Basolateral membrane potassium in rabbit cortical thick ascending ANNETTE Membrane Hurst, Lapointe.
Annette
M. HURST, Transport
M., Marcelle
MARCELLE
Research Duplain,
DUPLAIN,
Group, Universitk and Jean-Yves
Basolateralmembranepotassiumchannelsin rabbit cortical thick ascendinglimb. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F262-F267, 1992.-The nature of K exit acrossthe basolateral membrane of rabbit cortical thick ascendinglimb (CTAL) was investigated using the patch clamp technique. The basolateralmembranewasexposedby mild collagenasetreatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductancewas35.0 t 1.3 pS (n = 9) comparedwith an outward conductanceof 7.0 t 0.9 pS (n = 5), and the current reversedat a pipette potential of -63.5 & 3.1 mV (n = 9). The channel is strongly voltage dependent,showing an e-fold increasein openprobability per 1%mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channelcurrent amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excisedpatch recordingswere possible,which confirmed the presenceof a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possibleto obtain patch clamp recordingsfrom the rabbit CTAL basolateralmembrane using a very mild collagenasetreatment, and 2) the exit of K acrossthe basolateralmembraneis mediatedat least in part by the presenceof voltage-sensitive K channels. patch clamp; inward rectifier; voltage dependence;channel rundown
conductancesonthebasolateral membrane of renal epithelia has been established for many years (16). The effect of barium and peritubular ion substitutions suggests the presence of barium-sensitive, K-selective ionic channels in the rabbit proximal tubule (1, 2), in the hamster medullary thick ascending limb (26) and in the rabbit cortical collecting duct (15). Contrary to the idea of a ubiquitous basolateral K conductance in renal epithelia, there is evidence against the presence of a conductive pathway for K on the basolateral membrane of rabbit cortical thick ascending limb (CTAL). It has been shown that barium depolarizes the basolateral membrane of rabbit CTAL, but this effect is not accompanied by any change in transepithelial resistance or fractional resistance of the basolateral membrane (10). Although a Cl channel has been reported on this membrane (8) the presence of a functional K conductance has been questioned, and thus the current cell model favors the presence of an electroneutral barium-sensitive K-Cl cotransporter in parallel with a Cl conductance (9). Until relatively recently the presence of ionic channels on the basolateral membrane of mammalian renal epithelia had not been well documented using a direct approach. This is presumably related to the problem of restricted access to the basolateral membrane with a patch pipette because of the encircling basement mem-
THEEXISTENCE
F262
OFPOTASSIUM
0363-6127/92
$2.00
Copyright
channels limb AND JEAN-YVES de Montrkal,
Montreal,
LAPOINTE Quebec H3C 3J7, Canada
brane. K channels have now been identified in the rabbit proximal convoluted tubule (7, 18) and in the distal convoluted tubule (2 l), and a cation-selective, Ca-activated channel has been found in the CTAL of mouse (22). In all but one of these studies, where patch clamp recordings were obtained from the lateral membrane of torn tubules (7), the basement membrane was removed with a harsh collagenase treatment (collagenase concentration up to 400 U/ml for up to 60 min). Considering that the collagenase used is rarely pure, this aggressive treatment could cause permanent damage to the underlying membrane proteins. The aim of the present work was to investigate the nature of the ionic channels present on the basolateral membrane of rabbit CTAL using a much milder collagenase treatment than previously used for other nephron segments, in an effort to minimize damage to the underlying membrane. With this preparation, we found a K-selective, inwardly rectifying channel that may be involved in the recycling of K across the basolateral membrane. The direct observation of this K channel modifies considerably the cell model for ionic transport by the rabbit CTAL. METHODS Tubule preparation. New Zealand White rabbits were anesthetized with pentobarbitone sodium (35 mg/kg iv), and a laparotomy was performed. The left kidney was exposed,and the renal artery was cannulated; then the kidney was removed and flushed by gravity with 30 ml of ice-cold phosphate-buffered sucrosesolution (56 mM Na,HPO,, 13 mM NaH,PO,, and 140 mM sucrose;seeRef. 19). Kidney sliceswere cut coronally, and CTAL segmentswere dissectedmanually at 50x magnification. The dissectedtubule wasthen placed in a poly-L-lysine (Sigma Chemical, St. Louis, MO)-coated chamber on the microscope stage and incubated for 10 min at room temperature with Ringer solution [(in mM) 140 NaCl, 5 KCl, 2 CaCl,, 1 MgCl,, 5 glucose, and 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) adjusted to pH 7.4 with NaOH] containing 0.1 U/ml collagenasetype A (Boehringer Mannheim, Laval, Quebec) to remove the basementmembrane.After the incubation the solution bathing the tubule was replacedwith collagenase-freeRinger solution, and patch clamp studies (12) were performed at room temperature. Patch clamp studies. Patch pipettes (resistancesof 10 Ma) were fabricated from hematocrit glasstubing (Fisher, Ottawa, Ontario) using a vertical two-stage pipette puller (Narishige, Japan). Channel currents were amplified with a patch clamp amplifier (L/M-EPC7, List, FRG) and stored on videotape, after pulse-codemodulation (Neurodata DR-384, New York, NY). The applied potential, V, correspondsto Vbath - Vpipette. However, in cell-attached experiments the potential acrossthe patch is equal to the applied potential, V, plus the cell membrane potential, whereasin excisedpatches (becausethere is no contributing potential from the cell) the potential acrossthe patch is the applied potential, V.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
BASOLATERAL
For analysis, the channel records were filtered at 500 Hz and sampled at 1 kHz into computer memory (386 IBM-compatible PC) via a TL-1 DMA Labmaster interface (Axon Instruments, Foster City, CA). Channel current records were analyzed using pCLAMP (version 5.5.1; Axon Instruments). Channel conductance was estimated by linear regression between -40 mV and +40 mV for the inward currents and between +lOO and +160 mV for the outward currents. The voltage dependence of the channel was determined by measuring the open probability (P,) at different applied potentials. The best fit of the data to the Boltzmann distribution was determined using the Simplex algorithm (4), which gives the potential at which the channel PO was 0.5 (V,) and the equivalent gating charge (n). Solutions. In all the experiments the pipette contained (in mM) 140 KCl, 2 CaCl,, 1 MgCl,, 5 glucose, and 5 HEPES adjusted to pH 7.4 with KOH, and the tubule was bathed with Ringer solution. In some experiments the following were added to the bath solution: 1 mM barium chloride, 1 PM ionomycin, 0.1 mM ATP, 0.1 mM dibutyryladenosine 3’,5’-cyclic monophosphate (dibutyryl-CAMP) + 1 PM forskolin, and 20 U/ml catalytic subunit of protein kinase A. All of the preceding chemicals were purchased from Sigma. Statistics. Groups of paired data were analyzed using Student’s t test and are presented as means I SE. Mean values were assumed to be significantly different from each other when P < 0.05. RESULTS
A very mild collagenase treatment was used to loosen the basement membrane, which normally obstructs access with a patch pipette to the basolateral membrane of
Fig. 1. Photograph of microdissected rabbit cortical 10 min at room temperature. Bar = 20 ym.
