From the * Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520;
and Department of Medicine, University of Vermont, Burlington, Vermont 05401
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ABSTRACT |
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The potassium conductance of the basolateral membrane (BLM) of proximal tubule cells is a critical
regulator of transport since it is the major determinant of the negative cell membrane potential and is necessary
for pump-leak coupling to the Na+,K+-ATPase pump. Despite this pivotal physiological role, the properties of this
conductance have been incompletely characterized, in part due to difficulty gaining access to the BLM. We have
investigated the properties of this BLM K+ conductance in dissociated, polarized Ambystoma proximal tubule cells.
Nearly all seals made on Ambystoma cells contained inward rectifier K+ channels (slope, in = 24.5 ± 0.6 pS,
chord, out = 3.7 ± 0.4 pS). The rectification is mediated in part by internal Mg2+. The open probability of the channel increases modestly with hyperpolarization. The inward conducting properties are described by a saturating binding-unbinding model. The channel conducts Tl+ and K+, but there is no significant conductance for Na+, Rb+,
Cs+, Li+, NH4+, or Cl
. The channel is inhibited by barium and the sulfonylurea agent glibenclamide, but not by tetraethylammonium. Channel rundown typically occurs in the absence of ATP, but cytosolic addition of 0.2 mM
ATP (or any hydrolyzable nucleoside triphosphate) sustains channel activity indefinitely. Phosphorylation processes alone fail to sustain channel activity. Higher doses of ATP (or other nucleoside triphosphates) reversibly inhibit the channel. The K+ channel opener diazoxide opens the channel in the presence of 0.2 mM ATP, but does
not alleviate the inhibition of millimolar doses of ATP. We conclude that this K+ channel is the major ATP-sensitive basolateral K+ conductance in the proximal tubule.
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INTRODUCTION |
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The ability of the renal proximal tubule to maintain homeostatic electrolyte and water reabsorption in the face
of drastic changes in dietary solute and water intake
and renal hemodynamics implies that transtubular ion
transport is tightly regulated. Proximal tubule potassium channels, particularly in the basolateral membrane (BLM),1 play pivotal physiologic roles in the regulation of membrane voltage, potassium recycling, and
ultimately in transepithelial solute and water reabsorption. That the basolateral membrane potential of a
proximal tubule cell is dominated by the BLM K conductance is well established (Sackin and Boulpaep,
1983). The Na+,K+-ATPase pump in the BLM provides
the energy that makes ion transport thermodynamically favorable, but continuous operation of the pump requires there be a K+ exit pathway. The BLM K conductance provides such a pathway and thus steady state
intracellular K activity can be maintained in the face of
large transcellular fluxes of salt and water (Sackin and
Boulpaep, 1983
). Since a major portion of the transcellular Na+ flux is reabsorbed across the BLM by the action of the Na+,K+-ATPase, a population of BLM K+
channels working in concert with the pump would allow K+ to recycle in a regulated fashion. Moreover, hyperpolarization secondary to the opening of BLM K+
channels enhances the driving force for electrogenic
apical Na+ entry and basolateral Cl
efflux, resulting in
net NaCl reabsorption.
The Ambystoma proximal tubule exhibits a large K+
conductance in the basolateral membrane (Siebens
and Boron, 1987; Sackin and Boulpaep, 1981
) that has
been shown macroscopically to be sensitive to barium
and pH. However, previous studies of the "macroscopic" BLM K+ conductance (Boulpaep, 1976
) lack
the resolution to distinguish whether there is one population of imperfectly selective K+ channels or a set of
highly selective K+ channels coexisting with a population of nonselective cation channels. More recent "microscopic" single-channel patch-clamp studies of BLM
K+ channels have shown diversity in both experimental
design and findings (Tsuchiya et al., 1992
; Hunter,
1991
; Parent et al., 1988
; Kawahara et al., 1987
; Sackin
and Palmer, 1987
; Gögelein and Greger, 1987a
, 1987b
),
so a clear consensus has been elusive and details of the
properties and regulation are lacking.
We have now characterized the properties and regulation (see Mauerer et al., 1998) of the principal K+
channel in the BLM in a preparation of dissociated yet
polarized Ambystoma proximal tubule cells (Segal et al.,
1996
). Inwardly rectifying, ATP-sensitive K+ channels
were present in >95% of recordings from the BLM,
each containing from 2 to >25 KATP channels/patch.
Although the regulation of this proximal tubule BLM
K+ channel is similar to that of recently cloned KATP
channels in the apical membrane of the distal nephron
(ROMK1 and ROMK2), there are important differences, and ROMK has not been found in the proximal
tubule (Chepilko et al., 1995
; Zhou et al., 1994
; Lee and Hebert, 1995
; Boim et al., 1995
; Ho et al., 1993
).
The studies reported in this paper and the companion
paper elucidate the properties and the regulation, respectively, of the major K+ channel underlying the
BLM K conductance that is coupled to transport in the
proximal tubule.
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MATERIALS AND METHODS |
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Solutions and Drugs
The compositions of the solutions used are summarized in Table
I. After titration to pH 7.5 (710A; Orion Research, Boston, MA),
sucrose was added to adjust the osmolality of the solutions (3MO;
Advanced Instruments Inc., Needham Heights, MA). To determine channel conductance as a function of [K+], sucrose was
added to the standard pipette KCl to maintain osmolality. Chemicals used were of the highest quality and obtained from Sigma Chemical Co. (St. Louis, MO), except thallium acetate (Aldrich Chemical Co., Milwaukee, WI), diazoxide (Calbiochem Corp., La Jolla, CA), ATPS, and ADP (Boehringer-Mannheim Biochemicals, Indianapolis, IN). Nucleotides were prepared fresh daily as
20-50-mM stocks in bath solution. Glibenclamide was dissolved
in DMSO (100 mM stock).
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Cell Preparation
Dissociated proximal tubule cells were isolated from amphibian
kidneys as previously described (Segal et al., 1996). Briefly, aquatic phase Ambystoma tigrinum kept at 4°C were killed by submersion in 0.2% tricaine methanesulfonate. The kidneys were
rapidly removed and placed in iced HEPES-buffered NaCl at pH
7.5 (solution a). The adventitial tissue was removed by hand dissection, and the renal tissue was cut into 1-2 mm3 pieces and incubated in collagenase-dispase (0.2 U/ml of collagenase; Boehringer-Mannheim Biochemicals) on a gyratory shaker for 60 min
at 22°C. The enzyme reaction was stopped by washing with Ca2+-
and Mg2+-free NaCl (solution b). The cells were then mechanically dispersed into suspension by repeated trituration, and a pellet was obtained by centrifugation at ~1,600 rpm for 3 min. Finally, the cells were resuspended in 2.5 ml NaCl (solution a) in a
35-mm culture dish, and stored at 4°C until use. The dissociated
proximal tubule cells can retain their epithelial polarity for up to
14 d (Segal et al., 1996
). Cells were used for experiments from 2 to 12 d after dissociation. Representative cells as seen under light
microscopy (Fig. 1 A) and scanning electron microscopy (Fig. 1
B) are shown (for details of methods see Segal et al., 1996
).
