Novel Subunit Composition of a Renal Epithelial KATP
Channel*
Abdul
Ruknudin
§,
Dan H.
Schulze§,
Stephen K.
Sullivan
,
W. J.
Lederer¶, and
Paul A.
Welling
From the Departments of
Physiology and
§ Microbiology/Immunology, University of Maryland School of
Medicine, and ¶ Department of Molecular Biology and Biophysics,
University of Maryland Biotechnology Institute,
Baltimore, Maryland 21201
 |
ABSTRACT |
Unique ATP-inhibitable K+
channels (KATP) in the kidney determine the rate of urinary
K+ excretion and play an essential role in extracellular
K+ balance. Here, we demonstrate that functionally similar
low sulfonylurea affinity KATP channels are formed by two
heterologous molecules, products of Kir1.1a and cystic fibrosis
transmembrane conductance regulator (CFTR) genes. Co-injection of CFTR
and Kir1.1a cRNA into Xenopus oocytes lead to the
expression of K+ selective channels that retained the high
open probability behavior of Kir1.1a but acquired sulfonylurea
sensitivity and ATP-dependent gating properties. Similar to
the KATP channels in the kidney but different from
KATP channels in excitable tissues, the Kir1.1a/CFTR channel was inhibited by glibenclamide with micromolar affinity. Since
the expression of Kir1.1a and CFTR overlap at sites in the kidney where
the low sulfonylurea affinity KATP are expressed, our study
offers evidence that these native KATP channels are comprised of Kir1.1a and CFTR. The implication that Kir subunits can
interact with ABC proteins beyond the subfamily of sulfonylurea receptors provides an intriguing explanation for functional diversity in KATP channels.
 |
INTRODUCTION |
Extracellular potassium homeostasis, maintained through the
regulation of renal potassium excretion (1), is dependent on unique
epithelial ATP-sensitive K+ channels
(KATP)1 (2).
Providing the major, if not exclusive, route for potassium transport
into the distal nephron lumen, these channels constitute the final
regulated component of kidney's potassium secretory apparatus.
Physiological changes in secretory KATP channel activity subsequently determine the extent of urinary K+ excretion
in accord with the demands of potassium balance.
While the molecular basis of these physiologically important channels
has remained unresolved, elucidation of their biophysical nature by the
patch-clamp technique has provided important insights. Most telling are
the important similarities and differences exhibited between the
secretory channel and the ATP-sensitive K+ channels
identified in the cardiac myocyte (3) and the islet beta cell (4).
Characterized by the ability of cytoplasmic ATP to induce channel
closure (5), susceptibility to antidiabetic sulfonylurea inhibition (6)
and weak inward rectification (7), the distal nephron secretory channel
exhibits common functional features of all KATP channels
(8). However, the renal epithelial KATP channels are
readily distinguished from other ATP-sensitive K+ channels
by their relatively low affinity for sulfonylurea agents (6, 9) and
cytoplasmic ATP (5), suggesting the secretory KATP channel
might be encoded by particularly unique members of the multimeric
family of KATP proteins.
Several recent breakthroughs in the field offered tangible clues for
testing this hypothesis. Defining a new class of K+ channel
proteins, characterized by their inward rectifying properties (Kir)
(10) and a unique two-transmembrane structural motif, Ho et
al. (11) isolated a novel K+ channel cDNA, called
ROMK1 or Kir1.1a, from rat kidney. As required for a secretory
K+ channel gene, Kir 1.1a is specially expressed in the
distal nephron (12) on the apical membrane (13). While Kir1.1a shares
many functional features of the secretory channel, the notable absence of ATP (11) or sulfonylurea sensitivity suggested that the native channel was more complex than Kir 1.1a alone. Certainly, the recent discovery of Aguilar-Bryan et al. (14) gave credence to this notion. These investigators showed that the high affinity sulfonylurea receptor expressed in the pancreatic islet beta cell is encoded by a
unique ATP-binding cassette protein (ABC protein or traffic ATPase),
SUR1. The sulfonylurea binding protein does not exhibit any channel
activity itself. Instead, SUR1 interacts with a pancreatic inward
rectifying K+ channel subunit, Kir 6.2, to form a
KATP channel like those observed in beta-islet cells (15).
With the subsequent discovery that the cardiac KATP channel
is comprised of a related ATP-binding cassette protein, SUR2A, and the
inward-rectifying K+ channel isoform, Kir 6.2, a molecular
paradigm for other KATP channels began to be established
(16).
Having this multimeric Kir/ABC protein motif in mind, we explored the
possibility that the distal nephron KATP channel is encoded
by unique Kir and ATP-binding cassette proteins as a basis for its
distinct functional properties. While Kir 1.1a appeared to be an
excellent candidate for the inward rectifying K+ channel
subunit, the identity of the ABC subunit remained less certain. As
might be surmised from the low sulfonylurea sensitivity of renal
KATP channels, neither the pancreatic islet beta cell (SUR1) nor the cardiac myocyte (SUR2A) ABC proteins are expressed in
the kidney (14, 16). Another ABC protein, the cystic fibrosis transmembrane conductance regulator (CFTR), does standout as a plausible candidate. First, CFTR is expressed abundantly in the kidney
and is localized along the entire nephron (17) on the apical membrane
(18). Subsequently, the cellular and subcellular expression overlap
with Kir1.1a in the cortical collecting duct. Second, the cystic
fibrosis gene product is the only other member of the ABC superfamily
that is known to bind sulfonylurea agonists (19). Interestingly, the
sulfonylurea affinity of CFTR is similar to the renal epithelial
KATP channels. Finally, besides acting as a chloride
channel itself, there is a growing consensus that CFTR may also
regulate other channels (20).