thick
ascending
F263
K CHANNELS
limb
renal tubules. Figure 1 is a photograph of a microdissected rabbit CTAL that has been treated with collagenase. The tubule retains its structure, and the basolateral cell borders are clearly visible. The rate of seal formation with collagenase-treated tubules was 67% (n = 882). In cell-attached patches, we observed one type of channel repeatedly on the basolateral membrane, i.e., a lowconductance K channel. With a high-K solution in the patch pipette and Ringer solution bathing the tubule, inward currents of 2.2 + 0.07 pA (n = 7) were observed in the absence of an applied potential (Fig. 2). This is consistent with the movement of K from the patch pipette to the cell driven by the cell membrane potential. The current-voltage (I-V) relation for the channel is shown in Fig. 2. The channel displays inward rectification with an inward slope conductance of 35.0 f 1.3 pS (n = 9) and an outward conductance of 7.0 + 0.9 pS (n = 5). The potential at which the currents reverse was determined by extrapolation of the linear regression estimates of the inward conductance; this gave a mean value of 63.5 + 3.1 mV (n = 9), which is also consistent with a K-selective channel and a negative membrane potential. The channel is strongly voltage dependent (Fig. 3), opening with patch depolarization. With no applied potential to the patch pipette the channel P,, is 0.35 -C 0.04. From the Boltzmann equation it can be estimated that the channel is open for 50% of the time if the patch is depolarized by 9 mV, and the shape of the PO vs. voltage
after
treatment
with
0.1 U/ml
collagenase
for
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
F264
BASOLATERAL
K CHANNELS 2
1 (PA) 1
loo
120
140
160
closed state Fig. 2. Left: current (I) records at different applied potentials (where V = Vbath - Vpipette ). Arrows indicate of channel. Downward deflections are inward currents. Right: current-voltage relationship (I-V) for the channel, where different symbols represent different experiments. Solid line is best fit to a third-order polynomial.
PO 0.8. 4 PA barium
250
msecs
N.Po CA
0.2
i
(PA)
0.4 IO
9
/yf,/ -80
0 -60
q n
-40
control -20
1
I 20
I 40 ----&--
80
v (mv> Fig. 3. Voltage dependence of channel. Solid line is best fit line estimated from the Boltzmann equation (r = 0.99) where V, = 9 mV and n = 1.4. Different symbols represent different experiments. PO, open probability. V,, potential at which channel P, was 0.5.
curve is consistent with an equivalent gating charge of 1.4. The slope of the voltage dependence is 18 mV per e-fold change in P,. Figure 4 shows that the channel is sensitive to barium (1 mM) when added to the bathing solution. These data were obtained from cell-attached patches with no applied potential to the patch pipette. Barium causes a signifi-
barium
control
barium
Fig. 4. Top: representative trace showing effect of barium on channel activity. Bottom: effect of barium on mean open probability (NP,, left) and single-channel current amplitude (i, right) in cell-attached patches with no applied pipette potential. * P < 0.05.
cant reduction in NP, (where N is the number of channels in the patch and PO is the single-channel open probability) from 0.78 t 0.2 to 0.12 t 0.03 (n = 4, P = 0.03) as well as a reduction in the single-channel current amplitude from 2.2 t 0.1 to 1.23 t 0.01 pA (n = 4, P = 0.02). The channel does not seem to be sensitive to changes in intracellular Ca concentration (Fig. 5). In cell-attached patches (no applied potential) addition of the Ca ionophore ionomycin (1 PM) to the bath solution containing
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
BASOLATERAL
K CHANNELS
F265
not be reactivated by further addition of 20 U/ml catalytic subunit of protein kinase A (n = 3), and prestimulation of the tubule with 0.1 mM dibutyryl-CAMP plus 1 PM forskolin did not stimulate channel activity in cellattached patches (n = 6) or decrease the rapid rundown of the channel in excised patches (n = 3). Hence, as yet the factors that regulate this channel in vivo have not been identified. DISCUSSION
ionomycin
Fig. 5. Effect of ionomycin on channel open probability bath solution containing 2 mM Ca.
when added to
2 mM Ca did not significantly change the channel activity (0.55 t 0.1 in control and 0.54 t 0.1 in ionomycin solution, P = 0.7). On excision of the patch the K channel described exhibited a fast r Nundown (within seconds). In three pate hes it was possible to measure inwa .rd currents of 1.4 t 0.1 PA at 0 mV pipette potential, with a high- K solution in the pipette and Ringer solution bathing the intracellular face of the patch, and in two of these patches we were also able to change the pipette potential to obtain the I-V relation shown in Fig. 6. The direction of the current flow with no applied potential and the extrapolated reversal potential of the channel of 78 mV are again consistent with a highly K-selective channel. To prevent the rundown of the channels in excised patches to enable the study of the regulation of this channel, a number of experimental protocols were tried. The rundown of this channel could not be prevented by inclusion of 0.1 mM ATP in the bath solution (n = 3). It could 1
I
50
-1 , , ’I I’
I
I ,I
,,
,I
I
I II
I’
I ,’
,I
/’
100
Fig. 6. I- V relation in excised patches. Different symbols represent different experiments. Solid line, linear regression fit to the data; dotted line, extrapolation of the fit to estimate the reversal potential of this channel.