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Electrophysiology
A 5-µl aliquot of cell suspension in NaCl storage solution (solution a) was placed on a Cell-TakTM-coated glass coverslip in a recording chamber of our design (RC-5/25; Warner Instruments, Hamden, CT) mounted on an inverted microscope (Olympus IM; Olympus America, Inc., Melville, NY). The chamber has a bath volume of 500 µl, and solutions are perfused directly into an input multiplexer on the chamber at a gravity-driven flow rate of ~10 ml/min.
Nonadherent cells were washed off the coverslip with the recording solution (either Ca2+-free NaCl, solution c, or KCl, solution d, unless otherwise noted) and the cells were visualized under Hoffman modulation optics (Modulation Optics, Greenvale,
NY). Proximal tubule cells were readily recognized by their characteristic morphology (see Fig. 1; Segal et al., 1996). An individual proximal tubule cell was selected for an experiment only if it
fulfilled the following criteria (Segal et al., 1996
): (a) distinctly
bilobated structure, (b) clearly defined brush border sharply delimited on the apical surface, (c) relatively smooth appearing basolateral membrane, and (d) absence of large vacuoles.
Patch clamp.
The standard configurations for single-channel
and whole-cell tight seal patch-clamp technique (Hamill et al.,
1981) were used to record channel currents from the BLM. Patch
pipettes were fabricated from borosilicate glass capillaries (Warner
Instruments, Hamden, CT) on a two-step puller (PP-83; Narishige
Co., Ltd, Tokyo, Japan), coated with Sylgard 184TM (Dow-Corning
Corp., Midland, MI) to within 200 µm of the tip, and fire-polished
just before use. When filled with KCl, the open tip pipette resistance was 3-8 M
when placed in the initial bath solution. A hydraulic micromanipulator (Narishige) was used to guide the patch
microelectrode to the BLM of the cell. High resistance giga-ohm
seals (up to 50 G
) were obtained on the BLM in ~75% of attempts by applying gentle suction to the pipette just after it
touched the cell membrane. To achieve the whole-cell configuration, further suction was applied to rupture the cell-attached
patch. Data have not been corrected for liquid-junction potentials since for most solutions they were <4 mV when measured as
follows: the bath Ag-AgCl ground electrode was connected to the
control KCl bath through a 3% agar bridge made of KCl pipette
solution. A low resistance (<1 M
) pipette filled with 3 M KCl
was placed in a KCl bath and the DC offset was adjusted to 0 mV
in zero current clamp. The liquid-junction potentials were measured as the voltage offset resulting when the control KCl bath
was replaced by the test solution. Low [Cl] solutions in which
90% of Cl
was replaced by aspartate
had a liquid-junction potential of 13.3 mV.
Data Analysis
Current data were played back and low pass filtered at 400 Hz (902LPF eight-pole Bessel filter; Frequency Devices Inc., Haverhill, MA), digitized at 1,000 samples/s, and stored on the PDP-11/ 23. In some cases, currents were filtered at 40 Hz and digitized at 100 samples/s for current binning and averaging analysis. Datafiles were transferred to a Pentium computer (Gateway 2000, North Sioux City, SD) via Kermit (Columbia University, NY) for analysis. Custom software for data acquisition and analysis was written in our laboratory using BASIC-23, AxoBASIC 1.0 (Axon Instruments, Foster City, CA), and Matlab 4.0 (The Mathworks, Natick, MA).
Channel activity (nPo) was calculated over periods of 60-500 s as follows. The closed current level (ic) was taken as the mode of the distribution around closed events. This current was subtracted from the current of a given bin, and the difference was multiplied by the number of events in that bin. The sum of these products yields the open channel area of the histogram. nPo is given by dividing the open channel area by the single-channel current, isc. That is,
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(1) |
For kinetic analysis, currents were filtered at a corner frequency (fc) of 2 kHz and sampled at 5,000 s1. An event (transition) was counted each time a data point crossed the 50% level of
the unitary channel current. For our recording system, the patch-clamp has a 5-kHz step response, a 5-kHz tape bandwidth, and a
2-kHz eight-pole Bessel filter, yielding an effective bandwidth
(
3 dB point, fceff) of 1.74 kHz. With these settings, the "dead
time" of the recording system is given by Colquhoun and Sigworth (1983)
, Tdead = 0.179/fceff. The "50% delay time" of the
Bessel filter is T50% = 0.506/fc. For fceff = 1.74 kHz and fc = 2 kHz,
Tdead = 102.8 µs and T50% = 253 µs. Therefore, events in time histogram bins <500 s were cut off. Since an event lasting 253 µs would
be the margin of detection, all dwell lifetimes <253 µs would be
missed events.
Open and closed dwell-time kinetics were fit to a probability density function expressed as a sum of exponentials,
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(2) |
where n is the number of open or closed states, the Ai are the relative amplitudes, and the i are time constants. This function was
transformed according to x = ln(t) and logarithmically binned (Sigworth and Sine, 1987), such that
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This function was used to fit the data using the Levenberg-Marquardt nonlinear least-squares fitting algorithm (Origin 4.0; Microcal Software, Inc., Northampton, MA) to find the appropriate set of 's. Note that if the errors follow a Gaussian distribution, this nonlinear least-squares method is equivalent to the method of maximum likelihood (Colquhoun and Sigworth, 1983
).
Dose-response relations for a drug (D) were fitted to the Hill equation as
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(4) |
where I/Imax is the fractional inhibition, Ki is the concentration of drug giving 50% inhibition, [D] is the concentration of the drug, and nH is the Hill coefficient. When nH = 1, this equation reduces to the Langmuir adsorption isotherm.
In the text, the number of observations or experiments is reported, whereas n in the analysis denotes either the whole data set or the subset of total experiments in which precise quantitation could be reliably applied. In some figures, a running average (using a specified window width) of current versus time is displayed. Statistical values for the n elements are given as mean ± SEM. Student's t test was applied where appropriate.
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RESULTS |
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Properties of the BLM K+ Channel
Overview.
With [K+] = 95 mM in the patch pipette, we
found the K+ channel in 551 of 559 seals made on the
BLM (98.6%). This K+ channel was never detected in
seals made on the apical membrane (0 of 16, 0%), consistent with our previous finding that dissociated Ambystoma proximal tubule cells retain epithelial cell polarity (Segal et al., 1996). Seals on the BLM typically
contained from 2 to >25 K+ channels. Despite numerous attempts to minimize patch area using small-tipped
pipettes (resistance
30 M
), patches appearing to
have one and only one channel were very infrequent
(n = 4, only 0.7%).
Cell-attached patches.
