1 Division of Nephrology, Vanderbilt University Medical Center, Nashville, Tennessee 37232; 2 First Division of Internal Medicine, University of Tokyo, Tokyo 113; and 3 Department of Neurophysiology, Tohoku University School of Medicine, Sendai 980-8575, Japan
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Recent studies showed that coexpression of Kir6.1 or
Kir6.2 with the sulfonylurea receptor (SUR1, SUR2A, or SUR2B)
reconstituted an inwardly rectifying, ATP-sensitive K+
channel that was inhibited by glibenclamide (2, 15-17). Here we
report the isolation of a rat homolog of mouse SUR2B (denoted rSUR2B)
from a rat kidney cDNA library. The rSUR2B sequence contains a 4,635-bp
open reading frame that encodes a 1,545-amino acid polypeptide, showing
67% shared identity with SUR1 (a pancreatic -cell isoform) and 98%
with both SUR2A (a brain isoform) and SUR2B (a vascular smooth muscle
isoform). Consistent with the predicted structures of other members of
the ATP-binding cassette (ABC) superfamily, the sequence of rSUR2B
contains 17 putative membrane-spanning segments. Also, predicted Walker
A and B consensus binding motifs, present in other ABC members, are
conserved in the rSUR2B sequence. RT-PCR revealed that rSUR2B is widely
expressed in various rat tissues including brain, colon, heart, kidney, liver, skeletal muscle, and spleen. The intrarenal distribution of the
rSUR2B transcript was investigated using RT-PCR and Southern blot of
microdissected tubules. The rSUR2B transcript was detected in proximal
tubule, cortical thick ascending limb, distal collecting tubule,
cortical collecting duct, and outer medullary collecting duct, but not
medullary thick ascending limb. This distal distribution overlaps with
that of ROMK. Coexpression of rSUR2B with ROMK2 cRNA (in 1:10 ratio) in
Xenopus laevis oocytes resulted in whole cell
Ba2+-sensitive K+ currents that were inhibited
by glibenclamide (50% inhibition with 0.2 mM glibenclamide). In
contrast, rSUR2B did not confer significant glibenclamide sensitivity
to oocytes coinjected with ROMK1 or ROMK3. The interaction between
ROMK2 and rSUR2B was further studied by coimmunoprecipitation of in
vitro translated rSUR2B and ROMK2. In agreement with the functional
data, the rSUR2B protein was coimmunoprecipitated with ROMK2 in the
ROMK2-rSUR2B cotranslated samples. Our data demonstrate that ROMK2, but
not ROMK1 and ROMK3, can interact with rSUR2B to confer a
sulfonylurea-sensitive K+ channel, implicating SUR proteins
in forming and regulating renal ATP-sensitive K+ channels.
The ROMK isoform specificity of glibenclamide effects suggests that the
NH2 terminus of the ROMK protein mediates rSUR2B-ROMK2 interactions.
ROMK potassium channel; sulfonylurea receptor; glibenclamide; kidney
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
ROMK1 (KIR1.1A), CLONED FROM rat kidney outer medulla (14), belongs to a growing family of inwardly rectifying potassium (Kir) channels that are characterized by weak or strong inward rectification and high potassium selectivity (32). Renal tubule localization of the ROMK protein, together with the observed electrophysiological and regulatory properties of ROMK channels expressed in Xenopus laevis oocytes (14, 38, 39), suggest that ROMK forms the native 30-pS, ATP-sensitive K+ channel (KATP) identified previously in the apical membranes of thick ascending limb of the loop of Henle (TAL) and principal cells of cortical collecting duct (CCD) (26).