We have demonstrated that it is possible to obtain patch clamp recordings from the basolateral membrane of mildly collagenase-treated renal tubules. This may be of significant importance in the study of basolateral membrane ionic channels, as to date there have not been many studies of such conductances in mammalian tubules (18, 21, 22), probably largely due to the uncertainty of the damaging effects of harsh collagenase treatment. One group has successfully patched the lateral membrane of torn tubules (7, 8); however, there is a degree of uncertainty as to the membrane origin of the channel observed. In amphibian tubules it has been possible to manually remove the basement membrane with forceps (14, 20); but, given the difference in size between, for example, Necturus proximal tubule and rabbit CTAL, this technique is probably at the limits of feasibility for mammalian tubules. In our study the channel we have described is clearly K selective, as indicated from the direction of the current flow in both cell-attached and excised patches and from the potential at which the currents reverse in cell-attached patches and excised patches. From the I-V relation in excised patches we have estimated, from linear extrapolation, a reversal potential for the channel of 78 mV. From this value it can be calculated that this channel is at least 100 times more permeable to K than to Na. However, the estimate of the reversal potential, and hence the selectivity, are conservative, as one would expect some degree of Goldman rectification given the asymmetric composition of the solutions on either side of the membrane. The channel is strongly voltage dependent with an efold change in P, per 18-mV depolarization. Basolateral K channels in the rabbit proximal tubule are only mildly voltage sensitive, showing an e-fold change in P, per 56-mV depolarization (18), and the K channels on the basolateral membrane of the distal tubule are not sensitive to changes in membrane voltage (21). This strong voltage dependence could make this channel a good candidate for maintaining the basolateral membrane potential during large fluctuations in transepithelial NaCl transport. An increase in Cl load to the cell would tend to depolarize the cell, opening the basolateral K channel, which in turn would act to repolarize the membrane potential. Like many other K channels (3,13,24,25), the channel we have observed is inhibited by barium. It is interesting to note that barium addition to the bath can block this channel after the cell-attached patch configuration was achieved. If one assumes a tight seal between the pipette and the cell membrane, the simplest explanation would be that barium enters the cell and blocks the channel from
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
F266
BASOLATERAL
the intracellular face of the membrane. Also, in response to barium there is a fall in the single-channel current amplitude. If barium causes no change in the single-channel conductance, then this reduction is probably due to a reduction in the driving force for K entry into the cell, that is, the cell membrane potential. From microelectrode studies it has been shown that barium causes a variable depolarization averaging 22 mV (10); the reduction in single-channel current that we observed is consistent with a depolarization of this magnitude. This suggests that the tubules were able to maintain a significant cell membrane potential, although all the experiments were performed at room temperature. Also, it is unlikely that the reduction in channel P, with barium is due to the change in cell membrane potential, as depolarization of the membrane patch increases channel P, (Fig. 3). With the identification of a K-selective channel on the basolateral membrane of rabbit CTAL, an earlier observation, that the addition of barium caused a large depolarization of the cell potential without a significant change in fractional resistance of the basolateral membrane (lo), was puzzling. This observation led the authors to conclude that a functional K conductance was lacking on this membrane and that the large depolarization seen with barium was due to inhibition of a barium-sensitive electroneutral K-Cl cotransporter. Inhibition of such a transporter would presumably lead to a rise in intracellular Cl activity, an increase in the driving force for Cl exit across the basolateral membrane, and hence a cellular depolarization. The unexpected effect of barium could be explained by the following hypothesis. Barium could have inhibited a basolateral membrane K conductance, resulting in a large depolarization of the basolateral membrane potential. If this depolarization of the basolateral membrane then activated a voltage-dependent Cl conductance that compensated for the reduction in K conductance, then, under these experimental conditions, there would be no net change in membrane resistance. This hypothesis is further supported by the existence of outwardly rectifying voltage-dependent Cl channels that open on cell depolarization in the rabbit CTAL (8). The presence of a K channel on the basolateral membrane of rabbit CTAL changes the cell model recently proposed (9). We envisage that there are conductive pathways for both K and Cl (8) on the basolateral membrane (Fig. 7), and, although our data do not rule out the possibility of a basolateral K-Cl cotransporter, its presence
Na + 2 Cl-K+
Na + ATP
cell model of cortical work and Ref. 8.
thick
now needs to be verified experimentally. In hamster medullary thick ascending limb (26) and in Amphiuma early distal tubule (11) two cell populations have been reported. It is possible that a heterogeneous cell population exists in the rabbit CTAL, but this too is a subject for further study. To estimate the contribution made by this channel to the basolateral membrane conductance in the CTAL is difficult. However, since this channel was by far the most frequently observed during our studies (average number of channels per patch = 2) and since it is open for 40% of the time at the resting membrane potential, this channel could provide a significant means via which K leaves the cell across the basolateral membrane. The regulation of this channel, as yet, remains unclear. The fact that the channel activity runs down on excision seems to indicate that some intracellular factor keeps the channel open in normal conditions. Despite numerous attempts to keep the channel open in excised patches, we were unable to prevent the rapid rundown. In contrast to K channels in opossum kidney cells (17) and rat ventricular myocytes (6), where elevated Ca concentrations bathing the internal surface of the membrane promoted channel rundown, this K channel does not seem to be sensitive to changes in Ca concentration. The K channel found on the apical membrane of rabbit CTAL is also insensitive to changes in intracellular Ca concentration (25), although in the rat CTAL the apical K channel is sensitive to change in intracellular Ca concentration (3). This channel is not reactivated by low concentrations of ATP like the K channels found on the apical membrane of rat cortical collecting duct (23), nor can it be restimulated by further addition of catalytic subunit of protein kinase A or by prestimulation with dibutyryl-CAMP and forskolin, suggesting that, if the channel is activated by phosphorylation, then the pathway for activation is not via CAMP-dependent protein kinase. In summary, K exit across the basolateral membrane of rabbit CTAL is mediated, at least in part, by a K-selective ionic channel. This channel is strongly voltage dependent, insensitive to perturbations in intracellular Ca concentration, and exhibits rundown once the patch is excised, suggesting that the channel requires some intracellular factor(s) to be activated. It could be possible for this channel to play a role in the maintenance of the basolateral membrane potential, and it could be involved, as in the basolateral membrane of other epithelial cells (5), in the recycling of K taken up by the basolateral Na-K-ATPase. We thank Dr. Jon Beck for helpful discussion during the course of this work. A. M. Hurst is postdoctoral research fellow of the Science and Engineering Research Council-North Atlantic Treaty Organization. Address for reprint requests: A. M. Hurst, Groupe de Recherche en Transport Membranaire, Universite de Montreal, C.P. 6128, Succursale A, Montreal, Quebec H3C 357, Canada. Received
K+
23 January
1992; accepted
in final
form
12 March
1992.