When cell-attached (c/a) patches
were made in NaCl bath, spontaneous inward K+ currents were usually observed (95%) at 0 mV (Vpip)
command potential. By briefly switching to zero current clamp mode, the resting membrane potential
(Vm) of the cell can sometimes be estimated if Rseal >>
Rpatch. Using this method under these conditions, Vm
averaged
37.2 ± 2.1 mV (n = 16), in good agreement
with Vm =
40 mV as measured by conventional impalement (Segal et al., 1996
). The Vm of the dissociated
cells is ~15-20 mV less than that for a cell in the intact
tubule (Sackin and Boulpaep, 1983
), suggesting an anion conductance exists at either the apical or basolateral membrane. Indeed, we have characterized a
cAMP-activated Cl
channel in the BLM of these cells,
which often appears in the same membrane patch as
the K+ channel (not shown). Alternatively, the isolated
cells may have acquired a nonspecific leak pathway that
shunts the normally high K diffusion potential. Since
the K+ and Cl
activity of the pipette solution (aPK = 0.80*[95] = 76 mM and aPCl = 0.78*[92] = 71.8 mM)
is greater than the intracellular K+ and Cl
activity
(aiK = 0.80*[68] = 54.4 mM and aiCl = 0.78*[20.5] = 16 mM (Sackin and Boulpaep, 1983
), yielding a reversal potential (Erev) of
28.7 mV. Thus, at
Vpip = 0 mV, the inward current must be carried by K+ moving down its
electrochemical gradient from the pipette into the cell.
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Excised patches. When BLM membrane patches were excised in the inside-out (i/o) configuration into the standard bath solutions (solution c or d), channel activity typically began to decline and then disappear. Addition of 0.2 mM ATP to the bath before or just after patch excision prevented rundown and maintained channel activity indefinitely.
Measurements of single-channel current with [K+] = 95 mM on both sides of the membrane patch (plus 0.2 mM ATP on the cytosolic side) demonstrate that the BLM K+ channel is a true inward rectifier (Fig. 3, A and B). The I-V relation in symmetrical [K+] inwardly rectifies and reverses very close to EK = 0 mV. The channel has an inward slope conductance of
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Voltage dependence of nPo.
Since BLM membrane patches
almost always contained more than one K+ channel,
nPo (channel activity) was used to assess the relative voltage dependence of Po. This assumes that the number of active channel proteins in an excised patch remains constant as voltage varies. (We take n as the maximum number of simultaneously open channels observed, which places a minimum on the actual number of channel proteins in the patch.) As was the case for
cell-attached patches, channel activity with KCl on both
sides of the excised membrane patch increases with increasing hyperpolarization (Fig. 3 C). Although the absolute values of nPo differed significantly among patches,
relative nPo increased e-fold per ~83-mV hyperpolarization between 40 and
120 mV, reflecting a 27%
increase of nPo for every 20 mV of hyperpolarization.
Role of magnesium.
It has been shown that [Mg2+]i
mediates at least part of the inward rectification in
other inwardly rectifying K+ channels (Matsuda et al.,
1987; Horie et al., 1987
; Ficker et al., 1994
) by blocking
outward currents in a voltage-dependent manner.
When Mg2+i was removed from the "cytosolic" side of i/o
patches (solution e plus 0.2 mM Na2ATP), we observed
flickering, and then rundown of the channel. This is in
sharp contrast to the behavior of ROMK1 channels, in
which channel rundown is slowed in a Mg2+-free bath
(McNicholas et al., 1994
). Since rundown occurs in the
absence of free Mg2+i and the presence of 0.2 mM
Na2ATP (n = 4), it appears that at least the complex of
Mg-ATP is required to prevent rundown. Interestingly, it has been shown that both Mg-ATP and free Mg2+i are
required to sustain channel activity for an ATP-insensitive inward rectifier K+ channel (Fakler et al., 1994
).
However, since higher levels of ATP block the BLM K+
channel (see below), it is not possible for us to dissociate the role of free Mg2+i from that of the Mg-ATP complex. Millimolar concentrations of ATP (or indeed, any
nucleotide) block the BLM K+ channel, and this effect
is independent of both free Mg2+i and Mg-ATP.
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Concentration and voltage dependence of isc. To further investigate the biophysical properties of this channel, we asked the question of how varying extracellular [K+] would affect the current carried by the channel. To isolate the change in [K+] from any change in driving force across the patch, the same [KCl] was used in the pipette and bath, thus clamping Erev to 0 mV. This approach allowed us to measure the change in absolute conductance while maintaining a constant relative permeability (i.e., we varied the Goldman-Hodgkin-Katz current equation while holding the result of the Goldman-Hodgkin-Katz voltage equation constant). Under these conditions, the command potential is the only driving force for net K+ movement across the membrane patch. Pipette and bath [KCl] ranged from 5 to 205 mM while osmolality was kept at 400 mosm/kg using sucrose as necessary. Since isc for [KCl] = 95 mM was the same in both standard KCl solution (solution d, 200 mosm/kg) or 400 mosm/kg KCl solution, the tonicity change itself does not significantly alter the conducting properties of the channel.
Channel events (inward current) were analyzed at command potentials of
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Cation selectivity.
The cation to anion preference of
the total BLM conductance was determined by salt dilution experiments using i/o membrane patches. Starting in symmetrical 95 mM K+ and 94.5 mM Cl (EK = ECl = 0 mV) and holding at
Vpip = 0 mV, the bath was
changed to a 14 mM K+ and 13.5 mM Cl
solution (solution i, EK = +48.6 mV, ECl =
49.4 mV) plus sucrose to maintain isoosmolality. This maneuver resulted in
large inward currents (n = 3), reflecting the cation
(K+) moving down its chemical gradient. The reversal
potential for this membrane current (including leak)
was at least +40 mV (n = 3). While holding at this Erev,
outward current developed when the 10% KCl bath was
replaced with 100% KCl, due to K+ moving along its
electrical gradient. Thus, the BLM conductance is cation selective.
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Selectivity among cations. Two approaches were used to determine the selectivity of this inwardly rectifying K+ conductance on the BLM. (a) Using c/a and i/o patches, the K+ in the patch pipette was replaced with the chloride salt of Na+ (n = 48), Rb+ (n = 4), Li+ (n = 4), Cs+ (n = 2), or NH14 (n = 7). Each solution was adjusted to pH 7.5 with the respective hydroxide salt. In all cases, c/a and i/o patches failed to show inward channel currents. These results strongly suggest that the BLM K+ channel is highly selective for K+ and excludes these cations, since channel activity is seen in >98% of seals made on the BLM when K+ is in the pipette. (b) Outside-out patches were made to exclude the remote possibility that the c/a and i/o patches used above did not contain any channels. The pipette was filled with KCl, and 0.2 mM Mg-ATP was included to prevent channel rundown. After recording in a KCl bath, the test cation was introduced into the bath (as the chloride salt) and the voltage protocol was repeated. KCl bath exchanges were interposed between test cations.
The cation selectivity as determined from outside-out patches is exemplified in Fig. 5. For Na+ and Li+, the current at
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Thallium.
Many types of K+ channels have been
shown not only to conduct Tl+, but often better than
they conduct K+ (Hille, 1992). To assess the Tl+ conductance of the BLM K+ channel, the patch pipette was
filled with 90 mM Tl-acetate (solution g ; the Cl salt of
Tl+ was not used due to its low aqueous permeability)
and the bath was filled with K-acetate (solution h). Under these conditions, inward Tl+ currents were observed in both c/a and i/o patches. The kinetic behavior of these channel events was notably different from
those seen when the channel conducts K+. When conducting Tl+, the channel openings displayed more
bursting, with each opening interrupted by fast flickery
closures. The probability that this is actually a different
channel is low since (a) the frequency of finding the K+
channel exceeds 98%, (b) other cation-selective channels were rarely observed, (c) the disparate kinetics
were only seen when Tl+ was in the pipette, and (d) Tl+
currents were sensitive to glibenclamide (see below).