In agreement with this general hypothesis, McNicholas et al.
(21) have recently reported that CFTR confers glibenclamide sensitivity
on a closely related Kir1.1a isoform, ROMK2 (Kir1.1b). However, two
critical questions remained unanswered. First, does CFTR interact with
other members of the ROMK family? Second, and more importantly, does
coexpression of CFTR with any ROMK (Kir1.1) isoform reconstitute
ATP-sensitive K+ channels? In the present study, we have
specifically addressed these issues by measuring the functional
consequences of CFTR/Kir1.1a interaction in Xenopus oocytes
injected with CFTR and Kir1.1a cRNA. Our results demonstrate that CFTR
associates with Kir 1.1 to modify single channel conductance and to
confer both ATP and sulfonylurea sensitivity. The data are compatible
with the notion that CFTR and Kir1.1 physically associate to form a
hybrid channel with properties that are reminiscent of the distal
nephron KATP channel. The implication of this study that
Kir subunits can interact with ATP-binding cassette proteins beyond the
subfamily of sulfonylurea receptors provides an intriguing explanation
for functional diversity in KATP channels.
 |
EXPERIMENTAL PROCEDURES |
cRNA Synthesis--
Complementary RNA was transcribed in
vitro in the presence of capping analogue (G(5')ppp(5')G from 1)
NotI linearized Kir1.a-pSPORT (ROMK1 (11) and 2)
SmaI linearized CFTR-pBS vector (Genzyme). T7 or SP6 RNA
polymerase were used in the two reactions, respectively (Ambion,
mMESSAGE mMACHINETM). Following DNase treatment, cRNA was
purified by phenol-chloroform extraction and ammonium-acetate/ethanol
precipitation. The yield and concentration were quantified
spectrophotometrically.
Oocyte Injection--
Female Xenopus laevis frogs
were obtained from NASCO (Fort Atkinson, WI). Standard protocols were
followed for the isolation and care of X. laevis oocytes.
Briefly, frogs were anesthetized by immersion in 0.2% tricaine and a
partial oophorectomy was performed through an abdominal incision.
Oocyte aggregates were manually dissected from the ovarian lobes and
then were incubated in a calcium-free ORII medium (82.5 mM
NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH7.5) containing collagenase (Sigma type IA or
Worthington collagenase type 3, 2 mg/ml) for
2 h at room temperature
to remove the follicular layer. After washing the oocytes extensively
with collagenase-free ORII, they were placed in a modified L15 medium (50% Leibovitzi's medium, 10 mM HEPES, pH 7.5) and stored
at 19 °C. 12-24 h following isolation, healthy looking Dumont stage
V-VI oocytes were pneumatically injected with 50 nl of water
containing cRNA and then stored in L15 medium at 19 °C. Oocytes were
either injected with Kir1.1a cRNA alone (5 ng) or Kir1.1a (5 ng) and a
5-fold molar excess of CFTR cRNA to force the potential interaction by
mass action.
Electrophysiology--
Channel activity was assessed 2-6 days
post injection by patch-clamp under conditions where K+
channels could be discerned from chloride channels by differences in
reversal potential. For most studies, the pipette contained 140 mM KCl, 1 mM CaCl2, 5.0 mM HEPES, pH 7.4, while the bath solution was comprised of
120 mM sodium gluconate, 20 mM KCl, 1 mM CaCl2, 5.0 mM HEPES, pH 7.4 (EK = +50, ECl =
50
mV). For ion selectivity studies, bath sodium gluconate was replaced
with equal molar KCl. In all studies, we purposely excluded protein
kinase A to prevent CFTR activation in the excised cell-attached patch
(22). Glibenclamide was added from a freshly prepared alkaline stock as
before (9). The magnesium salt of ATP (vanadate-free, Sigma) was used
in the ATP studies. All ATP containing solutions were used immediately (
30 min) after preparation. After the addition of ATP or
glibenclamide the pH was titrated to 7.4. Solution exchange was
accomplished by an oil gate apparatus as described previously (23). All
ATP and glibenclamide studies were performed at
ECl (
50 mV cell relative to patch), so inward
K+ currents could be isolated.
In these studies, the vitelline membrane was removed from oocytes
following hyperosmototic shrinking (24). Patch-clamp electrodes, pulled
from filamented borsilicate glass, had resistances of 0.5-5 megohms.
Single channel currents were measured with Axopatch-1C patch clamp
amplifier and recorded on video tape via Neuro-corder (model DR-886).
Data were replayed and filtered by an eight-pole Bessel filter
(Frequency Devices 900) at a cut-off frequency 1 kHz. The sampling rate
for analog-to-digital conversion was
5 times the filtering frequency.
