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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Functional coupling of Na+,K+-ATPase pump activity to a basolateral membrane (BLM) K+ conductance is crucial for sustaining transport in the proximal tubule. Apical sodium entry stimulates pump activity, lowering cytosolic [ATP], which in turn disinhibits ATP-sensitive K+ (KATP) channels. Opening of these KATP channels mediates hyperpolarization of the BLM that facilitates Na+ reabsorption and K+ recycling required for continued Na+,K+-ATPase pump turnover. Despite its physiological importance, little is known about the regulation of this channel. The present study focuses on the regulation of the BLM KATP channel by second messengers and protein kinases using membrane patches from dissociated, polarized Ambystoma proximal tubule cells. The channel is regulated by protein kinases A and C, but in opposing directions. The channel is activated by forskolin in cell-attached (c/a) patches, and by PKA in inside-out (i/o) membrane patches. However, phosphorylation by PKA is not sufficient to prevent channel rundown. In contrast, the channel is inhibited by phorbol ester in c/a patches, and PKC decreases channel activity (nPo) in i/o patches. The channel is pH sensitive, and lowering cytosolic pH reduces nPo. Increasing intracellular [Ca2+] ([Ca2+]i) in c/a patches decreases nPo, and this effect is direct since [Ca2+]i inhibits nPo with a Ki of ~170 nM in i/o patches. Membrane stretch and hypotonic swelling do not significantly affect channel behavior, but the channel appears to be regulated by the actin cytoskeleton. Finally, the activity of this BLM KATP channel is coupled to transcellular transport. In c/a patches, maneuvers that inhibit turnover of the Na+,K+-ATPase pump reduce nPo, presumably due to a rise in intracellular [ATP], although the associated cell depolarization cannot be ruled out as the possible cause. Conversely, stimulation of transport (and thus pump turnover) leads to increases in nPo, presumably due to a fall in intracellular [ATP]. These results show that the inwardly rectifying KATP channel in the BLM of the proximal tubule is a key element in the feedback system that links cellular metabolism with transport activity. We conclude that coupling of this KATP channel to the activity of the Na+,K+-ATPase pump is a mechanism by which steady state NaCl reabsorption in the proximal tubule may be maintained.
Key words: ion channel; kidney; patch-clamp; sulfonylurea; epithelia ![]() |
INTRODUCTION |
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A consistent finding in essentially all Na+-reabsorbing
or Cl-secreting epithelia is an increase in basolateral
membrane (BLM)1 K conductance (GK) concurrent with
the activity of the Na+,K+-ATPase pump, and vice-versa
(Schultz, 1992
; Beck et al., 1994
). Using rabbit kidney
cortical tubule suspension, Balaban et al. (1980)
directly demonstrated that inhibition of the Na+,K+-ATPase
pump increased the ATP/ADP ratio, and concluded
that [ATP]i, [ADP]i, or their ratio could be the link
coupling cellular metabolism to transport. The demonstration of Type I KATP channels in cardiac muscle
(Noma, 1983
) opened the way to study the regulatory
role of ATP at the single-channel level. Since this topic
has been treated in several excellent reviews (e.g., see
Beck et al., 1994
; Lang and Rehwald, 1992
; Edwards
and Weston, 1993
; Ashcroft and Ashcroft, 1990
), as well
as in Mauerer et al. (1998)
, the following is limited to
renal K+ channels.
In the distal nephron, Hunter and Giebisch (1988)
reported a Ca2+-activated K+ channel in the apical
membrane of Amphiuma early distal tubule that was inhibited by ATP with a Ki of ~5 mM. This was the first ATP-sensitive K+ channel described in epithelia, and
was classified as Type 3 (Ashcroft and Ashcroft, 1990
)
because of its Ca2+ activation and lower sensitivity to
ATP. Moreover, this "maxi-K" channel was depolarization activated and did not exhibit rundown. Subsequently, Wang et al. (1990)
demonstrated that the K+
channel in the apical membrane of rat cortical collecting duct, first identified by Frindt and Palmer (1989)
,
was ATP sensitive. This 35-pS channel, which mediates
K+ secretion by principal cells, is Type 1-like (Ashcroft
and Ashcroft, 1990
) in that it is insensitive to Ca2+, voltage independent, and exhibits rundown (Wang and
Giebisch, 1991a
; Wang et al., 1990
). Thus, the single-channel findings in distal nephron segments were partially consistent with the notion that [ATP]i could link
cell metabolism and K+ conductance, although Schultz
(1981)
had specified that the relevant K conductance
was on the BLM. Two K+ channels have been identified
in the lateral membrane of rat cortical collecting duct
(Wang et al., 1994
), but they do not appear to be ATP
sensitive.