KATP channels are widely expressed (3, 4, 10, 29) and are
heteromultimers of an inwardly rectifying K+ channel
subunit (Kir6.1 or Kir6.2; Refs. 15, 16) that forms the pore and a
regulatory subunit SUR that belongs to the ATP-binding cassette (ABC)
superfamily. ATP sensitivity is thought to be conferred by the channel
subunit, and the sensitivity to sulfonylurea drugs (e.g.,
glibenclamide) is mediated by the SUR subunit. Three isoforms of SUR
have been cloned recently: SUR1 reconstitutes with Kir6.2 to form the
neuronal/pancreatic -cell-type KATP channel, showing high sulfonylurea sensitivity (2); and SUR2A and SUR2B, splice variants
of a single gene, reconstitute either the cardiac-type (SUR2A/Kir6.2)
(16) or the vascular smooth muscle-type (SUR2B/Kir6.1 or Kir6.2)
KATP channels, respectively. Both of the later
KATP channels exhibit moderate affinity to sulfonylurea
drugs compared with the pancreatic
-cell KATP channel
(18, 40). SUR also confers other physiological and pharmacological
properties to the KATP channels, including nucleotide
diphosphate stimulation, K+ channel-opening drug
stimulation, and burst channel kinetics characterized by spontaneous
bursting upon removal of intracellular ATP (2, 41). Given the
observation that coexpression of SUR1 with ROMK1, Kir2.1, or Kir3.4 in
X. laevis oocytes failed to confer sulfonylurea sensitivity to
these K+ channels (2), it appears that the SUR1 interacts
only with the Kir6.x subfamily of Kir channels. However, the recent
observations that another member of the ABC family, the cystic fibrosis
transmembrane conductance regulator (CFTR), can confer glibenclamide
sensitivity to ROMK2 in X. laevis oocytes (24, 25, 32) suggests
that ROMK may assemble the renal epithelial KATP channel
with other SUR isoform(s) or ABC family members.
In the present study, we report cloning and localization of a rat homolog of the mouse SUR2B (denoted rSUR2B). RT-PCR and Southern blot analyses showed that rSUR2B was expressed in various tissues and widely expressed in rat kidney nephron segments. Coexpression of rSUR2B with ROMK2 in X. laevis oocytes resulted in a glibenclamide-sensitive K+ channel, implicating SUR2B (in addition to CFTR) in forming and regulating the renal epithelial KATP channel.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
cDNA cloning a sulfonylurea receptor from rat kidney.
Total RNA (10 µg) from rat kidney was reverse transcribed
with an oligo(dT) primer and Moloney murine leukemia virus RT (GIBCO). The resulting cDNA was amplified in the PCR using several pairs of
oligonucleotide primers designed according to the DNA sequence of rat
SUR2A (16): sense primer, R2As: 5'-CGTGGCCATCGACTACTGGC-3'; alignment position 3011-3031 in rat SUR2A; antisense primer, R2Aa: 5'-GACAGCAGGAAGAGCGGTG-3'; alignment position
1738-1757 in rat SUR2A. The PCR fragments were subcloned into
BlueScript vector (Stratagene) and sequenced using the cycle sequencing
method (Perkin-Elmer). The PCR fragment homologous to SUR2A was random
prime-labeled using [-32P]dATP (>3,000
Ci/mmol, Amersham) and used as a probe to screen a rat kidney cDNA
library. Six distinct clonal positives were grouped by PCR and
restriction mapping; one representative clone was further sequenced by
cycle sequencing. The full length of rSUR2B was subcloned into the
pGEMHE vector, which contains a T7 RNA polymerase promoter for
eukaryotic in vitro translation, and a Xenopus
-globin gene
for functional expression (21).
Tissue distribution of rSUR2B expression. Expression of rSUR2B in different rat tissues was determined by RT-PCR. Total RNA isolated from various rat tissues was reverse transcribed as described above. The DNA products from each tissue were amplified by PCR (denaturation, 1 min at 94°C; annealing, 2 min at 50°C; and extension, 3 min at 72°C; 30 cycles) using a sense primer (2A-1: 5'-GACAGCCTTTGCGGATCG-3') paired with an antisense primer (2A-2: 5'-GCATCGAGACACAGGTGCTG-3'). These primers were designed to cross the 177-bp insert in SUR2A DNA sequence that gives rise to alternative spliced SUR2 variants (the primer pairs should generate PCR fragments of 211 bp for rSUR2B and 387 bp for rSUR2A).