Data were acquired and analyzed using pClamp 6.0.1 (Axon Instruments,
Foster City, CA). Single channel currents were estimated by fitting
Gaussian distributions to the current amplitude histograms or by
measuring the amplitudes directly from analog current traces. Inward
slope conductance was accessed from such current measurements at
100
to
20mV. In patches that contained multiple channels, activity was
accessed by integrating current flow at 10-s intervals with or without drug treatment. In patches that contained
3 channels, open
probability (Po) was determined from data by
Po = (1/N)
(t1 + t2 + ... tn), where
N is the number of channels, and t is the
fractional open time spent at each of the observed current levels.
 |
RESULTS |
Co-expression of CFTR Reduces the Single Channel Conductance of Kir
1.1a--
Since Kir 1.1a exhibits a larger conductance (35 pS) than
the values initially reported for the native secretory
KATP channel measured at the same temperature
(22-25 pS) (25), we first asked whether the potential interaction
between Kir 1.1a and CFTR might be revealed by a change in the unitary
conductance of Kir1.1a. As shown in Fig.
1, this hypothesis was borne out from
co-expression studies. Co-injection of Kir1.1a and a 5-fold molar
excess of CFTR cRNA into oocytes led to the expression of unique small
conductance K+ channels. The Kir1.1a/CFTR channel exhibited
similar high open probability kinetic properties as Kir1.1a
(Po = 0.77 ± 0.04,Kir1.1a/CFTR;
Po = 0.95 ± 0.02,Kir1.1a at
50 mV, Fig.
1A). Kir1.1a/CFTR was, however, easily distinguished from
Kir1.1a by a significantly smaller, albeit variable, unitary conductance (Fig. 1B). Compared with the inward slope
conductance of Kir 1.1a (35.71 ± 2.12 pS, as reported by others)
(11), the Kir 1.1a/CFTR channel (19.89 ± 6.0 pS
(p < 0.001) was significantly smaller (Fig.
1C). Consistent with a variable interaction stoichiometry, the smaller average unitary conductance of Kir 1.1a/CFTR
reflects the expression of a least four distinct channel types with
conductances ranging from 24 pS to 12 pS (24 ± 2 pS (13.8%),
20 ± 2 pS (23.1%), 16 ± 2 pS (36.92%) and 12 ± 2 pS
(9.23%) n = 65 channels/52 patches) (Fig.
2). K+ channels of this
smaller conductance fingerprint were never observed in uninjected
oocytes or in oocytes injected with either Kir 1.1a or CFTR cRNA alone.
The occasional detection a channel with the larger conductance
signature of the Kir 1.1a channel in co-injected oocytes (17% of
patches contained channels with a conductance of 35 ± 5 pS) is
consistent with unmodified Kir1.1a channels.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 1.
Co-expression of CFTR reduces the single
channel conductance of Kir 1.1a. A, representative single
channel recordings of Kir 1.1a and Kir 1.1a/CFTR channel in excised,
inside-out patches held at 50 mV (ECl). Inward
K+ currents are measured so upward deflections represent
channel closures. While both channels exhibit high open probability
kinetic properties, the current amplitude of Kir 1.1a/CFTR is 40-60%
smaller than the predominant conductive state of Kir 1.1a B,
all points histograms reveal a possible mechanism for the decrease in
the unitary channel conductance. The presence of an infrequent
subconductance state (*) in the Kir 1.1a channel constrains the best
fit (solid line) to three different Gaussian distributions
(dashed lines), reflecting the fully open state
(O), the subconductance state (*), and the fully closed
state (C). In contrast, the Kir 1.1a/CFTR channel, without
any detectable subconductance levels, requires only 2 different
Gaussian distributions (dashed lines) to account the one
fully open state level (O), and the one fully closed state
(C). The shift in the mean current amplitude of Kir
1.1a/CFTR to the subconductance level observed with Kir 1.1a is
consistent with the stabilization of the smaller conductance state.
C, current voltage relationships of the Kir 1.1a and the
Kir1.1a/CFTR channels confirm a bona fide reduction in the inward slope
conductance upon CFTR coexpression.
|
|

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 2.
Single channel conductance distribution in
Kir1.1a + CFTR coinjected oocytes compared with oocytes injected with
Kir1.1a alone. Illustrated are the number of channels
(N) that exhibited a particular single conductance ( ) for
both groups. The smaller average unitary conductance in Kir
1.1a/CFTR coinjected oocytes reflects the expression of a least four
distinct channel types. The histogram of the smaller conductance
channels (less than 26 pS) is best fit (solid line) by four
separate Gaussian distributions. While the smaller conductance channels
are unique to co-injected oocytes, the distribution of channels with a
larger single channel conductance (36 ± 2) is similar to that
observed in oocytes injected with Kir1.1a cRNA alone.
(n = 65 channels/52 patches for CFTR/ROMK1 and
n = 13 for ROMK1).
|
|
Observations that the predominant expression of the smaller conductance
K+ channel in co-injected oocytes was unaffected by patch
excision from the cell, imply that the interaction between CFTR and
Kir1.1a is membrane delimited. While we can not rule out the role of
unidentified linker proteins, such as cytoskeletal elements, these
observations suggest that the functional interaction between CFTR and
Kir1.1a may be direct and physical.
The Kir 1.1a/CFTR Channel Is K+ Selective--
As
predicted from our multimeric Kir1.1a/CFTR model of an epithelial
KATP, the small conductance Kir1.1a/CFTR channel is
K+ selective (Fig. 3). In the
excised inside-out configuration with 140 mM KCl in the
pipette, replacement of cytoplasmic KCl with equal molar sodium
gluconate (KCl + sodium gluconate = 140 mM) changed
the reversal potential of the small conductance channel from 1.4 ± 2.4 mV in 140 mM KCl to 22.6 ± 2.05 mV in 50 mM KCl (90 mM sodium gluconate) to 49.3 ± 4 mV in 20 mM KCl (120 mM sodium gluconate)
(n = 6). The shift of 51 mV per decade change in KCl is
in close agreement for the change in the equilibrium potential for
K+, indicating the small conductance Kir1.1a/CFTR channel
is highly K+ selective over sodium and chloride.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 3.