The situation in the proximal tubule, however, is
more compatible with homocellular regulation (Schultz,
1981). In their study of Necturus proximal tubule, Matsumura et al. (1984)
showed that inhibition of Na+,K+-ATPase activity using ouabain, low bath K+, or low luminal Na+ perfusate brought about a fall in BLM GK.
They suggested that this effect might be a metabolic
consequence of pump inhibition, but emphasized
[Ca2+]i as the proximate signal coupling pump activity
to BLM GK. Once it became evident that Type 1 KATP
channels were not Ca2+ activated, the focus shifted to
ATP itself as the link between pump activity and BLM
GK. Beck et al. (1991a)
quantitated the inverse relationship between Na+ transport and [ATP]i in perfused
rabbit proximal tubule and confirmed a qualitative correlation between [ATP]i and GK. Shortly thereafter, Tsuchiya et al. (1992)
observed that ATP reversibly inhibited BLM KATP channel activity in four of five inside-out patches pulled off the BLM of nonperfused rabbit
tubules, but regulation on the single-channel level was
not pursued in that study. Beck et al. (1993)
looked at
regulation of stretch-insensitive BLM K+ channels in
perfused rabbit tubules and found that the addition of
luminal substrates depolarized the cell and increased
channel activity. In a follow-up study, the same group
(Hurst et al., 1993
) showed that this channel could be
activated by diazoxide and inhibited by pump inhibition or direct application of ATP. These data, combined with our findings in nonperfused rabbit tubules and dissociated Ambystoma proximal tubule cells (see
Mauerer et al., 1998
) that maintain epithelial polarity
(Segal et al., 1996
), clearly show that a Type 1-like KATP
channel exists on the BLM of the proximal tubule.
In the present study, we have used the dissociated
Ambystoma proximal tubule cells (Segal et al., 1996) to
investigate the regulation of this BLM KATP channel by
protein kinases, intracellular nucleotides, pH (pHi),
Ca2+, and the cytoskeleton. We also show that regulation of the KATP channel is indirectly linked to transport
dynamics in the proximal tubule through changes in
intracellular [ATP], resulting from altered activity of
the Na+,K+-ATPase pump as transport is modulated.
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MATERIALS AND METHODS |
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Solutions and Drugs
The composition of the solutions used is 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). KCl solutions containing low levels (50, 100, 200, 500, and 1,000 nM) of
free Ca2+ were prepared by adding the appropriate amount of
CaCl2 (0.407, 0.579, 0.733, 0.873, and 0.933 mM, respectively) to
solution d (Table I). Free Mg2+ was maintained at ~1 mM except
in solution b (divalent-free NaCl). In solutions containing ATP,
the nucleotide was added as the Mg-salt to maintain the free
Mg2+ at ~1 mM (range 0.98-1.33 mM). Chemicals used were of
the highest quality and obtained from Sigma Chemical Co. (St.
Louis, MO), except ADP (Boehringer-Mannheim Biochemicals,
Indianapolis, IN). Nucleotides were prepared fresh daily as 20-
50-mM stocks in bath solution. Stock solutions of glibenclamide
(100 mM), PMA (10 mM), and 4-PMA (10 mM) were dissolved
in DMSO. Stock solutions (10 mM) of forskolin, 1,9-dideoxyforskolin (Calbiochem Corp., La Jolla, CA), and nigericin were dissolved in ethanol. The catalytic subunit of the cAMP-dependent
protein kinase A (Promega Corp., Madison, WI) or protein kinase C (0.5 U/ml; Promega Corp.) was pipetted directly into the
bath.
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Cell Preparation
Dissociated proximal tubule cells were isolated from amphibian
kidneys as previously described (Segal et al., 1996). Briefly, kidneys from Ambystoma tigrinum were rapidly removed and placed in iced HEPES-buffered NaCl at pH 7.5 (solution a). After incubation in collagenase-dispase (0.2 U/ml collagenase; Boehringer-Mannheim Biochemicals), the enzyme reaction was stopped by
washing with Ca2+- and Mg2+-free NaCl (solution b). Dissociated
cells were resuspended in 2.5 ml of NaCl (solution a) in a 35-mm
culture dish, and stored at 4°C until use. The dissociated proximal tubule cells 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. A representative cell is shown in Fig. 1 of
Mauerer et al. (1998)
.
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Electrophysiology, Data Acquisition, and Analysis
The methods are essentially as described in Mauerer et al.