REFERENCES
::
Fig. 7. Proposed based on present
K CHANNELS
ascending
limb
of rabbit,
1. Bello-Reuss, E. Electrical properties brane of the straight portion of the Physiol. Lond. 326: 49-63, 1982. 2. Biagi, B. A., T. Kubota, M. Sohtell, cellular potentials in rabbit proximal
of the basolateral memrabbit proximal tubule. J. and G. Giebisch. Intratubules perfused in vitro.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
BASOLATERAL Am. J. Physiol. F210, 1981. 3.
(Renal
Fluid
Electrolyte
Physiol.
F35-F47,
415: 449-460,
Bloomfield, Theory,
1990.
P., and W. L. Steiger.
Least
Absolute
Deuiations.
Boston: Birkhauser, 1983. cross-talk between epithelial cell
Applications
and Algorithms.
Diamond, J. M. Transcellular membranes. Nature Lond. 300: 683-685, 1982. 6. Findlay, I. ATP-sensitive potassium channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations. 5.
Pfluegers
Arch.
410: 313-320,
1987.
Gogelein, H., and R. Greger. A voltage dependent ionic channel in the basolateral membrane of late proximal tubules of the rabbit kidney. Pfluegers Arch. 407, Suppl. 2: Sl42-S148, 1986. 8. Greger, R., M. Bleich, and E. Schlatter. Ion channels in the thick ascending limb of Henle’s loop. Renal Physiol. Biochem. 13: 7.
37-50, 9.
ing duct. Am. J. Physiol.
9): F200-
Bleich, M., E. Schlatter, and R. Greger. The luminal potassium channel of the thick ascending limb of Henle’s loop. Pfluegers Arch.
4.
240
40, suppz.
33: s119-s124,
lg 2.
21
1991.
Greger, R., and E. Schlatter. Properties of the basolateral membrane of the cortical thick ascending limb of Henle’s loop of rabbit kidney. Pjluegers Arch. 396: 325-334, 1983. 11. Guggino, W. B. Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cland K+ transport across the basolateral cell membrane. Am. J. 10.
Physiol. 1986. 12
250
Physiol.
Arch.
391: 85-100,
19):
F430-F440,
1981.
Natl.
Acad.
Sci. USA
81: 4237-4239,
1984.
Kawahara, K., M. Hunter, and G. Giebisch. nels in Necturus proximal tubule. Am. J. Physiol. Electrolyte
15
Electrolyte
Hunter, M., A. G. Lopes, E. L. Boulpaep, and G. Giebisch. Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc.
14
Fluid
Hamill, 0. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch clamp techniques for high resolution current recording from cells and cell free membrane patches. Pfluegers
13
(Renal
Physiol.
22): F488-F494,
Potassium chan253 (Renal
Koeppen, B. M., B. A. Biagi, and G. H. Giebisch. Intracellular microelectrode characterization of the rabbit cortical collect-
Fluid
Electrolyte
Physiol.
13):
Physiol.
23): Fl05-Fl13,
1988.
Pirie, S. C., and D. J. Potts. Application of cold flush preser’ vation to in vitro microperfusion studies of kidney tubules. Kidney Int.
28: 982-984,
1985.
.
Sackin, H. Stretch-activated potassium channels in renal proximal tubule. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol.
.
Taniguchi, J., K. Yoshitomi, and M. Imai. K+ channel currents in basolateral membrane of distal convoluted tubule of rabbit kidney. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25):
22): Fl253-F1262,
F246-F254,
1987.
1989.
22* Teulon, J., M. Paulais, and M. Bouthier. A calcium-activated cation-selective channel in the basolateral membrane of the cortical thick ascending limb of Henle’s loop of the mouse. Biochim. Biophys.
Acta
905: 125-132,
1987.
Wang, W., and G. Giebisch. Dual effect of adenosine triphosphate on the apical small conductance K channel of the rat cortical collecting duct. J. Gen. Physiol. 98: 35-61, 1991. 24. Wang, W., A. Schwab, and G. Giebisch. Regulation of small conductance K+ channel in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 259 (Renal Fluid Electrolyte Phys-
23.
ioZ. 28): F494-F502,
1990.
25. Wang, W., S. White, J. Geibel, and G. Giebisch. A potassium channel in the apical membrane of rabbit thick ascending limb of Henle’s loop. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F244-F253,
Fluid
1987.
244 (Renal
1983.
16. Koeppen, B. M., and G. Giebisch. Electrophysiology of mammalian renal tubules: inferences from intracellular microelectrode studies. Annu. Rev. Physiol. 45: 497-517, 1983. 17. Ohno-Shosaku, T., T. Kubota, J. Yamaguchi, M. Fukase, T. Fujita, and M. Fujimoto. Reciprocal effects of calcium and Mg ATP on the rundown of the potassium channels in opossum kidney cells. Pfluegers Arch. 413: 562-564, 1989. 18. Parent, L., J. Cardinal, and R. Sauve. Single-channel analysis of a K channel at the basolateral membrane of rabbit proximal convoluted tubule. Am. J. Physiol. 254 (Renal FZuid Electrolyte
1990.
Greger, R., M. Bleich, and E. Schlatter. Ion channel regulation in the thick ascending limb of the loop of Henle. Kidney Int.
F267
K CHANNELS
26.
1990.