High K+ selectivity of Kir 1.1a
is not altered by CFTR interaction. Current-voltage relationships
of Kir1.1a/CFTR channels in excised inside-out patches with 140 mM KCl in the pipette and either 20 mM KCl
( ), 50 mM KCl ( ), or 140 mM KCl ( ) in
the bath using sodium gluconate as the substitute (KCl + sodium
gluconate = 140 mM). The shift in reversal potential
parallels the predicted change in the Nernst potential for
K+ (51 mV per decade change in KCl, n = 6)
|
|
CFTR-dependent Acquisition of Sulfonylurea
Sensitivity--
Having shown that CFTR interacts with Kir 1.1a to
form a K+ channel with conduction properties similar to the
secretory channel, we asked whether this channel also acquires the
characteristic features of KATP, such as sulfonylurea
sensitivity. An oil gate was employed in these studies to ensure
immediate and complete solution exchange. This experimental design
allowed an obvious distinction between the specific inhibitory effects
of glibenclamide and spontaneous channel rundown, a property of Kir1.1
(11, 26), the native secretory channel (5) and Kir1.1/CFTR. The high open probability behavior of the Kir 1.1a was not affected by exposure
up to 100 µM glibenclamide (1.5 ± 1.7% decrease in
channel activity, n = 4), incompatible with properties
of the native secretory KATP channel (Fig.
4A). In dramatic contrast to Kir 1.1a and
as expected for KATP, exposure to the same concentration of
glibenclamide inhibited Kir1.1a/CFTR channels in a rapid, reversible
and repeatable fashion; 100 µM glibenclamide caused a
88.49 ± 9.38% reduction of channel activity (n = 7) in less than 30 s of exposure.

View larger version (16K):
[in this window]
[in a new window]
|
Fig. 4.
Co-expression of CFTR confers
sulfonylurea-sensitivity on Kir 1.1a. Representative single
channel recordings in excised inside-out patches of Xenopus
oocytes injected with either A, Kir 1.1a cRNA alone or
B, Kir1.1a and CFTR cRNA (140 mM KCl in the
pipette and 20 mM KCl in the bath, Vm = 50 mV (ECl)). Inward K+ currents
are measured under these conditions so that upward deflections
represent channel closures. Rapid and complete solution exchange was
accomplished using a modified oil gate bath. A, Kir 1.1a
alone is not sensitive to glibenclamide. Exposure to 100 µM glibenclamide has no effect on Kir 1.1a channel
activity. At expanded time resolution (1 and 2),
no change in the open probability of the channel can be detected upon
glibenclamide exposure. B, Kir 1.1a/CFTR is sensitive to
glibenclamide. The upper tracing illustrates reversible and
repeatable inhibition of Kir1.1a + CFTR K+ channel activity
upon exposure to 100 µM glibenclamide. At higher gain,
unitary events are observed with the small conductance fingerprint of
the Kir 1.1a/CFTR channel. C, sulfonylurea inhibition occurs
with relatively low affinity. Inhibition (1 I/Imax) of
integrated Kir1.1a + CFTR and Kir1.1a channel activity are shown.
|
|
Channel activity was restored to 53.5 ± 14.1% of control within
10 s of glibenclamide washout. The reversible effect, in direct contrast to a recent observation of McNicholas et al. (21)
with ROMK2/CFTR, illustrates bona fide sulfonylurea sensitivity rather that unspecific channel rundown. Moreover, the inhibitory effects of
glibenclamide occurred with low affinity, almost identical to the
effects on CFTR alone (19) and similar to the native secretory kidney
KATP channel. Indeed dose-dependent
glibenclamide inhibition of the Kir 1.1a/CFTR channel reveal a
Ki of 33 µM (n = 6),
compared with the nanomolar sulfonylurea-sensitivity of the
KATP channels expressed in the pancreatic beta cell (Kir 6.2/SUR1, Ki = 8.6 nM) (16) and cardiac
myocytes (Kir6.2/SUR2A, Ki = 350 nM)
(16).
Kir 1.1a/CFTR Is a KATP Channel--
Cytoplasmic ATP
exposure had no inhibitory effect on Kir1.1a activity (Fig.
5A), confirming initial
observations (11). In excised inside-out patches, Kir1.1a exhibited
high open probability behavior in the absence (Po = 0.94 ± 0.04) or presence of cytoplasmic 5 mM ATP
(Po = 0.88 ± 0.09, n = 6). In dramatic contrast, the same concentration of cytoplasmic ATP inhibited the small conductance Kir1.1a/CFTR channel (12-26 pS) by 84 ± 1.2% (9/11 patches) within 30 s of ATP exposure with recovery after ATP washout (Fig. 5B). Repeated exposures of 5 mM ATP resulted in repeated bouts of channel closure
followed by reopening to 34 ± 3.2% of control levels upon return
to zero ATP. The response is consistent with a allosteric effect,
rather than phosphorylation/dephosphorylation mechanism of action (23).
If channel required rephosphorylation to open, the relative constant
activity on return to zero ATP would not have been observed.