(1998). These include the mounting and selection of single proximal cells for electrophysiological study. The standard tight-seal patch-clamp configurations for single-channel recording were
used (Hamill et al., 1981
), and channel currents were acquired
and analyzed as described in the accompanying paper. Briefly,
channel activity (nPo) was calculated over periods of 60-500 s,
and open and closed dwell lifetimes were determined as reported
elsewhere (Mauerer et al., 1998
).
The number of observations or experiments is reported in the text, 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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The regulation of the BLM KATP channel by PKA, PKC, [Ca2+]i, and pH was studied in cell-attached (c/a) and inside-out (i/o) patches. Channel activity in response to perturbations of cell volume was examined in c/a patches, and the effect of membrane stretch and the role of the cytoskeleton was also tested. Finally, the coupling of channel behavior to changes in cellular energy levels and transport activity was investigated.
Forskolin activates the BLM KATP channel.
The cAMP second messenger system was studied in c/a patches using
forskolin (FK), which increases [cAMP]i by activating
adenylyl cyclase. In each experiment, a cell served as its
own control. Fig. 1, A and B shows a representative experiment. Under control conditions with KCl in the pipette (solution d) in a NaCl bath (solution c), steady
state channel activity was recorded for 3-5 min before
the application of 10 µM FK at a command potential of
60 mV. Within 1-2 min, the single-channel current
begins to increase, consistent with hyperpolarization of
the cell membrane potential (Vm). This is most likely
due to the opening of K+ channels on the cell membrane that precede those under the patch pipette itself.
Within 5-10 min, FK increases the nPo of the BLM KATP
channels in the patch of membrane under study.
PKA directly activates the BLM KATP channel.
To test the
hypothesis that the channel is activated by the cAMP-dependent protein kinase, the effect of the catalytic
subunit of PKA was tested in i/o patches. To prevent
channel rundown, i/o patches were excised in a bath
containing 0.2 mM ATP (see Mauerer et al., 1998). After a control period in 0.2 mM ATP, 50-100 U/ml PKA
was directly added to the bath in the continued presence of ATP (n = 29). Channel activation occurred after a delay of 0.5-3 min (n = 26). This delay is most
likely due to the time necessary for the reagents to diffuse to the patch, and then phosphorylate the channel(s). In some experiments, a low [Cl] bath was used to isolate the effect of PKA on the K+ channel since
phosphorylation via PKA also activates a cystic fibrosis
transmembrane regulator (CFTR)-like Cl
channel
present in the BLM (Segal et al., 1996
). The mean nPo increase was 5.1 ± 0.7-fold in patches containing K+
channels exclusively (n = 6).
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PKA phosphorylation is necessary but not sufficient for
channel activation.
ATP can serve as a substrate for (a)
a kinase in a phosphorylation reaction, and/or (b) an
ATPase in a hydrolysis reaction. It is possible that either
or both are required for channel activation, but the use
of ATP does not allow the two to be dissociated. To test
the hypothesis that phosphorylated channels remain
active after the removal of hydrolyzable nucleotides,
the poorly hydrolyzable nucleotide ATP-S (0.2 mM)
was substituted for 0.2 mM ATP in the presence of
PKA. In this condition, the patch can be fully phosphorylated since ATP-
S is a substrate for PKA. Indeed, thiophosphorylation may be even more resistant to phosphatases than phosphorylation (Eckstein, 1985
). Fig. 3
shows that, after a period of steady channel activity in
0.2 mM ATP, activity begins to decrease upon substitution of ATP with 0.2 mM ATP-
S. Subsequent addition
of csPKA to thiophosphorylate the patch did not restore activity; in fact, the channels run down. Thus, because ATP-
S and PKA were not sufficient to restore or
even sustain channel activity (n = 7), it appears that
phosphorylation per se is necessary but not sufficient to
maximally activate the BLM KATP channel. That channel rundown occurs despite the presence of ATP-
S
and PKA confirms our previous finding that the BLM
KATP channel also requires submillimolar levels of a hydrolyzable nucleotide (see Mauerer et al., 1998
).
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Kinetics of activated channels.
Kinetic analysis was performed on the two patches that contained only one
channel both before and after stimulation with FK (c/a)
or PKA (i/o with 0.2 mM ATP in the bath). This analysis reveals that the major effect of PKA phosphorylation
is to activate the channel by shortening, or possibly
eliminating, the dwell time of the longest closed state.