Kinetics.
Due to the high density of this K+ channel
in the BLM, a patch apparently containing only one
channel is extremely rare. In over 550 seals, only four
membrane patches appeared to contain only one channel (0.7%). Since the open probability (Po) of the channel is only 0.05 ± 0.01 (n = 4), long recordings
were required to accumulate enough transitions for
meaningful analysis of the long closed state. Kinetic
analyses from such patches show that under resting
state conditions at Vpip =
60 mV, the BLM K+ channel has two apparent open states and two apparent
closed states. Parameters from one c/a patch and one
i/o patch show that the open dwell lifetimes are (ms):
o1 = 0.78 (c/a), 1.21 (i/o), and
o2 = 4.7 (c/a), 6.6 (i/o).
The closed dwell lifetimes are (ms):
c1 = 1.27 (c/a),
0.72 (i/o), and
c2 = 397 (c/a), 502 (i/o). Fig. 6 shows
the open and closed time histograms for a c/a patch.
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Inhibitor Profile
The BLM K+ channel is inhibited by barium and glibenclamide. The channel is insensitive to tetraethylammonium (up to 10 mM) applied either to the extracellular or cytoplasmic side.
Barium.
We have previously shown by recording single-cell membrane potential that the whole cell conductance is dominated by a barium-sensitive K+ conductance (Segal et al., 1996). Perforated patch whole-cell recordings show that the barium-sensitive whole
cell conductance inwardly rectifies. When 2 mM Ba2+
was included in the KCl pipette solution (unpaired experiments), channel openings were rare and a flickery
state was noted. In contrast to the voltage dependence
of channel activity without Ba2+ in the pipette (see Fig.
3 C), steady depolarization now has the effect of increasing nPo, and subsequent hyperpolarization reduced activity. This is presumably due to the voltage dependence of the Ba2+ block of the channel (data not
shown).
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Glibenclamide.
This sulfonylurea is known to inhibit
ATP-sensitive K+ channels in a number of epithelial tissues by binding to the sulfonylurea receptor (SUR).
SUR1 has recently been cloned (Aguilar-Bryan et al.,
1995) and is thought to associate with the KATP channel, thereby conferring sulfonylurea sensitivity. However, SUR1 may not be present in the kidney (Inagaki
et al., 1995
), which may explain in part the much
higher dose of glibenclamide required to inhibit renal
KATP channels (Hebert and Ho, 1994
). In this context,
the BLM K+ channel is glibenclamide sensitive, albeit
at "renal doses." We treated 22 patches with glibenclamide; 16 excised inside-out patches were exposed to
500 µM, while 6 cell-attached patches were exposed to
low (0.01-10 µM) concentrations. In 7 of 16 inside-out patches, 500 µM glibenclamide inhibited activity by
42.3 ± 5.6% (Fig. 7 B). Remarkably, the inhibition was
much more potent in the cell-attached patches: 10 µM
exerted an 83 ± 2% inhibition in three patches, and
100 nM exerted a 70 ± 3% inhibition in three other
patches (data not shown).
ATP Sensitivity
Similar to other KATP channels, low doses of ATP are required to prevent Ambystoma BLM K+ channel rundown, whereas millimolar doses inhibit channel activity. At a dose of 5 mM ATP, >90% of channel activity is inhibited. This effect is reversible as nPo returns to baseline when the bath [ATP] is returned to 0.2 mM (Fig. 8 A). The dose-response curve for ATP has a Ki ~ 2.4 mM (Fig. 8 B). The Hill coefficient of ~4 may suggest that the channel has a tetrameric structure, with each subunit possessing an ATP binding site. Thus, in the Ambystoma proximal tubule, the BLM K+ channel that appears to be the major K+ conductance of the cell is ATP sensitive.
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Among the nucleoside diphosphates, ADP is less potent than ATP (ADP inhibits by 65.6 ± 8.1%, n = 4, P < 0.01), but more potent than CDP, GDP, IDP, TDP, or UDP (data not shown). This suggests that the putative nucleotide binding site(s) recognize NDPs as well as NTPs, and that nucleotide hydrolysis is probably not occurring at this site. Indeed, even nucleoside monophosphates have a moderate inhibitory effect, although nucleosides themselves are without effect. The relative potency of the adenosine nucleosides (at 5 mM) in inhibiting the BLM KATP channel is ATP (93.3 ± 1.9%) > ADP (65.6 ± 8.1%) > AMP (38.7 ± 3.7%) > adenosine (1 ± 2%) (Fig. 8 C, n = 3-9).
Other nucleotides. The effect of nucleotides was tested in excised i/o patches. All the NTPs tested reversibly inhibited BLM K+ channel activity at a dose of 5 mM (ATP 93.3 ± 1.9%, n = 9; CTP 70.3 ± 10.0%, n = 5; GTP 62.3 ± 7.5%, n = 3; ITP 61.1 ± 0.8%, n = 2; TTP 53.7 ± 6.7%, n = 2; UTP 71.7 ± 5.7%, n = 2) (Fig. 8 C). These results suggest that each compound probably interacts with common cytoplasmic nucleotide binding site(s). Note that ATP is significantly more potent than the other nucleoside triphosphates (P < 0.02), but there is no significant difference among the nonadenosine nucleotides.
Rundown of the BLM K+ Channel
One characteristic of KATP channels is "rundown," a
gradual loss of activity when the membrane patch is deprived of cytosolic ATP (Findlay and Dunne, 1986).
Typically, both Mg2+ and ATP are required to prevent
rundown in KATP channels (Ashcroft and Ashcroft,
1990
). Likewise, the BLM K+ channel runs down in the
absence of either Mg2+ or ATP (or both). Lower concentrations of ATP (100-200 µM) will prevent or "rescue" channel rundown. The experiment shown in Fig. 9 A summarizes the characteristics of BLM K+ channel
rundown. Channel activity typically begins to decrease (rundown) upon excision of the membrane patch into
a nucleotide-free bath. If this process is allowed to continue, channel activity will cease, usually irreversibly.
When 0.2 mM of ATP is added back, channel activity
can be restored. When ATP is removed, all channels
rapidly close. In the continued presence of ATP-
S, readdition of ATP is again able to rescue rundown, and
activity returns to baseline upon washout of the ATP-
S. Frequently (but not invariably), ATP-
S has an inhibitory effect on single channel activity when added in
the presence of ATP, which is reversible as long as the exposure is not prolonged (n = 6), as shown in Fig. 9
B. When ATP-
S is added in the absence of ATP, channel activity runs down very quickly, usually irreversibly
(n = 4, data not shown).
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Thus, ATP-S cannot substitute for ATP in sustaining
channel activity, suggesting that phosphorylation itself
is not sufficient to prevent rundown. Although phosphorylation may be necessary, it appears that the nucleoside triphosphate must be hydrolyzable to maintain
channel activity. This hypothesis is supported by the
finding that CTP, GTP, ITP, TTP, and UTP could all
prevent or rescue rundown, but the corresponding
NDPs could not. Rescue does not appear to require the
cAMP-dependent protein kinase, since channel activity
can be restored even in the presence of a high concentration of protein kinase inhibitor (PKI, 1 µg/ml, P-0300;
Sigma Chemical Co.).