Yoshitomi, K., C. Koseki, J. Taniguchi, and M. Imai. Functional heterogeneity in the hamster medullary thick ascending limb of Henle’s loop. Pfluegers Arch. 408: 600-608, 1987.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
Annette
M. HURST, Transport
M., Marcelle
MARCELLE
Research Duplain,
DUPLAIN,
Group, Universitk and Jean-Yves
Basolateralmembranepotassiumchannelsin rabbit cortical thick ascendinglimb. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F262-F267, 1992.-The nature of K exit acrossthe basolateral membrane of rabbit cortical thick ascendinglimb (CTAL) was investigated using the patch clamp technique. The basolateralmembranewasexposedby mild collagenasetreatment (0.1 U/ml), and a K-selective inwardly rectifying channel was identified. In cell-attached patches (140 mM K pipette) the inward conductancewas35.0 t 1.3 pS (n = 9) comparedwith an outward conductanceof 7.0 t 0.9 pS (n = 5), and the current reversedat a pipette potential of -63.5 & 3.1 mV (n = 9). The channel is strongly voltage dependent,showing an e-fold increasein openprobability per 1%mV depolarization. Barium blocked the channel, reducing both mean open probability and single-channelcurrent amplitude; however, the channel was not Ca sensitive. On excision the channel exhibited rundown, which could not be prevented by 0.1 mM ATP or ATP plus 20 U/ml catalytic subunit of protein kinase A. A few excisedpatch recordingswere possible,which confirmed the presenceof a highly K-selective channel with a K-to-Na permeability ratio of 100. In conclusion, 1) it is possibleto obtain patch clamp recordingsfrom the rabbit CTAL basolateralmembrane using a very mild collagenasetreatment, and 2) the exit of K acrossthe basolateralmembraneis mediatedat least in part by the presenceof voltage-sensitive K channels. patch clamp; inward rectifier; voltage dependence;channel rundown
conductancesonthebasolateral membrane of renal epithelia has been established for many years (16). The effect of barium and peritubular ion substitutions suggests the presence of barium-sensitive, K-selective ionic channels in the rabbit proximal tubule (1, 2), in the hamster medullary thick ascending limb (26) and in the rabbit cortical collecting duct (15). Contrary to the idea of a ubiquitous basolateral K conductance in renal epithelia, there is evidence against the presence of a conductive pathway for K on the basolateral membrane of rabbit cortical thick ascending limb (CTAL). It has been shown that barium depolarizes the basolateral membrane of rabbit CTAL, but this effect is not accompanied by any change in transepithelial resistance or fractional resistance of the basolateral membrane (10). Although a Cl channel has been reported on this membrane (8) the presence of a functional K conductance has been questioned, and thus the current cell model favors the presence of an electroneutral barium-sensitive K-Cl cotransporter in parallel with a Cl conductance (9). Until relatively recently the presence of ionic channels on the basolateral membrane of mammalian renal epithelia had not been well documented using a direct approach. This is presumably related to the problem of restricted access to the basolateral membrane with a patch pipette because of the encircling basement mem-
THEEXISTENCE
F262
OFPOTASSIUM
0363-6127/92
$2.00
Copyright
channels limb AND JEAN-YVES de Montrkal,
Montreal,
LAPOINTE Quebec H3C 3J7, Canada
brane. K channels have now been identified in the rabbit proximal convoluted tubule (7, 18) and in the distal convoluted tubule (2 l), and a cation-selective, Ca-activated channel has been found in the CTAL of mouse (22). In all but one of these studies, where patch clamp recordings were obtained from the lateral membrane of torn tubules (7), the basement membrane was removed with a harsh collagenase treatment (collagenase concentration up to 400 U/ml for up to 60 min). Considering that the collagenase used is rarely pure, this aggressive treatment could cause permanent damage to the underlying membrane proteins. The aim of the present work was to investigate the nature of the ionic channels present on the basolateral membrane of rabbit CTAL using a much milder collagenase treatment than previously used for other nephron segments, in an effort to minimize damage to the underlying membrane. With this preparation, we found a K-selective, inwardly rectifying channel that may be involved in the recycling of K across the basolateral membrane. The direct observation of this K channel modifies considerably the cell model for ionic transport by the rabbit CTAL. METHODS Tubule preparation. New Zealand White rabbits were anesthetized with pentobarbitone sodium (35 mg/kg iv), and a laparotomy was performed. The left kidney was exposed,and the renal artery was cannulated; then the kidney was removed and flushed by gravity with 30 ml of ice-cold phosphate-buffered sucrosesolution (56 mM Na,HPO,, 13 mM NaH,PO,, and 140 mM sucrose;seeRef. 19). Kidney sliceswere cut coronally, and CTAL segmentswere dissectedmanually at 50x magnification. The dissectedtubule wasthen placed in a poly-L-lysine (Sigma Chemical, St. Louis, MO)-coated chamber on the microscope stage and incubated for 10 min at room temperature with Ringer solution [(in mM) 140 NaCl, 5 KCl, 2 CaCl,, 1 MgCl,, 5 glucose, and 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid (HEPES) adjusted to pH 7.4 with NaOH] containing 0.1 U/ml collagenasetype A (Boehringer Mannheim, Laval, Quebec) to remove the basementmembrane.After the incubation the solution bathing the tubule was replacedwith collagenase-freeRinger solution, and patch clamp studies (12) were performed at room temperature. Patch clamp studies. Patch pipettes (resistancesof 10 Ma) were fabricated from hematocrit glasstubing (Fisher, Ottawa, Ontario) using a vertical two-stage pipette puller (Narishige, Japan). Channel currents were amplified with a patch clamp amplifier (L/M-EPC7, List, FRG) and stored on videotape, after pulse-codemodulation (Neurodata DR-384, New York, NY). The applied potential, V, correspondsto Vbath - Vpipette. However, in cell-attached experiments the potential acrossthe patch is equal to the applied potential, V, plus the cell membrane potential, whereasin excisedpatches (becausethere is no contributing potential from the cell) the potential acrossthe patch is the applied potential, V.
0 1992 the American
Physiological
Society
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
BASOLATERAL
For analysis, the channel records were filtered at 500 Hz and sampled at 1 kHz into computer memory (386 IBM-compatible PC) via a TL-1 DMA Labmaster interface (Axon Instruments, Foster City, CA). Channel current records were analyzed using pCLAMP (version 5.5.1; Axon Instruments). Channel conductance was estimated by linear regression between -40 mV and +40 mV for the inward currents and between +lOO and +160 mV for the outward currents. The voltage dependence of the channel was determined by measuring the open probability (P,) at different applied potentials. The best fit of the data to the Boltzmann distribution was determined using the Simplex algorithm (4), which gives the potential at which the channel PO was 0.5 (V,) and the equivalent gating charge (n). Solutions. In all the experiments the pipette contained (in mM) 140 KCl, 2 CaCl,, 1 MgCl,, 5 glucose, and 5 HEPES adjusted to pH 7.4 with KOH, and the tubule was bathed with Ringer solution. In some experiments the following were added to the bath solution: 1 mM barium chloride, 1 PM ionomycin, 0.1 mM ATP, 0.1 mM dibutyryladenosine 3’,5’-cyclic monophosphate (dibutyryl-CAMP) + 1 PM forskolin, and 20 U/ml catalytic subunit of protein kinase A. All of the preceding chemicals were purchased from Sigma. Statistics. Groups of paired data were analyzed using Student’s t test and are presented as means I SE. Mean values were assumed to be significantly different from each other when P < 0.05. RESULTS
A very mild collagenase treatment was used to loosen the basement membrane, which normally obstructs access with a patch pipette to the basolateral membrane of
Fig. 1. Photograph of microdissected rabbit cortical 10 min at room temperature. Bar = 20 ym.