Under control conditions, the mean lifetime of the
longest closed state is ~397 (c/a, Fig. 4 A, top) and 502 (i/o, Fig. 4 B, top) ms (see Mauerer et al., 1998). After
activation by FK or the catalytic subunit of PKA, the
mean dwell time of this state decreases to 7 (c/a, Fig. 4
A, bottom) and 100 (i/o, Fig. 4 B, bottom) ms, respectively. Note that there is more pronounced shortening
occurring in the c/a mode (98%) compared with the
i/o mode (80%). Activation by FK or PKA did not affect the open state dwell times.
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Stimulation of PKC inhibits the BLM KATP channel.
To determine whether PKC is involved in the regulation of
this K+ channel, c/a patches were treated with the
phorbol ester PMA. As shown in Fig. 5, the application
of 10 µM PMA markedly decreases channel activity concomitant with a reduction in isc, consistent with depolarization of Vm. Similar results were obtained in eight cells with an average reduction in nPo of 63 ± 11% (n = 6). Based on an inward slope conductance of 22.2 pS
under similar conditions (see Mauerer et al., 1998), the
mean decrease of isc of 0.28 ± 0.11 pA (n = 6) reflects
an average depolarization (
Vm) of 12.0 ± 4.6 mV. A
phorbol ester that does not activate PKC was used as a
control. In five cell-attached patches, the addition of 10 µM 4
-PMA did not affect nPo (Fig. 5).
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pH Sensitivity
The activity of the BLM KATP channel is pH sensitive.
Directly lowering the pH on the cytoplasmic side of an
i/o patch from 7.5 to 6.8 (a fivefold increase in [H+]i)
decreases channel activity by 81 ± 2% (n = 3, Fig. 7 A).
To assess whether this effect occurs under more physiological conditions, an intracellular acidosis was generated during cell-attached recording using the K+/H+
exchanger nigericin (Margolis et al., 1989) in a low K+
bath (solution c, Fig. 7 B). We have previously shown
that under these conditions, pHi decreases by ~0.4 pH
units in the presence of 10 µM nigericin (Segal et al.,
1992
). This protocol decreases the nPo of the BLM KATP
channel by 61 ± 3% (n = 3).
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Effect of Calcium
The BLM KATP channel does not require Ca2+ for activity since excising membrane patches into an EGTA-buffered Ca2+-free solution (solution c) does not alter channel current. However, increases in [Ca2+]i inhibit the channel. The addition of the calcium ionophore ionomycin has no effect on cell-attached channel currents recorded from cells bathed in Ca2+-free solution (Fig. 8, A and B). However, in the continued presence of ionomycin, raising bath Ca2+ ([Ca2+]o) to 1 µM (solution e) promptly and significantly reduces nPo by 63 ± 9% (n = 4). This effect appears to be direct since exposing the cytoplasmic side of excised patches to varying [Ca2+]i produces a dose-dependent decrease in channel activity with a Ki = 166 nM (Figs. 8, C and D). However, since the excised membrane may still contain elements of Ca2+-activated signal transduction pathways (e.g., Ca2+/calmodulin kinase, PKC), a possible indirect effect cannot be fully ruled out. Indeed, the Hill coefficient of 0.73 may suggest that Ca2+ has both direct and indirect effects. In any case, an increase in [Ca2+]i leads to a decrease in channel activity.
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Membrane stretch.
Several classes of cationic channels
are known to be stretch-activated, including a K+ channel on the BLM of Necturus proximal tubule (Sackin,
1989). Application of negative pressure up to
60 mm
Hg to the patch pipette using a calibrated electronic
negative pressure generator (DPM-1B; Bio-Tek Instruments, Inc., Winooski, VT) had no detectable effect on
the nPo of this BLM KATP channel in either c/a or i/o patches (n > 100). Thus, the BLM KATP channel of Ambystoma proximal tubule is not stretch activated and is
distinct from the SA-K found on the BLM of Necturus
proximal tubule (Filipovic and Sackin, 1992
).
Cell swelling.
To assess whether the BLM KATP channel might be involved in cell volume regulation, nPo in
c/a patches was monitored as measured bath tonicity
was reduced by 30% from 200 (solution g) to 140 (solution h) mosM/kg. Despite clearly observable cell swelling due to the hypotonic shock, KATP channel activity
did not increase and actually tended to decrease. This
slight inhibition may be mediated either by an increase
in [Ca2+]i and/or activation of PKC, since cell swelling
is often associated with an increase in [Ca2+]i (Robson
and Hunter, 1994a, 1994b
; Ubl et al., 1988
). Nonetheless, measurements made in zero current clamp showed
that Vm hyperpolarizes (by 15-20 mV) during hypotonicity, probably due to the opening of a swelling-activated K+ exit pathway. It does not appear that the BLM
KATP channel mediates swelling-activated K+ efflux.