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 5.
Kir 1.1a/CFTR is a KATP
channel. Representative single channel recordings in excised
inside-out patches of Xenopus oocytes injected with either
A, Kir 1.1a cRNA alone or B, Kir1.1a and CFTR
cRNA (140 mM KCl in the pipette and 20 mM KCl
in the bath, Vm = 50 mV
(ECl)). Inward K+ currents are
measured under these conditions so that upward deflections represent
channel closures. Rapid and complete solution exchange was accomplished
using a modified oil gate bath. A, Kir 1.1a alone is not
inhibited by ATP. Exposure to 5 mM MgATP has no effect on
Kir 1.1a channel activity. At expanded time resolution (1 and 2), no change in the open probability of the channel can
be detected upon ATP exposure. B, like the native channel,
Kir 1.1a/CFTR is inhibited by cytoplasmic ATP. The upper
tracing illustrates reversible inhibition of Kir1.1a + CFTR
K+ channel activity upon exposure to 5 mM
MgATP. At higher gain, unitary events are observed with the small
conductance fingerprint of the Kir 1.1a/CFTR channel. C,
dose response to cytoplasmic ATP in Kir 1.1a/CFTR (n 5 for each point, mean ± SD).
|
|
Dose-response studies (Fig. 5C) revealed that the ATP
sensitivity of Kir1.1a/CFTR is more similar to the native kidney
KATP channel (Ki = 0.5 mM)
(5) than the higher affinity KATP channels in the
pancreatic islet beta cell (Kir6.2/SUR1, EC50
10 µM (16), or heart (Kir6.2/SUR2A, EC50
100 µM (16). Indeed, ATP-dependent channel
inhibition of Kir1.1a/CFTR occurred with an EC50 of 0.6 mM. Furthermore, at concentrations less than 100 µM, ATP had the tendency to increase channel activity as
has been described for the native channel (5).
Also reminiscent of the native channel, cytoplasmic ADP antagonized the
inhibitory effect of ATP (Fig. 6) on
Kir1.1a/CFTR. In contrast to the 67.64 ± 2.45% decrease in
channel activity observed in 1 mM ATP, cytoplasmic addition
of 1 mM ATP + 1 mM ADP caused channel activity
to decrease by 45.29±7.3% (p < 0.005, n = 6). In this regard and as have been and have been
shown for the native kidney channel, the ratio of ATP/ADP would appear
to be a more important regulator of Kir1.1a/CFTR activity than ATP alone.

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 6.
ADP antagonizes the inhibitory effect of ATP
on Kir 1.1a/CFTR. A, representative single channel recording
in an excised inside-out patches of Xenopus oocyte injected
with Kir1.1a and CFTR cRNA (140 mM KCl in the pipette and
20 mM KCl in the bath, Vm = 50 mV
(ECl)) upon exposure to ATP, or ATP and ADP.
B, mean ± S.E., n = 6, of the
inhibition in 1 mM ATP or 1 mM ADP + 1 mM ATP relative to control (0.01 mM ATP).
|
|
In most but not all studies (compare Figs. 5B and 6), the
inhibitory effect that was observed in Kir1.1a/CFTR upon elevation of
cytoplasmic ATP occurred with a slower time course than would be
anticipated from the immediate step increase in cytoplasmic ATP (
20
ms) (27). Currents relaxed to near zero upon exposure to ATP with a
half-time of
15 s. While we can not be certain of the mechanism
responsible for the time course, in low channel density patches where
single channel conductance could be precisely resolved, we frequently
observed the transient emergence of the 35-40-pS channel upon exposure
to ATP. Such a response may reflect the activation of non interacting
ROMK channels or the dissociation of putative CFTR/ROMK1 subunits
before ATP-dependent inhibition.
 |
DISCUSSION |
Unique KATP channels in the renal cortical collecting
duct, comprising the final regulated component of kidney's potassium secretory machinery, play an essential role in the regulation of
extracellular K+ balance (28). In the present study, we
demonstrate that functionally similar low sulfonylurea affinity
KATP channels are formed by two heterologous molecules,
products of Kir1.1a and CFTR genes. Co-injection of CFTR and Kir1.1a
cRNA into Xenopus oocytes lead to the expression of unique
K+ selective channels that retained the high open
probability behavior of all Kir1.1a channels but acquired sulfonylurea
sensitivity and ATP-dependent gating properties, the
sine qua non of KATP. The Kir1.1a/CFTR channel
was inhibited by glibenclamide with a much lower affinity than those
expressed in excitable tissues (endocrine pancreas, Kir 6.2/SUR1 or
heart, Kir6.2/SUR2), similar to the unique KATP channels in
the kidney (6). With observations that the expression patterns of
Kir1.1a (12) and CFTR (18) overlap in the distal nephron along the
apical membrane, our study offers compelling evidence that the native
potassium secretory KATP channels may be comprised of
Kir1.1a and CFTR.
Several important aspects of our study differ from a recent report of
McNicholas et al. (21) with Kir1.1b(ROMK2) and CFTR. By
employing an oil gate for rapid and efficient solution exchange, we
demonstrated that glibenclamide directly inhibits Kir1.1a/CFTR as
required for a KATP channel. Subsequently it is not
necessary to evoke a dephosphorylation-dependent rundown
process to explain the glibenclamide effect on Kir1.1a/CFTR as
suggested by McNicholas et al. for Kir1.1b/CFTR. In addition
to extending and clarifying the mechanism of sulfonylurea sensitivity,
we have discovered that co-expression CFTR with Kir1.1a was required to
reconstitute ATP-dependent gating properties. In these
regards, the renal epithelial KATP channel appears to mimic
the basic Kir/ATP binding cassette protein paradigm exhibited by other
KATP channels.