thick
ascending
F263
K CHANNELS
limb
renal tubules. Figure 1 is a photograph of a microdissected rabbit CTAL that has been treated with collagenase. The tubule retains its structure, and the basolateral cell borders are clearly visible. The rate of seal formation with collagenase-treated tubules was 67% (n = 882). In cell-attached patches, we observed one type of channel repeatedly on the basolateral membrane, i.e., a lowconductance K channel. With a high-K solution in the patch pipette and Ringer solution bathing the tubule, inward currents of 2.2 + 0.07 pA (n = 7) were observed in the absence of an applied potential (Fig. 2). This is consistent with the movement of K from the patch pipette to the cell driven by the cell membrane potential. The current-voltage (I-V) relation for the channel is shown in Fig. 2. The channel displays inward rectification with an inward slope conductance of 35.0 f 1.3 pS (n = 9) and an outward conductance of 7.0 + 0.9 pS (n = 5). The potential at which the currents reverse was determined by extrapolation of the linear regression estimates of the inward conductance; this gave a mean value of 63.5 + 3.1 mV (n = 9), which is also consistent with a K-selective channel and a negative membrane potential. The channel is strongly voltage dependent (Fig. 3), opening with patch depolarization. With no applied potential to the patch pipette the channel P,, is 0.35 -C 0.04. From the Boltzmann equation it can be estimated that the channel is open for 50% of the time if the patch is depolarized by 9 mV, and the shape of the PO vs. voltage
after
treatment
with
0.1 U/ml
collagenase
for
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
F264
BASOLATERAL
K CHANNELS 2
1 (PA) 1
loo
120
140
160
closed state Fig. 2. Left: current (I) records at different applied potentials (where V = Vbath - Vpipette ). Arrows indicate of channel. Downward deflections are inward currents. Right: current-voltage relationship (I-V) for the channel, where different symbols represent different experiments. Solid line is best fit to a third-order polynomial.
PO 0.8. 4 PA barium
250
msecs
N.Po CA
0.2
i
(PA)
0.4 IO
9
/yf,/ -80
0 -60
q n
-40
control -20
1
I 20
I 40 ----&--
80
v (mv> Fig. 3. Voltage dependence of channel. Solid line is best fit line estimated from the Boltzmann equation (r = 0.99) where V, = 9 mV and n = 1.4. Different symbols represent different experiments. PO, open probability. V,, potential at which channel P, was 0.5.
curve is consistent with an equivalent gating charge of 1.4. The slope of the voltage dependence is 18 mV per e-fold change in P,. Figure 4 shows that the channel is sensitive to barium (1 mM) when added to the bathing solution. These data were obtained from cell-attached patches with no applied potential to the patch pipette. Barium causes a signifi-
barium
control
barium
Fig. 4. Top: representative trace showing effect of barium on channel activity. Bottom: effect of barium on mean open probability (NP,, left) and single-channel current amplitude (i, right) in cell-attached patches with no applied pipette potential. * P < 0.05.
cant reduction in NP, (where N is the number of channels in the patch and PO is the single-channel open probability) from 0.78 t 0.2 to 0.12 t 0.03 (n = 4, P = 0.03) as well as a reduction in the single-channel current amplitude from 2.2 t 0.1 to 1.23 t 0.01 pA (n = 4, P = 0.02). The channel does not seem to be sensitive to changes in intracellular Ca concentration (Fig. 5). In cell-attached patches (no applied potential) addition of the Ca ionophore ionomycin (1 PM) to the bath solution containing
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
BASOLATERAL
K CHANNELS
F265
not be reactivated by further addition of 20 U/ml catalytic subunit of protein kinase A (n = 3), and prestimulation of the tubule with 0.1 mM dibutyryl-CAMP plus 1 PM forskolin did not stimulate channel activity in cellattached patches (n = 6) or decrease the rapid rundown of the channel in excised patches (n = 3). Hence, as yet the factors that regulate this channel in vivo have not been identified. DISCUSSION
ionomycin
Fig. 5. Effect of ionomycin on channel open probability bath solution containing 2 mM Ca.
when added to
2 mM Ca did not significantly change the channel activity (0.55 t 0.1 in control and 0.54 t 0.1 in ionomycin solution, P = 0.7). On excision of the patch the K channel described exhibited a fast r Nundown (within seconds). In three pate hes it was possible to measure inwa .rd currents of 1.4 t 0.1 PA at 0 mV pipette potential, with a high- K solution in the pipette and Ringer solution bathing the intracellular face of the patch, and in two of these patches we were also able to change the pipette potential to obtain the I-V relation shown in Fig. 6. The direction of the current flow with no applied potential and the extrapolated reversal potential of the channel of 78 mV are again consistent with a highly K-selective channel. To prevent the rundown of the channels in excised patches to enable the study of the regulation of this channel, a number of experimental protocols were tried. The rundown of this channel could not be prevented by inclusion of 0.1 mM ATP in the bath solution (n = 3). It could 1
I
50
-1 , , ’I I’
I
I ,I
,,
,I
I
I II
I’
I ,’
,I
/’
100
Fig. 6. I- V relation in excised patches. Different symbols represent different experiments. Solid line, linear regression fit to the data; dotted line, extrapolation of the fit to estimate the reversal potential of this channel.