Cytoskeleton.
The cytoskeleton is a major factor in
maintaining epithelial polarity in the dissociated cells
(Segal et al., 1996). It is also well established that cytoskeletal elements are important in anchoring some
proteins to their membrane (Morrow et al., 1989
). More recently, it has been demonstrated that Na+ (Prat
et al., 1993
), K+ (Wang et al., 1994
), and CFTR Cl
channels (Prat et al., 1995
) can be regulated by the cytoskeleton.
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Functional Coupling of the BLM KATP Channel to Na+,K+-ATPase Pump Activity
Sustained reabsorption of salt and water in the proximal tubule requires the action of the Na+,K+-ATPase pump on the BLM. As K+ ions are pumped into the cell, a basolateral exit pathway must exist to allow K+ to recycle, thus permitting continuous operation of the pump. The functional coupling of these two transport elements may be linked by the intracellular [ATP] ([ATP]i) level. This was examined in c/a patches.
Inhibition of transport.
Retarding pump activity should
result in less ATP consumption and a rise in [ATP]i.
This was achieved either using either (a) cardiac glycosides (ouabain or strophanthidin), or (b) removal of
extracellular K+. Both methods were used in an attempt to dissociate the rise in [ATP]i from a voltage-dependent effect, since the former depolarizes Vm while
the latter (at least initially) hyperpolarizes Vm. However, it must be noted that these maneuvers result in
concomitant changes in Vm, [ATP]i, and even [K]i.
Since nPo of the KATP channel decreases with depolarization (see Fig. 3 C in Mauerer et al., 1998), it is not
possible to be certain how much of the fall in nPo is due to
Vm versus
[ATP]i. Applying any of these maneuvers resulted in a gradual decrease of BLM KATP channel activity, as illustrated in Fig. 10.
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Stimulation of transport. A rise in intracellular [Na+] is the primary physiological stimulus for the Na+,K+-ATPase pump. Na+ entry via apical membrane Na-coupled cotransporters was enhanced by the addition of glucose (5 mM), alanine (5 mM), or both (2.5 mM each) to a NaCl bath while recording KATP channel activity from the BLM. Fig. 11 depicts a representative experiment in which 5 mM alanine was added to the bath. Within a minute of substrate addition, BLM KATP channel activity markedly increased despite an initial depolarization due to Na+ entry. Comparable effects were seen in 17 of 19 (89%) cells exposed to glucose and/or alanine, with an approximately twofold increase in nPo (1.95 ± 0.20, n = 5).
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ATP:ADP ratio. Stimulation of transport leads to activation of the Na+,K+-ATPase pump while the resultant fall in [ATP]i disinhibits the BLM KATP channel. However, the increased turnover of the Na+,K+-ATPase pump produces a rise in [ADP]i and/or a fall in the [ATP]i:[ADP]i ratio. It is not clear which of these signals is the primary determinant of BLM KATP channel activity in intact cells. This issue was examined in i/o patches in two ways: (a) varying the ratio of [ATP]: [ADP] while keeping the [ATP] constant, and (b) varying the ratio of [ATP]:[ADP] while keeping the sum of [ATP] + [ADP] constant. The latter was performed for sums of 0.5, 2.5, and 5 mM.
To test whether ADP is able to relieve the block by ATP on the BLM KATP channel, increasing [ADP] were added to the cytoplasmic side of i/o patches in the presence of 5 mM ATP (Fig. 13 A). Channel activity was almost completely and reversibly blocked by 5 mM ATP alone (Fig. 13 A, middle), and addition of 5 (left) or 10 (right) mM ADP did not relieve the block. Indeed, the block became more pronounced as the total concentration of nucleotide was raised.
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DISCUSSION |
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The properties of the BLM K+ (KATP) channel were the
subject of the companion paper (Mauerer et al., 1998).
The regulation of basolateral K+ channels was not examined in most previous studies (Hunter, 1991
; Sackin
and Palmer, 1987
; Kawahara et al., 1987
; Parent et al., 1988
; Gögelein and Greger, 1987
). In the present
study, we have investigated the regulation of this Type
1-like KATP channel in the BLM of dissociated Ambystoma proximal tubule cells that maintain epithelial
polarity (Segal et al., 1996
).