Since it has been reported that removal of free Mg2+
nearly abolishes rundown of KATP in cultured CRI-G1
insulin-secreting cells (Kozlowski and Ashford, 1990)
and partially inhibits rundown of ATP-regulated
ROMK1 channels excised in an ATP-free bath (McNicholas et al., 1994
), we assessed BLM KATP channel activity
under these conditions. The representative experiment
shown in Fig. 9 C shows that rundown of the BLM KATP
channel still occurs in the absence of ATP despite excision of the patch into a Mg2+-free bath.
Diazoxide.
The synthetic KATP channel opener diazoxide was applied to the cytoplasmic side of i/o patches.
It has been shown that this benzothiadiazine can open
KATP channels in the presence of Mg-ATP, but it may
have an inhibitory effect in the absence of Mg-ATP (Kozlowski et al., 1989). Initial excision of the patch into an ATP-free bath leads to channel rundown as discussed
above, and 200 µM diazoxide alone does not rescue
rundown. However, addition of 0.2 mM Mg-ATP in the
continued presence of, or after exposure to, diazoxide
increases channel activity well in excess of that before
rundown (n = 3, Fig. 10). The inhibitory effect of 5 mM ATP is not diminished in the presence of, or by
previous exposure to, diazoxide (n = 5, data not
shown).
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DISCUSSION |
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The proximal tubule (a leaky epithelium) can be considered to function within the general scheme of the
epithelial transport model first proposed by Koefoed-Johnsen and Ussing (1958) (KJU), in which the apical
membrane is primarily Na+ selective and the BLM is
primarily K+ selective. Although this model was first applied to tight epithelia such as frog skin (Koefoed
Johnsen and Ussing, 1958) and urinary bladder (Davis
and Finn, 1982), its essence holds for leaky epithelia such as small intestine (Gunter-Smith et al., 1982
) and
the proximal tubule (Matsumura et al., 1984
). In the
KJU model, maintenance of unidirectional Na transport requires that K moves in a closed circuit (recycling) across the BLM. That is, barring intracellular accumulation of K (or K+ secretion), the K+ pumped into
the cell by the pump must be matched by an outward K+ current across the BLM.
Steady state vectorial transport in the proximal tubule thus requires continuous activity of the basolateral
Na+,K+-ATPase pump, which consumes ATP and obligates intracellular accumulation of K+. A conductance
for K+ is necessary both to allow this K+ to recycle and
to maintain Vbl. Experiments performed by Matsumura et al. (1984) on perfused Necturus proximal tubules first
demonstrated that the BLM GK varies as a function of
pump activity, and they suggested that the regulation of
BLM GK was linked to cellular metabolism, as had been
previously proposed for red cells (Romero, 1978
) and
suspensions of rabbit cortical tubules (Balaban et al., 1980
).
This hypothesis was bolstered when the first single-channel records of an ATP-sensitive K+ channel (Ki = 0.1 mM) from cardiac muscle were published (Noma, 1983). Similar ATP-sensitive K+ (KATP) channels were
subsequently found in pancreatic
-islet cells (Cook
and Hales, 1984
), skeletal muscle (Spruce et al., 1985
),
and smooth muscle (Standen et al., 1989
). The Ki for
ATP is 10-100 µM for all these Type I KATP channels
(Ashcroft and Ashcroft, 1990
), and they are inhibited
by sulfonylurea agents (Edwards and Weston, 1993
). A
KATP channel with these Type I properties has not been
identified in epithelial tissues. However, a related KATP
channel was recently demonstrated in nonperfused
(Tsuchiya et al., 1992
) and perfused (Hurst et al., 1993
;
Beck et al., 1993
) rabbit proximal tubules. In preliminary experiments, we have also found this channel in
the BLM of nonperfused rabbit proximal tubule, and
in the present study we provide the first detailed description of the analogous BLM KATP channel in an amphibian using a novel preparation of dissociated proximal tubule cells that retain epithelial polarity (see Fig.
1 and Segal et al., 1996
).
Investigators have used several techniques to patch
the BLM, including mechanically stripping off the
basement membrane (Sackin and Palmer, 1987; Kawahara et al., 1987
) and tearing tubules to access the lateral membrane (Gögelein and Greger, 1987b
). However, enzymatic treatment of nonperfused tubules (Tsuchiya
et al., 1992
; Parent et al., 1988
), perfused tubules
(Hurst et al., 1993
; Beck et al., 1993
), and single cells
(Hunter, 1991
) has been employed most commonly.
We have previously shown that the dissociated Ambystoma proximal tubule cells (see Fig. 1) retain epithelial cell polarity (Segal et al., 1996). Indeed, the K+
channel described in the present study was present in
>98% of seals made on the BLM, but was never detected in the apical membrane. With high [K] on both
sides of the membrane patch, this BLM K+ channel exhibits inward rectification with
slope, in = 24.5 ± 0.6 pS
and
chord, out = 3.7 ± 0.4 pS. This non-ohmic behavior in both cell-attached and -free membrane patches is
due at least in part to a voltage-dependent open channel block by cytosolic Mg2+ (Matsuda et al., 1987
). Removal of Mg2+ from the cytosolic side of i/o patches
leads to an increase in outward conductance without affecting inward conductance. In c/a patches made on
frog BLM (NaCl pipette in a KCl bath), Hunter (1991)
estimated that the blocking site was 51.3% across the
membrane from the cytosolic side (i.e., 0.513). This
compares well to the value of 0.57 obtained for the Ambystoma BLM K+ channel in the present study.
Inward rectification of current is not necessarily due
to a decrease in the intrinsic outward conductance of
the channel pore. Indeed, Matsuda et al. (1987) first
described how ohmic single channel units could behave as rectifiers. They showed that inward rectification
of a cardiac myocyte K+ channel was mediated by two
effects: (a) Mg2+-independent voltage gating in which
ohmic channels are open at hyperpolarized voltages
and essentially inactivate upon depolarization, and (b)
a Mg2+-induced voltage-dependent open channel (fast)
block. Our data for the BLM K+ channel are in keeping
with these observations since (a) the conductance is
ohmic in the absence of cytosolic Mg2+, and (b) Mg2+
appears to cause a flickery open channel block of the
outward current in the steady state (e.g., see Fig. 3 A).
Naturally occurring polyamines (e.g., spermine) have
also been shown to mediate inward rectification of
some K+ channels (Ficker et al., 1994
). However, spermine (up to 5 mM) does not affect the Ambystoma BLM
K+ channel.
Properties of K+ conduction through the pore.
Constant-field
theory predicts that net K+ flux across the channel
should increase linearly with rising [K+]. Most channels do not exhibit this behavior due to "competition" for binding sites within the pore, and saturation occurs
at higher concentrations of the permeant ion (Hille,
1992). In our study, conductance versus [K+] was fit
with the Hill equation (Table II). Hill coefficients of
~1 suggest that one K+ ion interacts with the channel
at a time, allowing permeation to be modeled as follows:
![]() |
Voltage dependence.
Hyperpolarization increases nPo.