We have demonstrated that it is possible to obtain patch clamp recordings from the basolateral membrane of mildly collagenase-treated renal tubules. This may be of significant importance in the study of basolateral membrane ionic channels, as to date there have not been many studies of such conductances in mammalian tubules (18, 21, 22), probably largely due to the uncertainty of the damaging effects of harsh collagenase treatment. One group has successfully patched the lateral membrane of torn tubules (7, 8); however, there is a degree of uncertainty as to the membrane origin of the channel observed. In amphibian tubules it has been possible to manually remove the basement membrane with forceps (14, 20); but, given the difference in size between, for example, Necturus proximal tubule and rabbit CTAL, this technique is probably at the limits of feasibility for mammalian tubules. In our study the channel we have described is clearly K selective, as indicated from the direction of the current flow in both cell-attached and excised patches and from the potential at which the currents reverse in cell-attached patches and excised patches. From the I-V relation in excised patches we have estimated, from linear extrapolation, a reversal potential for the channel of 78 mV. From this value it can be calculated that this channel is at least 100 times more permeable to K than to Na. However, the estimate of the reversal potential, and hence the selectivity, are conservative, as one would expect some degree of Goldman rectification given the asymmetric composition of the solutions on either side of the membrane. The channel is strongly voltage dependent with an efold change in P, per 18-mV depolarization. Basolateral K channels in the rabbit proximal tubule are only mildly voltage sensitive, showing an e-fold change in P, per 56-mV depolarization (18), and the K channels on the basolateral membrane of the distal tubule are not sensitive to changes in membrane voltage (21). This strong voltage dependence could make this channel a good candidate for maintaining the basolateral membrane potential during large fluctuations in transepithelial NaCl transport. An increase in Cl load to the cell would tend to depolarize the cell, opening the basolateral K channel, which in turn would act to repolarize the membrane potential. Like many other K channels (3,13,24,25), the channel we have observed is inhibited by barium. It is interesting to note that barium addition to the bath can block this channel after the cell-attached patch configuration was achieved. If one assumes a tight seal between the pipette and the cell membrane, the simplest explanation would be that barium enters the cell and blocks the channel from
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
F266
BASOLATERAL
the intracellular face of the membrane. Also, in response to barium there is a fall in the single-channel current amplitude. If barium causes no change in the single-channel conductance, then this reduction is probably due to a reduction in the driving force for K entry into the cell, that is, the cell membrane potential. From microelectrode studies it has been shown that barium causes a variable depolarization averaging 22 mV (10); the reduction in single-channel current that we observed is consistent with a depolarization of this magnitude. This suggests that the tubules were able to maintain a significant cell membrane potential, although all the experiments were performed at room temperature. Also, it is unlikely that the reduction in channel P, with barium is due to the change in cell membrane potential, as depolarization of the membrane patch increases channel P, (Fig. 3). With the identification of a K-selective channel on the basolateral membrane of rabbit CTAL, an earlier observation, that the addition of barium caused a large depolarization of the cell potential without a significant change in fractional resistance of the basolateral membrane (lo), was puzzling. This observation led the authors to conclude that a functional K conductance was lacking on this membrane and that the large depolarization seen with barium was due to inhibition of a barium-sensitive electroneutral K-Cl cotransporter. Inhibition of such a transporter would presumably lead to a rise in intracellular Cl activity, an increase in the driving force for Cl exit across the basolateral membrane, and hence a cellular depolarization. The unexpected effect of barium could be explained by the following hypothesis. Barium could have inhibited a basolateral membrane K conductance, resulting in a large depolarization of the basolateral membrane potential. If this depolarization of the basolateral membrane then activated a voltage-dependent Cl conductance that compensated for the reduction in K conductance, then, under these experimental conditions, there would be no net change in membrane resistance. This hypothesis is further supported by the existence of outwardly rectifying voltage-dependent Cl channels that open on cell depolarization in the rabbit CTAL (8). The presence of a K channel on the basolateral membrane of rabbit CTAL changes the cell model recently proposed (9). We envisage that there are conductive pathways for both K and Cl (8) on the basolateral membrane (Fig. 7), and, although our data do not rule out the possibility of a basolateral K-Cl cotransporter, its presence
Na + 2 Cl-K+
Na + ATP
cell model of cortical work and Ref. 8.
thick
now needs to be verified experimentally. In hamster medullary thick ascending limb (26) and in Amphiuma early distal tubule (11) two cell populations have been reported. It is possible that a heterogeneous cell population exists in the rabbit CTAL, but this too is a subject for further study. To estimate the contribution made by this channel to the basolateral membrane conductance in the CTAL is difficult. However, since this channel was by far the most frequently observed during our studies (average number of channels per patch = 2) and since it is open for 40% of the time at the resting membrane potential, this channel could provide a significant means via which K leaves the cell across the basolateral membrane. The regulation of this channel, as yet, remains unclear. The fact that the channel activity runs down on excision seems to indicate that some intracellular factor keeps the channel open in normal conditions. Despite numerous attempts to keep the channel open in excised patches, we were unable to prevent the rapid rundown. In contrast to K channels in opossum kidney cells (17) and rat ventricular myocytes (6), where elevated Ca concentrations bathing the internal surface of the membrane promoted channel rundown, this K channel does not seem to be sensitive to changes in Ca concentration. The K channel found on the apical membrane of rabbit CTAL is also insensitive to changes in intracellular Ca concentration (25), although in the rat CTAL the apical K channel is sensitive to change in intracellular Ca concentration (3). This channel is not reactivated by low concentrations of ATP like the K channels found on the apical membrane of rat cortical collecting duct (23), nor can it be restimulated by further addition of catalytic subunit of protein kinase A or by prestimulation with dibutyryl-CAMP and forskolin, suggesting that, if the channel is activated by phosphorylation, then the pathway for activation is not via CAMP-dependent protein kinase. In summary, K exit across the basolateral membrane of rabbit CTAL is mediated, at least in part, by a K-selective ionic channel. This channel is strongly voltage dependent, insensitive to perturbations in intracellular Ca concentration, and exhibits rundown once the patch is excised, suggesting that the channel requires some intracellular factor(s) to be activated. It could be possible for this channel to play a role in the maintenance of the basolateral membrane potential, and it could be involved, as in the basolateral membrane of other epithelial cells (5), in the recycling of K taken up by the basolateral Na-K-ATPase. We thank Dr. Jon Beck for helpful discussion during the course of this work. A. M. Hurst is postdoctoral research fellow of the Science and Engineering Research Council-North Atlantic Treaty Organization. Address for reprint requests: A. M. Hurst, Groupe de Recherche en Transport Membranaire, Universite de Montreal, C.P. 6128, Succursale A, Montreal, Quebec H3C 357, Canada. Received
K+
23 January
1992; accepted
in final
form
12 March
1992.