Although the acquisition of ATP and low-affinity sulfonylurea
sensitivity are compatible with the reconstitution of the renal secretory KATP channel, the single channel conductance of
Kir1.1a/CFTR deserves some comment. In the present study, we
demonstrate a variable downward shift in the single channel conductance
of Kir1.1a with co-expression of CFTR, from 36 pS in Kir1.1a alone to
an average of 20 pS in Kir1.1a/CFTR. Sulfonylurea sensitivity and ATP-dependent gating properties appeared to be unique
characteristic of the smaller conductance channel, making it likely
that these particular channels reflect the interaction among CFTR and
Kir1.1a subunits. The shift in the mean current amplitude of Kir
1.1a/CFTR to the subconductance level observed with Kir 1.1a is
consistent with the CFTR-mediated stabilization of the smaller and more
rare conductance state in the Kir 1.1a-only channels. A mutation in the
Kir1.1 channel that eliminates a site for protein kinase A phosphorylation also stabilizes the subconductance state (29), suggesting a possible mechanism for the CFTR induced change in the
conductive properties.
In any regard, the conductive properties of Kir1.1a/CFTR are more
similar to those initially reported for the native secretory channel
than Kir1.1a alone. Frindt and Palmer (25) found that the apical
membrane K+ channel in the cortical collecting duct
exhibited an inward slope conductance of 22-25 pS at room temperature,
smaller than Kir1.1a but more similar to Kir1.1a/CFTR. In agreement
with these early observations and as predicted by the temperature
dependence of aqueous diffusion (Q10 = 1.3-1.8)
(30), latter measurements at 37 °C placed the value at 36 ps (7).
Although all these observations are more compatible with Kir1.1a/CFTR
than the Kir1.1a channel, it should be pointed out that Palmer et
al. (31) have recently come to a different conclusion in a
systematic comparison of the native channel to Kir1.1b (ROMK2), a
splice variant that lacks the first 19 amino acid residues of Kir1.1a.
These investigators now report that the conductive properties of the
two channels are more similar (31). At room temperature both channels
exhibit a inward slope conductance of 36 pS. At present we can not
wholly account for the disconcordance between these more recent
measurements in the native secretory KATP channel and our
own with Kir1.1a/CFTR. However, co-expression of CFTR appears to be
absolutely required for the acquisition of ATP and
sulfonylurea-sensitivity in Kir1.1a.
The time dependence of ATP inhibition in the native channel has never
been systematically studied under ideal conditions (i.e. an
oil gate). Nevertheless, based on what can be inferred from work with
the native channel, it would appear that inhibition of the Kir1.1a/CFTR
channel often occurs at a slower rate than the kidney channel. At
present, the mechanism underlying the kinetics of
ATP-dependent inhibition in Kir1.1a/CFTR, or any
KATP channel for that matter, are unknown. Based on the
variability of the response, however, we speculate that
ATP-dependent modulation may be dependent on a particular
conformational state of Kir1.1a/CFTR. Obviously, further work to
elucidate the mechanism by which CFTR interacts with Kir1.1a to form a
KATP channel will be necessarily before a mechanism is
established with any certainty.
CFTR-dependent modulation of other ion channels, involving
either direct or indirect interaction mechanisms (20), provides some
clues, however. While still controversial, activation of the outward
rectifying chloride channel in respiratory epithelia is perhaps the
best characterized example of indirect regulation (32). In this
system, CFTR is thought to mediate the efflux of ATP which in turn
increases outward rectifying chloride channel activity through a
purinergic receptor signaling pathway (33). In contrast to this
autocrine mechanism of interaction, our observations point to a
membrane delimited pathway. CFTR-dependent modulation of
Kir1.1a, revealed by the decrease in single channel conductance, acquisition of sulfonylurea sensitivity and development
ATP-dependent gating properties, were observed in the
excised patch configuration. Furthermore, the interaction between CFTR
and Kir1.1a occurred in the apparent absence of any autocrine
regulators, addition of kinases or nucleotides for phosphorylation,
making an indirect pathway unlikely. While we can not rule out a role
of undefined linker proteins, such as cytoskeletal elements, our
observations strongly imply that CFTR modifies Kir1.1a by direct
protein-protein interactions, similar to that proposed for
CFTR-dependent modulation of the amiloride-sensitive sodium
channel (34) and Kir6.2/SUR1 (35). Recently, the pancreatic islet beta
cell KATP channel has recently been shown to consist of an
(SUR1-Kir6.2)4 octamer, comprised of a Kir 6.2 tetramer,
forming the K+ selective pore, surrounded by four SUR1
subunits (35). Based on homology, it seems plausible that the
organization of CFTR and Kir1.1a subunits is similar. While the
distribution of several different conductance states in CFTR/Kir1.1a
may suggest the CFTR interaction stoichiometry is variable, further
experimentation is required before any definitive conclusions can
be made.