The BLM KATP Channel Is Regulated by PKA and PKC, but in Opposing Directions
The proximal tubule BLM KATP channel is activated by
PKA, both in cell-attached patches via stimulation of
adenylyl cyclase-elevating cAMP levels, or directly by
PKA itself in inside-out patches. In contrast, this channel is inhibited by PKC, either by treating cell-attached
patches with phorbol ester, or directly by PKC in excised
patches. Opposite regulation by PKA and PKC has been
reported for other K channels (Chen and Yu, 1994;
Fakler et al., 1994
) including KATP channels (Bonev and
Nelson, 1993
; Honore and Lazdunski, 1993
; Zhang et
al., 1994
) in the kidney (Wang and Giebisch, 1991b
).
Activation by PKA.
In cell-attached experiments, stimulation of endogenous cAMP-dependent protein kinase
(PKA) increases channel activity and single-channel current, the latter most likely due to hyperpolarization of
the cell secondary to K+ channel opening. Activation of
channel by direct exposure of cell-free membrane patches
to the catalytic subunit of PKA strongly suggests that phosphorylation of a PKA site on the channel itself (or a
closely associated protein) increases the mean open time. PKA increased channel activity by 5.1 ± 0.7-fold, compared with 7.50 ± 1.55-fold in the case of forskolin.
This difference is at least in part due to the additional
stimulatory effect of membrane hyperpolarization (see
Mauerer et al., 1998) in the cell-attached experiments.
Kinetic analysis limited to patches containing one channel reveals that PKA phosphorylation does not significantly affect the open states, rather the major effect appears to be shortening of the longest closed state.
Inhibition by PKC.
In contrast to the effect of FK,
treatment of cell-attached patches with PMA, a phorbol
ester known to stimulate protein kinase C, results in a
decrease in channel activity and depolarization as K+
channels close. Channel activity did not change in control experiments with 4-PMA, a phorbol ester that does
not stimulate PKC. Subsequent removal of PMA did not
restore channel activity, suggesting that phosphorylation
by PKC is long lasting or that adequate specific phosphatase activity was not present. In inside-out patches
from rabbit ventricular myocytes, Light et al. (1995)
found PKC reversibly inhibited the KATP channel, and
that a phosphatase inhibitor (okadaic acid) prevented
channel recovery. These observations strongly suggested
that the membrane patch contained an endogenous
protein phosphatase. For the BLM KATP channel, the irreversible inhibitory effect of PKC in inside-out patches
strongly suggests that such a phosphatase was not present in our membrane patches. Future experiments will
be aimed at determining the role of phosphatases in
the regulation of the BLM KATP channel.
Metabolic Regulation of the BLM KATP Channel
Schultz (1981) emphasized the implications of the Koefoed-Johnsen and Ussing model originally developed
for frog skin (Koefoed-Johnsen and Ussing, 1958
) as
applied to Na-transporting epithelia operating over a
wide range of transport rates, and reviewed the data
then emerging that the K conductance of the basolateral membrane was correlated to pump activity, an example of what Schultz termed a "homocellular regulatory mechanism." Accordingly, Na+ entry across the
apical membrane should be matched by basolateral Na+ efflux, thus maintaining a low intracellular [Na+]
as Na+ is transported across the epithelium. This process is mediated by cross talk between opposing membranes. Schultz's group reported cross talk operating in
leaky epithelia when apical Na+ entry was increased in
amphibian small intestine via rheogenic Na-coupled
cotransport (Grasset et al., 1983
; Gunter-Smith et al.,
1982
). They found that addition of alanine to the mucosal solution promptly increased the Na conductance
of the apical membrane (with depolarization), and secondarily brought about an increase in K conductance
of the BLM (with hyperpolarization). In the Koefoed-Johnsen and Ussing model, maintenance of unidirectional Na transport requires that K moves in a closed
circuit (recycling) across the BLM. Coordination of
pump activity and basolateral K conductance is necessary to avoid large fluctuations in intracellular [K+]
and volume, and to maintain a driving force for Na entry across the apical membrane.