Such voltage sensitivity renders the channel susceptible
to changes in membrane voltage, a potential regulator
of the BLM K+ conductance. On the BLM in the Necturus proximal tubule, hyperpolarization increased K+
channel activity (Sackin and Palmer, 1987; Kawahara et
al., 1987
). In frog kidney, one study showed that nPo increased with hyperpolarization (Kawahara, 1990
), whereas
in another study, no voltage dependence of nPo was observed (Hunter, 1991
). In preliminary experiments using rabbit proximal tubule, we again found that hyperpolarization increases nPo, although Parent et al.
(1988)
reported the opposite. Note that for the cloned
renal K+ channels, ROMK1 (Ho et al., 1993
) and
ROMK2 (Chepilko et al., 1995
), which have not been
detected in proximal tubule, nPo decreases with hyperpolarization.
Selectivity and blockade.
The channel is highly selective
for K+ over a number of cations (Na+, Rb+, Li+, NH4+,
and Cs+) as well as over Cl. When K+ in the patch pipette was replaced by these cations, c/a and i/o patches failed to show inward channel currents. Paired
experiments in outside-out patches show that gK:gNa exceeds 30:1, and that the cation selectivity of the BLM
K+ channel is K+ >> Rb+
Cs+
NH4+ > Na+
Li+
(see Fig. 5). Detailed selectivity data are lacking in
other studies on BLM K+ channels in the proximal tubule, but more distal K+ channels, including ROMK,
conduct Rb+. With Rb+ in the patch pipette, gRb/gK = 0.36 for ROMK2 (Chepilko et al., 1995
). Under similar
conditions, we did not observe inward Rb+ currents
through the BLM K+ channel.
Sensitivity to Nucleotides, Sulfonylureas, and Activation by Diazoxide
Nucleotides.
The Ambystoma BLM K+ channel is sensitive to ATP, albeit at millimolar levels. Since the first
demonstration of an ATP-sensitive K (KATP) channel in
cardiac muscle (Noma, 1983) and pancreatic cells
(Cook and Hales, 1984
), potassium channels inhibited
by nucleotides have been described in a wide variety of
tissues (Ashcroft and Ashcroft, 1990
). In pancreatic
cells, KATP channels function in the regulation of insulin secretion. Ashcroft and Ashcroft (1990)
have classified such inwardly rectifying KATP channels as Type I
based on their exquisite sensitivity to ATP (Ki ~ 10-100 µM) and sulfonylureas.
Sulfonylureas.
In cell-free patches, the Ambystoma and
rabbit BLM K+ channels are (variably) inhibited by submillimolar levels of glibenclamide, whereas native cell
Type I KATP channels are sensitive to nanomolar concentrations of glibenclamide (Ashcroft and Ashcroft,
1990). Indeed, no renal and perhaps no epithelial K+
channel has the sensitivity to sulfonylureas (or ATP)
that Type I KATP channels possess. This may suggest that
either Type I KATP channels are only found in specific
tissues, or that associated molecules (e.g., the sulfonylurea receptor) conferring sulfonylurea sensitivity are
cell specific. In this regard, it is of interest that all but
one of the cloned KATP channels fail to exhibit the degree of sulfonylurea sensitivity seen in the native
cell
KATP channels. The exception is the KATP channel composed of Kir6.2 and the sulfonylurea receptor itself (Inagaki et al., 1995
).
Diazoxide.
Diazoxide is a benzothiadiazine K channel opener (KCO) that activates several types of KATP
channels. In many tissues, it is thought that activation
by KCOs requires Mg-ATP (Edwards and Weston, 1993;
Ashcroft and Ashcroft, 1990
), as illustrated for the BLM
K+ channel in Fig. 10. Diazoxide also does not alter the
requirement for Mg-ATP to prevent BLM K+ channel
rundown (see below). Indeed, diazoxide can be inhibitory in the absence of Mg-ATP, perhaps by accelerating
channel rundown (Kozlowski et al., 1989
). Therefore,
it is not likely that diazoxide exerts its effect on the rundown site of the BLM K+ channel.
Rundown of the BLM K+ Channel
A hallmark of KATP channels is that they exhibit the
phenomenon of rundown, a gradual loss of activity
when the membrane patch is deprived of cytosolic
ATP. As first noted by Findlay and Dunne (1986), rundown is "a paradoxical situation in that K+ channels
that are inhibited by intracellular ATP require intracellular ATP to retain the ability to open." In a recent
study on frog proximal tubule cells (Robson and
Hunter, 1997
), washout of intracellular ATP reduced
the whole-cell barium-sensitive conductance by ~60% over 10 min. Inclusion of 2 mM ATP in the patch pipette not only prevented this rundown, but the barium-sensitive conductance increased by 54% over 10 min.
Accordingly, the BLM K+ channel runs down in the
absence of ATP, and low concentrations of ATP (100-
200 µM) are required to prevent or rescue channel
rundown in cell-free patches. Once rundown has begun, application of ATP will restore channel activity,
but the ATP-restorable current decreases as the time
between rundown and ATP application lengthens. The
BLM K+ channel specifically requires Mg-ATP to support activity as rundown occurs if either ATP, Mg2+, or
both are removed (see Table III). Typically, both Mg2+
and ATP are required to prevent rundown in KATP
channels (Ashcroft and Ashcroft, 1990). Mg2+ is required as a cofactor, as channel rundown occurs in a
Mg2+-free solution despite the presence of Na-ATP.
Free Mg2+ concentrations as low as 200 nM are sufficient to prevent BLM K+ channel rundown in the presence of ATP. The possibility that free Mg2+ itself
(rather than the Mg-ATP complex) plays a distinct role in the rundown of the BLM K+ channel cannot be excluded, but is difficult to test since rundown occurs if
either Mg2+ or ATP is removed. Interestingly, free
Mg2+ itself (
10 µM) can prevent rundown of Kir2.1,
an ATP-insensitive inward rectifier (Fakler et al., 1994
).
|
Paradoxically, removal of free Mg2+ nearly abolishes
rundown of KATP in cultured CRI-G1 insulin-secreting
cells (Kozlowski and Ashford, 1990) and partially inhibits rundown of ATP-regulated ROMK1 channels excised in an ATP-free bath (McNicholas et al., 1994
).
The latter effect is presumed to occur via the inhibition of a Mg2+-dependent phosphatase (McNicholas et al.,
1994
). Thus, removal of Mg2+ inhibits rundown in
ROMK1, but actually produces rundown in the BLM
K+ channel (see Fig. 9 C). We conclude that the Mg2+-dependent phosphatase thought to be involved in the
rundown of ROMK1 (McNicholas et al., 1994
) does not
mediate rundown of the BLM K+ channel.
ATP can serve as a substrate in hydrolysis reactions
mediated by an ATPase and in phosphorylation reactions mediated by a kinase. We asked whether phosphorylation itself (without hydrolysis) can prevent BLM
K+ channel rundown, as has been proposed for other
channels, including ROMK1 (McNicholas et al., 1994).