The domains of the Kir1.1a/CFTR complex that confer ATP and
sulfonylurea sensitivity are presently unknown. The molecular homology
of CFTR to sulfonylurea receptors, all members of the ATP-binding
cassette family of proteins, suggests that CFTR acts as the
sulfonylurea receptor in the Kir1.1a/CFTR channel. Certainly, the close
concordance in the affinity of the Kir1.1a/CFTR channel for
glibenclamide and that of CFTR alone (19) support this view. The
functional domains responsible for ATP-sensitivity remain more obscure.
Because CFTR contains two functional nucleotide binding domains (36),
similar to the nucleotide binding domains in SUR1 that have been
implicated nucleotide-dependent modulation of the islet
beta cell KATP (37), it is possible that these sites play
some role. Alternatively, a recent report by Tucker et al.
(38) with Kir 6.2 suggests that the critical ATP binding site resides
in the Kir subunit. In agreement with this notion with ROMK channels,
Kir1.1a contains a functional nucleotide binding domain (39, 40).
Subsequently, CFTR may stabilize a more favorable gating conformation
in Kir1.1a, allowing direct ATP-binding with Kir1.1a to induce channel
closure. Obviously, further study is required to determine the relative
importance of each of the nucleotide-binding domains in the two
different subunits of the renal epithelial KATP
channel.
In summary, we have shown that co-expression of Kir1.1a (ROMK1) with
CFTR in Xenopus oocytes reconstitute unique low-sulfonylurea affinity KATP similar to those involved in renal
K+ excretion and K+ homeostasis. The
implication of this study that Kir subunits can interact with ATP
binding cassette proteins beyond the subfamily of sulfonylurea
receptors provides an intriguing explanation for functional diversity
in KATP channels.
 |
ACKNOWLEDGEMENTS |
We are grateful to Dr. Steven Hebert for the
ROMK1 (Kir1.1a) cDNA and to Genzyme for the CFTR cDNA. We thank
Drs. G. Frindt and L. G. Palmer for discussion and a preprint of
their article.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants DK48271 (to P. A. W.) and HL25675 and HL36974 (to W. J. L.) and an American Heart grant-in-aid (to D. H. S.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Physiology, University of Maryland, School of Medicine, 655 W. Baltimore St., Baltimore, MD 21201. Tel.: 410-706-3851; Fax:
410-706-8341.
1
The abbreviations used are: KATP,
ATP-sensitive K+ channel; CFTR, cystic fibrosis
transmembrane conductance regulator.
 |
REFERENCES |
-
Wright, F. S.,
and Giebisch, G.
(1993)
in
The Kidney: Physiology and Pathophysiology (Seldin, D. W., and Giebisch, G., eds), pp. 2249-2278, Raven Press, New York
-
Wang, W.,
Sackin, H.,
and Giebisch, G.
(1992)
Annu. Rev. Physiol.
54,
81-96[CrossRef][Medline]
[Order article via Infotrieve]
-
Noma, A.
(1983)
Nature
305,
147-148[Medline]
[Order article via Infotrieve]
-
Cook, D. L.,
and Hales, C. N.
(1984)
Nature
311,
271-273[Medline]
[Order article via Infotrieve]
-
Wang, W.,
and Giebisch, G.
(1991)
J. Gen. Physiol.
98,
35-61[Abstract]
-
Wang, T.,
Wang, W. H.,
Klein-Robbenhaar, G.,
and Giebisch, G.
(1995)
Renal Physiol. Biochem.
18,
169-182[Medline]
[Order article via Infotrieve]
-
Wang, W.,
Schwab, A.,
and Giebisch, G.
(1990)
Am. J. Physiol.
259,
F494-F502[Abstract/Free Full Text]
-
Ashcroft, F.
(1981)
Annu. Rev. Neurosci.
11,
97[CrossRef][Medline]
[Order article via Infotrieve]
-
Tsuchiya, K.,
Wang, W.,
Giebisch, G.,
and Welling, P. A.
(1992)
Proc. Natl. Acad. Sci. U. S. A.
89,
6418-6422[Abstract]
-
Doupnik, C. A.,
Davidson, N.,
and Lester, H. A.
(1995)
Curr. Opin. Neurobiol.
5,
268-277[CrossRef][Medline]
[Order article via Infotrieve]
-
Ho, K.,
Nichols, C. G.,
Lederer, W. J.,
Lytton, J.,
Vassilev, P. M.,
Kanazirska, M. V.,
and Hebert, S. C.
(1993)
Nature
362,
31-38[CrossRef][Medline]
[Order article via Infotrieve]
-
Lee, W. S.,
and Hebert, S. C.
(1995)
Am. J. Physiol.
268,
F1124-F1131[Abstract/Free Full Text]
-
Austin, N.,
Li, Q.,
Horby, D.,
and White, S.
(1997)
J. Am. Soc. Nephrol.
7,
1275 (abstr.)
-
Aguilar-Bryan, L.,
Nichols, C. G.,
Wechsler, S. W.,
Clement, J. P.,
Boyd, A. E.,
Herrera-Sosa, H.,
Nguy, K.,
Bryan, J.,
and Nelson, D. A.
(1995)
Science
268,
423-426[Medline]
[Order article via Infotrieve]
-
Inagaki, N.,
Gonoi, T.,
Clement, J. P.,
Namba, N.,
Inazawa, J.,
Gonzalez, G.,
Seino, S.,
and Bryan, J.