REFERENCES
::
Fig. 7. Proposed based on present
K CHANNELS
ascending
limb
of rabbit,
1. Bello-Reuss, E. Electrical properties brane of the straight portion of the Physiol. Lond. 326: 49-63, 1982. 2. Biagi, B. A., T. Kubota, M. Sohtell, cellular potentials in rabbit proximal
of the basolateral memrabbit proximal tubule. J. and G. Giebisch. Intratubules perfused in vitro.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
BASOLATERAL Am. J. Physiol. F210, 1981. 3.
(Renal
Fluid
Electrolyte
Physiol.
F35-F47,
415: 449-460,
Bloomfield, Theory,
1990.
P., and W. L. Steiger.
Least
Absolute
Deuiations.
Boston: Birkhauser, 1983. cross-talk between epithelial cell
Applications
and Algorithms.
Diamond, J. M. Transcellular membranes. Nature Lond. 300: 683-685, 1982. 6. Findlay, I. ATP-sensitive potassium channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations. 5.
Pfluegers
Arch.
410: 313-320,
1987.
Gogelein, H., and R. Greger. A voltage dependent ionic channel in the basolateral membrane of late proximal tubules of the rabbit kidney. Pfluegers Arch. 407, Suppl. 2: Sl42-S148, 1986. 8. Greger, R., M. Bleich, and E. Schlatter. Ion channels in the thick ascending limb of Henle’s loop. Renal Physiol. Biochem. 13: 7.
37-50, 9.
ing duct. Am. J. Physiol.
9): F200-
Bleich, M., E. Schlatter, and R. Greger. The luminal potassium channel of the thick ascending limb of Henle’s loop. Pfluegers Arch.
4.
240
40, suppz.
33: s119-s124,
lg 2.
21
1991.
Greger, R., and E. Schlatter. Properties of the basolateral membrane of the cortical thick ascending limb of Henle’s loop of rabbit kidney. Pjluegers Arch. 396: 325-334, 1983. 11. Guggino, W. B. Functional heterogeneity in the early distal tubule of the Amphiuma kidney: evidence for two modes of Cland K+ transport across the basolateral cell membrane. Am. J. 10.
Physiol. 1986. 12
250
Physiol.
Arch.
391: 85-100,
19):
F430-F440,
1981.
Natl.
Acad.
Sci. USA
81: 4237-4239,
1984.
Kawahara, K., M. Hunter, and G. Giebisch. nels in Necturus proximal tubule. Am. J. Physiol. Electrolyte
15
Electrolyte
Hunter, M., A. G. Lopes, E. L. Boulpaep, and G. Giebisch. Single channel recordings of calcium-activated potassium channels in the apical membrane of rabbit cortical collecting tubules. Proc.
14
Fluid
Hamill, 0. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. Improved patch clamp techniques for high resolution current recording from cells and cell free membrane patches. Pfluegers
13
(Renal
Physiol.
22): F488-F494,
Potassium chan253 (Renal
Koeppen, B. M., B. A. Biagi, and G. H. Giebisch. Intracellular microelectrode characterization of the rabbit cortical collect-
Fluid
Electrolyte
Physiol.
13):
Physiol.
23): Fl05-Fl13,
1988.
Pirie, S. C., and D. J. Potts. Application of cold flush preser’ vation to in vitro microperfusion studies of kidney tubules. Kidney Int.
28: 982-984,
1985.
.
Sackin, H. Stretch-activated potassium channels in renal proximal tubule. Am. J. Physiol. 253 (Renal Fluid Electrolyte Physiol.
.
Taniguchi, J., K. Yoshitomi, and M. Imai. K+ channel currents in basolateral membrane of distal convoluted tubule of rabbit kidney. Am. J. Physiol. 256 (Renal Fluid Electrolyte Physiol. 25):
22): Fl253-F1262,
F246-F254,
1987.
1989.
22* Teulon, J., M. Paulais, and M. Bouthier. A calcium-activated cation-selective channel in the basolateral membrane of the cortical thick ascending limb of Henle’s loop of the mouse. Biochim. Biophys.
Acta
905: 125-132,
1987.
Wang, W., and G. Giebisch. Dual effect of adenosine triphosphate on the apical small conductance K channel of the rat cortical collecting duct. J. Gen. Physiol. 98: 35-61, 1991. 24. Wang, W., A. Schwab, and G. Giebisch. Regulation of small conductance K+ channel in apical membrane of rat cortical collecting tubule. Am. J. Physiol. 259 (Renal Fluid Electrolyte Phys-
23.
ioZ. 28): F494-F502,
1990.
25. Wang, W., S. White, J. Geibel, and G. Giebisch. A potassium channel in the apical membrane of rabbit thick ascending limb of Henle’s loop. Am. J. Physiol. 258 (Renal Fluid Electrolyte Physiol. 27): F244-F253,
Fluid
1987.
244 (Renal
1983.
16. Koeppen, B. M., and G. Giebisch. Electrophysiology of mammalian renal tubules: inferences from intracellular microelectrode studies. Annu. Rev. Physiol. 45: 497-517, 1983. 17. Ohno-Shosaku, T., T. Kubota, J. Yamaguchi, M. Fukase, T. Fujita, and M. Fujimoto. Reciprocal effects of calcium and Mg ATP on the rundown of the potassium channels in opossum kidney cells. Pfluegers Arch. 413: 562-564, 1989. 18. Parent, L., J. Cardinal, and R. Sauve. Single-channel analysis of a K channel at the basolateral membrane of rabbit proximal convoluted tubule. Am. J. Physiol. 254 (Renal FZuid Electrolyte
1990.
Greger, R., M. Bleich, and E. Schlatter. Ion channel regulation in the thick ascending limb of the loop of Henle. Kidney Int.
F267
K CHANNELS
26.
1990.
Yoshitomi, K., C. Koseki, J. Taniguchi, and M. Imai. Functional heterogeneity in the hamster medullary thick ascending limb of Henle’s loop. Pfluegers Arch. 408: 600-608, 1987.
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.186.138.035) on January 18, 2019.