To address this issue, we used ATP-
S, a poorly hydrolyzable ATP analogue that is an effective substrate for
most kinases (e.g., PKA) in (thio)phosphorylation reactions (Eckstein, 1985
). Similar to results reported for
the cell KATP channel (Ohno-Shosaku et al., 1987
), our
findings (see Fig. 9 and Table III) indicate that ATP-
S
cannot substitute for ATP in sustaining channel activity, whereas all hydrolyzable nucleoside triphosphates
we tested do prevent or rescue rundown. The corresponding nucleoside diphosphates were also ineffective.
In summary, rundown of the BLM K+ channel in excised patches seems to be prevented by a high affinity
nucleotide binding site, which hydrolyzes nucleoside
triphosphates in the presence of Mg2+. Rundown is
prevented by ATP even in the presence of high concentrations of a protein kinase inhibitor, strongly suggesting that typical protein phosphorylation processes
alone are insufficient to prevent/rescue channel rundown. Fakler et al. (1994) obtained similar results in
Kir2.1 channels.
Comparison with Other Studies
Gögelein and Greger (1987b) found a very weak inward
rectifier K+ channel on the lateral membrane of rabbit
proximal straight tubule. In symmetrical KCl with 1 mM Mg2+ in the bath, this channel had a limiting gin of
~45 pS and a limiting gout of ~40 pS. In contrast to the
results of the present study, their channel's open probability did not increase with hyperpolarization, and nPo
was unaffected by Ca2+ (<1 nM-1 µM) applied to inside-out patches (Mauerer et al., 1998
). They did not
observe channel rundown in excised patches, the channel was tetraethylammonium sensitive, and the effect of
ATP was not tested. Thus, it is not likely that their channel was the BLM K+ channel we find in Ambystoma proximal tubule.
Kawahara et al. (1987) found a 31-pS K+ channel in
42% of cell-attached patches made on the BLM of Necturus proximal tubule. They also found saturation of inward channel conductance at ~50 pS (compared with
~35 pS for Ambystoma) and an apparent Km of 65.5 mM
(compared with 77 mM in Ambystoma). Whereas we observed inward rectification for all [K], their I-V plots
were restricted to inward currents. Similar to our findings in Ambystoma, the K+ channel in Necturus showed
high selectivity (PK:PNa
10:1 in Necturus versus
30:1
in Ambystoma), channel activity increased with hyperpolarization, and external Na+ did not influence channel
behavior. They also found that hyperpolarization did
not affect channel open time; rather, the increase in
nPo was due to shortening a closed state lifetime. Significantly, Kawahara et al. (1987)
were unable to maintain
channel activity in excised patches, stating that "patches
became unstable and noisy after excision." It is likely
that this was channel rundown since this description is
reminiscent of the channel "choking" and rundown we
observe in an ATP-free bath.
Similarly, Sackin and Palmer (1987) observed two
BLM K+ channels in Necturus, a short open-time channel that they studied in detail, and a long open-time
channel that disappeared after excision, perhaps because of rundown. The short open-time channel is different from the BLM K+ channel of Ambystoma since it
was nonrectifying and did not rundown when excised
in an ATP-free bath. The long open-time channel was thought to be the same as that studied in cell-attached
patches by Kawahara et al. (1987)
, and probably corresponds to the BLM K+ channel in Ambystoma except the
channel in Necturus had a mean open time of ~60 ms,
much longer than that seen in our study.
Parent et al. (1988) made cell-attached patches on
rabbit BLM and found an inward-rectifier K+ channel
that had different properties than both the Ambystoma BLM K+ channels in the present study. Their voltage
dependence was opposite to ours, as they concluded
that hyperpolarization induced a long closed state.
Hunter (1991) made cell-attached patches on the
BLM of single frog proximal tubule cells and found
only one kind of K+ channel, an inward rectifier with
an inward gslope = 32.4 pS, and an outward gchord = 6.2 pS at +80 mV, similar to the one we studied on the
BLM of Ambystoma proximal tubule. Depolarization reduces the macroscopic BLM K+ conductance and single-channel conductance in both preparations. A Boltzmann fit of the g-V curves (rederived using Fig. 5 of
Hunter, 1991
, and the inset of Fig. 3 B in the present
study) shows a V1/2 = +31.7 mV and a width of 14.5 mV
for the BLM K+ channel in c/a patches of frog, compared with a V1/2 =
14.9 mV and a width of 21.3 mV
for the BLM K+ channel in i/o patches of Ambystoma.
Thus, the sensitivity of conductance versus voltage is
such that there is an e-fold change in g per 14.5 mV in
frog (c/a), and per 21.3 mV in Ambystoma (i/o). The
discrepancy in V1/2 may be due to different experimental conditions and levels of cytosolic Mg2+. Hunter
(1991)
also reported that the addition of glucose and/ or alanine did not affect BLM K+ channel activity in the
frog cells. Although addition of alanine depolarized Vm
> 30 mV, he was unable to explain the repolarization of the BLM under his experimental conditions. In contrast, the Ambystoma BLM K+ channel under discussion
is activated by these substrates, which can explain the
repolarization of the BLM (Mauerer et al., 1998
). In a
recent follow-up study, Robson and Hunter (1997)
proposed that, based on differential sensitivity to barium
and quinidine, the frog cells have two separate K+ conductances. The inwardly rectifying conductance that
was inhibited by both agents appears similar to the Ambystoma BLM KATP channel, although direct sensitivity
to ATP or glibenclamide was not tested in their study.
Tsuchiya et al. (1992) found an ATP-sensitive K+
channel in five patches made on the BLM of nonperfused rabbit S1 and S2 segments. With 145 mM K+ in
the patch pipette and a bath containing 140 mM Na+
and 5 mM K+, they reported an inward gslope = 56 pS,
although an I-V plot was not given. In c/a patches at a
command potential of 0 mV, they found a baseline nPo
of 0.72, much higher than that found in the present
study and the two studies in which perfused tubules were patched (see below) (Hurst et al., 1993
; Beck et
al., 1993
). Although 1 mM ATP applied to the cytoplasmic side of an inside-out patch reversibly reduced nPo
by 77% (from 0.70 to 0.16), the sensitivity of the basolateral conductance was reduced in a perfused tubule when 1 mM ATP was added to the bath.
Beck et al. (1993) were the first to patch clamp the
BLM of (collagenase-treated) perfused rabbit proximal
tubules, and found a K+ channel whose activity correlated to transport activity. This channel showed inward
rectification in c/a patches (with gin = 61 pS and gout = 17 pS) and Po was voltage independent. In a follow-up study (Hurst et al., 1993
), the same group showed that
2 mM ATP (i/o) inhibited channel activity by ~80%
and 100 µM diazoxide (c/a) effectively opened this
BLM K+ channel.
The ROMK family of K+ channels is, to date, the only
cloned ATP-regulated renal K+ channel (Ho et al.,
1993). The ROMK1 channel cloned from rat kidney is
an inwardly rectifying K+ channel with gin = 39 pS and
a high open probability (0.8-0.9) at voltages more depolarized than
60 mV, and is not activated by hyperpolarization. ROMK1 exhibits channel rundown when
membrane patches are excised in ATP-free bath, although ROMK1 is not sensitive to ATP or glibenclamide (Ho et al., 1993
). In situ hybridization studies
show that transcripts (mRNA) for ROMK are absent
from the proximal tubule (Lee and Hebert, 1995
).
