potassium transport by the cortical collecting duct. In" Graves JS (ed) Regulation and development of mem- brane transport processes. John Wiley and Sons, Inc, pp 8 9 - 104

10. Koeppen BM, Biagi BA, Giebisch GH (1983a) Intracellular microelectrode characterization of the rabbit cortical collecting duct. Am J Physiol 244:F35 - F47

11. Koeppen BM, Biagi BA, Giebisch G (1983 b) Electrophysiology of mammalian renal tubules: Inferences from intracellular microelectrode studies. Ann Rev Physiol 45 : 497- 517

12, Koeppen BM, Beyenbach KW, Helman SI (1984) Single- channel currents in renal tubules. Am J Physiol 247:F380- F384

13. Laski ME, Kurtzman NE (1983) Characterization of acidifica- tion in the cortical and medullary collecting tubule of the rabbit. J Clin Invest 72: 2050- 2059

14. Lombard WE, Kokko JP, Jacobson HR (1983) Bicarbonate transport in cortical and outer medullary collecting tubules. Am J Physiol 244: F289- F296

15. Madsen KM, Tisher CC (1983) Cellular response to acute respiratory acidosis in rat medullary collecting duct. Am J Physiol 245 : F670- F676

16. Marver D, Kokko JP (1983) Renal target sites and the mecha- ulsm of action of aldosterone. Min Elec Metab 9:1 - 18

17. O'Neil RG, Helman SI (1977) Transport characteristics of renal collecting tubules: influences of DOCA and diet. Am J Physiol 233 : F544- F558

18. O'Neil R, Sansom SC (1984) Characterization of apical cell membranes Na + and K + conductance of cortical collecting duct using microelectrode techniques. Am J Physiol 247: F I 4 - F24

19. Sansom SC, O'Neil RG (1984) Effect 0f mineralocorticoids on cellular conductive properties of the cortical collecting duct. Fed Proc 43 : 302

20. Schwartz GJ, Burg MB (1978) Mineralocorticoid effects on cation transport by cortical collecting tubules in vitro. Am J Physiol 235 : F576- F585

21. Stanton BA (1984) Regulation of ion transport in epithelia: Role of membrane recruitment from cytoplasmic vesicles. Lab Invest 51:255-257

22. Stanton BA, Biemesderfer D, Wade JB, Giebisch G (1981) Structural and functional study of the rat distal nephron: effects of potassium adaptation and depletion. Kidney Int 19:36-48

23. Stetson DL, Wade JB, Giebisch G (1980) Morphologic alterations in the rat medullary collecting duct following potassium depletion. Kidney Int 17:45- 56

24. Stokes JB, Ingram MJ, Williams AD, Ingrain D (1981) Hetero- geneity of the rabbit collecting tubule: Localization of mineralocorticoid hormone action to the cortical portion. Kidney Int 20: 340- 347

Audience discussion

Oberleithner: Gerhard, the data on the collecting duct re- mined me very much of the resistance measurements you and Bill and I did on the diluting segment in the K adapted animals, in which the absolute conductance of the luminal membrane for K increased. Do you have any evidence that there also might be something like a Na /H exchange mediating these effects in the luminal membrane o f the collecting duct?

Giebisch: We have not studied acidification properties under these conditions, but f rom all the available evidence I doubt whether a N a / H exchange is involved in the collecting duct because the transport of H there is electrogenic and Na independent. However, in the thick ascending limb of Henle's loop and the early part of the distal tubule there may be a N a / H exchange. In late distal tubular segments where there are intercalated cells, there is ATPase-driven electrogenic H ion transport.

Greger: Gerhard, did you perform any experiments in which the patch pipettes were filled with a KC1 solution, so as to study whether there is any rectification o f this channel?

Giebisch: No, we do not have any evidence for rectification and we have not yet used different K concentrations in the patch pipette to examine this.

Kinne: What is the evidence that K movement in DOCA- treated animals at the basolateral membrane is into the cell rather than out of the cell as under normal conditions?

Giebisch: The evidence is that after D O C A the membrane potential hyperpolarizes to values that would make it osmotically impossible to get the K activity that high. In other words, you would need a K activity in excess of 200 mEq/1 cell water. We have not yet measured the K activities.

Wiederholt: We measured the K activities in the distal tubule of the rat with and without aldosterone. After aldosterone the cell membrane hyperpolarized, K activity decreased, but was not in equilibrium, as you postulated.

Rick: What is the Na to K flux ratio across the epithelium under these conditions? It should be influenced if the passive K flux across the basolateral membrane is reversed.

Giebisch: The Na to K flux ratio drops sharply after DOCA. That is exactly what is expected.