It is important to recognize that pump activity and
BLM K conductance can be coordinated in parallel or
in series. For instance, if the initial perturbation is considered to be the incremental change in cell Na+ content due to increased apical Na+ entry, pump activity
and BLM K conductance could be coupled via: (a) an
increase in cell volume (Sackin, 1987, 1989
; Cemerikic and Sackin, 1993
; Kawahara, 1990
), (b) increased [Ca2+]i
secondary to either a decrease in Na/Ca exchange
(Yang et al., 1988
) or swelling-induced opening of Ca2+
channels (McCarty and O'Neil, 1992
), (c) an increase
in pHi dependent on the relative fractions of Na entering via the Na/H exchanger and Na-coupled cotransporters (Ohno-Shosaku et al., 1990
; Beck et al., 1993
),
(d) increased [K+]i due to pump activation (Messner et
al., 1985b
), (e) hyperpolarization of Vm due to pump activation (Lapointe and Duplain, 1991
), and/or (f) decreased [ATP]i due to pump activation. Note that the
first set of three signals simultaneously mediate
changes in both pump activity and BLM K conductance
in parallel; the latter three signals are already the result
of activation of the pump and mediate, in series, the increase in BLM K conductance (GK). The behavior of
the BLM KATP channel in regards to the first set of three
will be considered separately, while the last set of three
will be discussed together in the context of pump activity.
Cell volume, stretch, and the cytoskeleton.
An increase in
apical Na+ entry will increase steady state cell volume
(Beck et al., 1991a), which has been shown to lead to a
rise in BLM GK (Beck et al., 1991b
; Macri et al., 1993
).
It is likely that multiple factors are involved in this response, including signal transduction via the cytoskeleton and effects of membrane deformation (e.g., membrane stretch; for review, see Sackin, 1995
). Swelling-
(Sackin, 1989
) and stretch-activated (Kawahara, 1990
)
K channels on the BLM of Xenopus, Rana (Cemerikic and Sackin, 1993
), and Necturus (Sackin, 1989
) proximal tubule cells exist, but they are not ATP sensitive.
Although we have detected stretch-activated channels
(Segal and Boulpaep, 1994
) on the BLM of the Ambystoma cells, they are not K selective. We and others
(Parent et al., 1988
; Beck et al., 1993
) have shown that
rabbit BLM K+ channels are not stretch activated. The
Ambystoma BLM KATP channel reported in the present
study was not affected by stretch or hypotonic swelling
and thus is analogous to the KATP channels described in
mammalian preparations, which are also insensitive to
membrane stretch (Beck et al., 1993
; Tsuchiya et al.,
1992
).
[Ca2+]i.
Single-channel data on the calcium sensitivity of proximal tubule BLM K+ channels is limited to a
few studies (Parent et al., 1988; Gögelein and Greger,
1987
), and, as discussed in Mauerer et al. (1998)
, it is
not likely that these were the KATP channels we studied. The Ambystoma BLM KATP channel was sensitive to increases in [Ca2+]i in both cell-attached and excised
patches. In a Ca2+-free bath, ionomycin itself had no effect on KATP channel in cell-attached patches, but raising bath Ca2+ to 1 µM reduced channel activity by 63%.
Experiments using inside-out patches showed that Ca2+
inhibited channel activity with a Ki of 166 nM, within
the physiologic range. This effect in excised patches
suggests that Ca2+ is acting directly on the channel, but
does not exclude the involvement of Ca2+-activated kinases such as PKC or Ca2+/calmodulin-dependent protein kinase.
pHi.
Extracellular acidosis is known to depolarize
proximal tubule cells, a finding attributed to a decrease
in GK (Steels and Boulpaep, 1987; Kuwahara et al.,
1989
; Bello-Reuss, 1982
; Biagi and Sohtell, 1986
), probably mediated by a corresponding decrease in pHi (Kuwahara et al., 1989
). Cell-attached recordings from the
BLM of perfused rabbit proximal tubules showed that
lowering pH from 7.4 to 6.5 in either the lumen or
bath decreased K channel activity (Beck et al., 1993
). In
the present study, nigericin was used to produce an intracellular acidosis in the dissociated Ambystoma cells
without altering external pH (Segal et al., 1992
) during
cell-attached recordings, and BLM KATP channel activity fell by 61%. Since acidosis may reduce intracellular
K+ activity (Cemerikic et al., 1982
), the effect of pHi
was studied in excised patches where K+ activity remains constant. These experiments showed that lowering pHi from 7.5 to 6.8 reduced BLM KATP channel activity by 81%, confirming a direct effect of acidosis on
the BLM KATP channel.
Modulation of BLM KATP Channel Activity by Signals Dependent on Pump Activation
In many tissues, KATP channels serve to couple cell metabolism to electrical activity. In nerve and muscle, the
opening of KATP channels hyperpolarizes the cell and
thus reduces electrical excitability. In the pancreatic
cell, inhibition of glycolysis during hypoglycemia leads
to a fall in [ATP]i and a rise in [ADP]i. These metabolic
changes activate KATP channels and the resultant hyperpolarization leads to a decrease in insulin secretion
(Ashcroft and Ashcroft, 1990). Recently, a close coupling between Na+,K+-ATPase pump activity and KATP
channel current was demonstrated in cardiac myocytes
(Priebe et al., 1996
).