Combined with the differences in mechanism of channel rundown (see above and Table III), it is highly unlikely that the BLM K+ channel is ROMK1, although it
may be within the same family of inwardly rectifying K+
channels with two transmembrane domains (Ho et al.,
1993
; Kubo et al., 1993
).
Contribution of the BLM K+ Channel to Total BLM Conductance
The component of BLM conductance (GBLM) due to the BLM K+ channel (GK) can be estimated as:
![]() |
(10) |
where g is the "physiologic" single channel conductance, n is the average number of channels in a patch, Po is the mean open time of one channel, f is the incidence of finding a channel in the patch, and a is the area of the membrane patch.
In the present study, the conductance (g) for outward currents was ~4 pS, the average nPo extrapolated
to 0 mV was 0.20, and channel activity was found in
98.6% of patches. Given that the average inner diameter of our patch pipette was 1.5 µm, the minimal membrane area of a patch is 1.77 µm2. Using these estimates
in Ambystoma, the value for GK is (4 pS*0.20*0.986)/ 1.77 µm2 = 0.446 pS/µm2 or 44.6 S/cm2 (RK = 22.4 k · cm2). Considering the estimate of GBLM = 51.3 µS/cm2
(RBLM = 19.5 k
· cm2) in Ambystoma proximal tubule
(Maunsbach and Boulpaep, 1984
), GK/GBLM = 44.6/
51.3 = 0.87. Thus, under the conditions used in our experiments, GK may account for 87% of GBLM. However,
to the extent that the area of the membrane patch is
larger than our estimate, GK will be smaller.
The basolateral membrane area of dissociated Ambystoma proximal tubule cells exhibits less infoldings
than these cells in situ (Segal et al., 1996). Assuming a
whole-cell membrane capacitance of ~120 pF (Segal et
al., 1996
) and a specific capacitance of 1 pF per 100 µm2, we can estimate that the surface of a single isolated cell is ~12,000 µm2. Using a BLM surface area of
10,000 µm2 (100 × 10
6 cm2) and a GBLM = 51.3 µS/
cm2, the total BLM conductance is 5.13 nS. Using the
transport number GK/GBLM of 0.87 from Table IV, GK
= 4.46 nS. Despite possible changes in membrane area
(e.g., due to membrane recycling) and/or channel
density that could occur in the dissociated cells over time, this is a reasonable estimate since perforated-patch whole-cell studies with KCl pipette and NaCl bath
(EK =
84 mV) show current that reverses at Vm =
40
mV with ~240 pA of outward current at 0 mV command potential, and a whole-cell conductance of 6 nS. Thus, the total BLM conductance is 5.13 nS, of which
4.46 nS is K conductance, and the total apical conductance is 0.87 nS (the difference of 6-5.13).
|
Similar comparisons between GK derived from single-channel data and GBLM based on cable analysis are shown in Table IV for Necturus, Rana, and rabbit proximal tubule, respectively. With the exception of the rabbit, the transport numbers show that the BLM conductance of all other species is dominated by K+ and, specifically for Ambystoma, KATP channels. From this information, one can estimate the K+ channel density on the BLM of dissociated Ambystoma proximal tubule cells. If Po was unity, the number of channels would be given by 4.46 nS/4 pS = 1,115 channels. However, since Po = 0.05, the number of K+ channels on the BLM is ~22,300 per cell; a channel density of 2.23 channels/µm2. Using the estimate of membrane patch area of 1.77 µm2, one would expect on average approximately four channels per patch, in good agreement with our experience.
Physiological Role of the BLM KATP Channel
The task of the proximal tubule under physiologic conditions is to effect transport; i.e., to maintain ionic flux
in the direction of reabsorption. The electrochemical
difference across the BLM of proximal tubule, given by
Vbl EK, is an invariably positive driving force promoting K+ efflux (Boulpaep, 1979
). The presence of other
conductances in the BLM with reversal potentials less
negative than EK provides a nonzero difference between EK and Vbl that maintains K efflux. The BLM
Na+,K+-ATPase pump loads the cell with two K+ ions to
effect the transcellular transport of three Na+ ions. An
efflux pathway for K+ is necessary to recycle K+ across
the BLM, thus permitting continuous operation of the
pump. Pump activity and the opening of these K+ channels hyperpolarize the BLM, favoring apical Na+ entry.
An interesting contrast is that the negative shift in membrane potential consequent to the opening of
KATP channels tends to depress cell function in excitable cells, but tends to promote transport in epithelia.
The magnitude of BLM K+ efflux must match the K+
current of the pump. The regulatory effects of hyperpolarization and intracellular ATP levels on the BLM
KATP channels thus subserve this role.
This system of a variable BLM K+ conductance coupled to changes in pump rate in a single cell has further implications when extrapolated to the level of an
epithelium with a paracellular shunt pathway. Such an
epithelial model should include all current pathways
(see Fig. 11, adapted from Fig. 5 of Sackin and Boulpaep, 1983); namely, (a) the active Na+ and K+ pump
currents, (b) the basolateral K+ leak current, (c) the
other basolateral leak currents, principally carried by
Cl
, and (d) the shunt current carried by Cl
and, to a
lesser extent, by Na+. In this equivalent electrical cell/
circuit, Kirchoff's current law at node A gives
![]() |
(11) |
|
where is
the transcellular component of Cl
absorption, and
Itotalshunt=IClshunt+INashunt (i.e., the sum of the paracellular
component of Cl
absorption and any Na+ backleak).
If the outward current through BLM K channels could be exactly matched to IKpump
, then Eq. 11 becomes
![]() |
(12) |
When Eq. 12 is satisfied, all actively transported Na+
is accompanied by Cl absorption (sum of transcellular
and paracellular), except for the backleak, INashunt
. Under these optimal conditions, the proximal tubule accomplishes NaCl reabsorption at maximum efficiency.
Note that if GKbl
did not increase with pump rate, IKbl
would rise only slightly due to the small increase in
driving force. Under these conditions, INapump>IClbl+ (IClshunt+INashunt)
and the efficiency of NaCl reabsorption
deteriorates. That is, the incremental increase in NaCl
reabsorption would be less than that of the pump.
Therefore, the tight linkage between the Na+,K+-ATPase pump and the K+ leak through the BLM KATP channels is especially important in leaky epithelia that absorb large, fluctuating quantities of NaCl such as the
proximal tubule. The regulation of the BLM KATP channels and how they are coupled to the pump during
transport is the subject of the companion paper
(Mauerer et al., 1998).
![]() |
FOOTNOTES |
---|
Address correspondence to Alan S. Segal, Department of Medicine, University of Vermont, 55A South Park Drive, Colchester, VT 05446. Fax: 802-656-8915; E-mail: asegal{at}zoo.uvm.edu
Received for publication 16 January 1997 and accepted in revised form 30 October 1997.
This work is dedicated to the memory of Dr. Roman Mauerer (father of Ulrich Mauerer), who passed away during the preparation of the manuscript. The authors thank Ms. Christine Macol for excellent technical assistance.This work was supported by grant DK-17433 from the National Institutes of Health (NIH). Dr. A. Segal is a recipient of a Physician-Scientist Award from the NIH (DK-02103).
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