(1995)
Science
270,
1166-1170[Abstract]
-
Inagaki, N.,
Gonoi, T.,
Clement, J. P.,
Wang, C-Z.,
Aguilar-Bryan, L.,
Bryan, J.,
and Seino, S.
(1996)
Neuron
16,
1011-1017[Medline]
[Order article via Infotrieve]
-
Morales, M. M.,
Carroll, T. P.,
Morita, T.,
Schwiebert, E. M.,
Devuyst, O.,
Wilson, P. D.,
Lopes, A. G.,
Stanton, B. A.,
Dietz, H. C.,
Cutting, G. R.,
and Guggino, W. B.
(1996)
Am. J. Physiol.
270,
F1038-F1048[Abstract/Free Full Text]
-
Crawford, I.,
Maloney, P. C.,
Zeitlin, P. L.,
Guggino, W. B.,
Hyde, S. C.,
Turley, H.,
Harris, A.,
and Higgins, C. F.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
9262-9266[Abstract]
-
Sheppard, D. N.,
and Welsh, M. J.
(1992)
J. Gen. Physiol.
100,
573-591[Abstract]
-
Higgins, C. F.
(1995)
Cell
82,
693-696[Medline]
[Order article via Infotrieve]
-
McNicholas, C. M.,
Guggino, W. B.,
Schwiebert, E. M.,
Hebert, S. C.,
Giebisch, G.,
and Egan, M. E.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8083-8088[Abstract/Free Full Text]
-
Anderson, M. P.,
Berger, H. A.,
Rich, D. P.,
Gregory, R. J.,
Smith, A. E.,
and Welsh, M. J.
(1991)
Cell
67,
775-784[Medline]
[Order article via Infotrieve]
-
Nichols, C. G.,
and Lederer, W. J.
(1991)
J. Gen. Physiol.
97,
1095-1098[Medline]
[Order article via Infotrieve]
-
Methfessel, C.,
Witzemann, V.,
Takahashi, T.,
Mishina, M.,
Numa, S.,
and Sakmann, B.
(1986)
Pflugers Arch.
407,
577-588[Medline]
[Order article via Infotrieve]
-
Frindt, G.,
and Palmer, L. G.
(1989)
Am. J. Physiol.
256,
F143-F151[Abstract/Free Full Text]
-
McNicholas, C. M.,
Wang, W.,
Ho, K.,
Hebert, S. C.,
and Giebisch, G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
8077-8081[Abstract]
-
Nichols, C. G.,
Lederer, W. J.,
and Cannell, M. B.
(1991)
Biophys. J.
60,
1164-1177[Abstract]
-
Wang, W.-H.
(1995)
Kidney Int.
48,
1024-1030[Medline]
[Order article via Infotrieve]
-
MacGregor, G. G.,
Xu, Z-C.,
Yang, Y.,
McNicholas, C. M.,
Hebert, S. C.,
and Giebisch, G.
(1996)
J. Am. Soc. Nephrol.
7,
1285 (abstr.)
-
Hille, B.
(1992)
Ionic Channels of Excitable Membranes, p. 329, Sinauer Associates, Sunderland, MA
-
Palmer, L. G.,
Choe, H.,
and Frindt, G.
(1997)
Am. J. Physiol.
273,
F404-F410[Abstract/Free Full Text]
-
Egan, M.,
Flotte, T.,
Afione, S.,
Solow, R.,
Zeitlin, P. L.,
Carter, B. J.,
and Guggino, W. B.
(1992)
Nature
358,
581-584[CrossRef][Medline]
[Order article via Infotrieve]
-
Schwiebert, E. M.,
Egan, M. E.,
Hwang, T. H.,
Fulmer, S. B.,
Allen, S. S.,
and Cutting, G. R.
(1995)
Cell
81,
1063-1073[Medline]
[Order article via Infotrieve]
-
Stutts, M. J.,
Canessa, C. M.,
Olsen, J. C.,
Hamrick, M.,
Cohn, J. A.,
Rossier, B. C.,
and Boucher, R. C.
(1995)
Science
269,
847-850[Medline]
[Order article via Infotrieve]
-
Clement, J. P.,
Kunjilwar, K.,
Gonzalez, G.,
Schwanstecher, M.,
Panten, U.,
Aguilar-Bryan, L.,
and Bryan, J.
(1997)
Neuron
18,
827-838[Medline]
[Order article via Infotrieve]
-
Smit, L. S.,
Wilkinson, D. J.,
Mansoura, M. K.,
Collins, F. S.,
and Dawson, D. C.
(1993)
Proc. Natl. Acad. Sci. U. S. A.
90,
9963-9967[Abstract]
-
Nichols, C. G.,
Shyng, S. L.,
Nestorowicz, A.,
Glaser, B.,
Clement, J. P.,
Aguilar-Bryan, L.,
Permutt, M. A.,
and Bryan, J.
(1996)
Science
272,
1785-1787[Abstract]
-
Tucker, S. J.,
Gribble, F. M.,
Zhao, C.,
Trapp, S.,
and Ashcroft, F. M.
(1997)
Nature
387,
179-183[CrossRef][Medline]
[Order article via Infotrieve]
-
McNicholas, C. M.,
Yang, Y.,
Giebisch, G.,
and Hebert, S. C.
(1996)
Am. J. Physiol.
271,
F275-F285[Abstract/Free Full Text]
-
Welling, P. A.,
and Salyer, L.
(1996)
J. Am. Soc. Nephrol.
7,
1294 (abstr.)
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.