Likewise in epithelial cells, transport-induced changes
in cell metabolism as a result of pump activity may regulate KATP channels to optimize efficiency of transport,
as discussed in Mauerer et al. (1998) and elsewhere
(Schultz, 1992
; Tsuchiya et al., 1992
; Beck et al., 1994
).
BLM K+ conductance responds to changes in apical sodium entry (Messner et al., 1985a
; Matsumura et al.,
1984
) as well as alterations in basolateral sodium exit
(Beck et al., 1991a
; Messner et al., 1985b
; Matsumura et
al., 1984
). There is evidence from studies on rabbit
proximal tubule that [ATP]i is the signal that couples pump activity to channel activity (Beck et al., 1993
;
Tsuchiya et al., 1992
). In the present study, modulation
of transport in dissociated Ambystoma cells yielded results consistent with this proposal.
Retarding pump activity should lead to less ATP consumption and a rise in [ATP]i. Experimentally, this was
attempted by using either cardiac glycosides or by removal of extracellular K+. A possible downside of the
former is the introduction of nonspecific glycoside effects, while the latter has the disadvantage of altering Vm, which by itself changes channel activity. In both
cases, inhibition of pump activity resulted in a decrease
of BLM KATP channel activity. In the case of extracellular K+ removal, the initial increase of channel current
(isc) and nPo suggests that hyperpolarization of the cell
membrane precedes the eventual decrease in nPo. A
similar transient increase in KATP channel current also
occurs in cardiac myocytes (Priebe et al., 1996). However, since inhibition of the pump also tends to depolarize Vm, a fall in BLM KATP channel activity due to a
voltage-dependent effect cannot be excluded. Conversely, stimulation of transport leads to an increase of
BLM KATP channel activity. Although Vm was not monitored in this study, the addition of a substrate to the
bath is known to increase Na-coupled cotransport across
the apical membrane in the proximal tubule, thus stimulating Na+,K+-ATPase pump activity (Beck et al., 1993
).
In an attempt to examine the effect of changes in Vm
alone, we made efforts to clamp [ATP]i since proximal
tubule cells are known to take up ATP from the bath
(Tsuchiya et al., 1992; see Fig. 12). This maneuver significantly curtailed the increase in BLM KATP channel
activity when alanine was subsequently added, suggesting that changes in [ATP]i are coupled to BLM KATP
channel activity. Taken together, the BLM KATP channel responds to alterations in transport activity in both
directions, most likely mediated by changes in Vm and
[ATP]i (Beck et al., 1993
; Tsuchiya et al., 1992
).
Direct evidence for the functional coupling of KATP
channels to changes in cytosolic ATP directly relating
to Na+,K+-ATPase pump activity was recently demonstrated in guinea pig heart myocytes (Priebe et al.,
1996). Under conditions in which the pump was running forward, addition of strophanthidin halted ATP
consumption, leading to closure of KATP channels. Conversely, under conditions in which the pump was running backwards (by removal of extracellular K), addition of strophanthidin halted ATP production, leading
to opening of KATP channels. These authors proposed
that KATP current is modulated by cytosolic ATP within
an "ATP window" that can be approached from both
sides. They concluded that in cardiac myocytes (a)
Na+,K+-ATPase pump activity and KATP channels are
closely coupled, and (b) pump activity is the major determinant of cytosolic ATP. Our findings in renal proximal tubule cells are consistent with these conclusions.
Channel Regulation by ADP.
Activity of the Na+,K+-ATPase pump results in a fall of [ATP]i, while [ADP]i
rises, and there is substantial evidence that the ATP:
ADP ratio is an important regulator of KATP activity
(Wang and Giebisch, 1991a; Ashcroft and Ashcroft,
1990
). Nucleotide diphosphates have several modulatory actions on KATP channels, often dependent on
whether Mg2+ is present. In the absence of Mg2+, ADP
inhibits KATP channels (Ashcroft and Ashcroft, 1990
; Allard and Lazdunski, 1992
), including the BLM KATP
channel (Mauerer et al., 1998
). Mg-ADP can restore activity to some KATP channels that are running down
(Tung and Kurachi, 1991
). Mg-ADP (but not ADP3
)
can also relieve channel inhibition by ATP (Nichols et
al., 1996
; Allard and Lazdunski, 1992
) by shifting the Ki
for ATP to the right.
|
![]() |
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. Segal is a recipient of a Physician-Scientist Award from the NIH (DK-02103).
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