Renal tubule distribution of rSUR2B expression.
Expression of rSUR2B in renal tubules was determined by RT-PCR
and Southern blotting. Young pathogen-free male Sprague-Dawley rats
(80-100 g) were anesthetized, and kidneys were perfused initially with 10 ml PBS and then with 10 ml digestion solution [DMEM
(GIBCO) containing 0.5 mg/ml collagenase (type I, Sigma), 0.5 mg/ml
pronase E (Sigma), and 0.1% antifoam B (Sigma)]. After death of
the rat, the perfused kidney was removed, and individual nephron
segment microdissection was performed as described previously (7, 20, 38). Average length of dissected tubules was 0.5-1 mm, and three tubules were combined as one sample. Dissected tubules were directly reverse transcribed and amplified by the PCR using primers 2A-1 and
2A-2. The PCR products were displayed on a Southern blot and visualized
by hybridization to a 32P-labeled cDNA probe of the full
length of rSUR2B coding sequence. Specific hybridization of the rSUR2B
probe was quantified by the intensity of the -actin ethidium
bromide-stained bands. Each PCR reaction also included a pair of
-actin primers that span one intron to control for genomic DNA contamination.
Two-electrode voltage clamp.
Ba2+-sensitive K+ currents in X. laevis oocytes injected with cRNA from each of the three ROMK
splice variants (ROMK1, -2, and -3; 5 ng), or with rSUR2B (50 ng), or
coinjected with rSUR2B and each of the ROMK splice variants (5 ng ROMK,
50 ng rSUR2B; molar ratio = 1 ROMK: 3 rSUR2B) were functionally examined by two-electrode voltage clamping as
described (7). Electrophysiological recordings were performed 96 h
after RNA injection at 22 ± 2°C by two-electrode voltage clamp at
a holding potential of 80 mV (Axoclamp 2A, Axon Instruments).
The bath solution was as follows (in mM): 96 NaCl, 1 KCl, 1.8 CaCl2, 1 MgCl2, and 5 HEPES (pH 7.4), with 5 mM
Ba2+ or various concentrations (0.1, 0.2, and 0.4 mM) of
glibenclamide. The average resting potential
was
100 mV (range
70 to
104 mV) at
anexternal K+ concentration of 1 mM. The oocyte holding potential was
90 mV. The oocytes were
pulsed over the range of
160 to ±40 mV every 20 mV for 50 ms.
Currents ranged from 0.06 to 0.20 µA at
140 mV with 1 mM
external K+. As indicated in our previous publication (24),
glibenclamide sensitivity is best observed with low external
K+. Glibenclamide was diluted from a 1,000× stock
solution dissolved in DMSO (Sigma). Equal amounts of DMSO were added to
the control bath solutions. The EC50 value for
glibenclamide block was calculated from the
one-site nonlinear regression model performed
using GraphPad Prism version 3.00 for
Windows (GraphPad Software, San
Diego, CA; www.graphpad.com).
In vitro translation of ROMK2 and rSUR2B and immunoprecipitation . Hemagglutinin (HA)-tagged ROMK1, HA-tagged ROMK2 (39) and wild-type rSUR2B cDNA were translated in vitro either separately or together using TNT®-coupled reticulocyte lysate system and [35S]methionine in the presence or absence of canine microsomal membranes as per the manufacturer's instruction (Promega). Reaction mixtures were incubated at 30°C for 120 min after addition of [35S]methionine. Immunoprecipitation was performed according to Xu et al. (39) using anti-HA monoclonal antibody (clone 12CA5, Boehringer Mannheim). Protein products were resolved by 8% SDS-PAGE, and the [35S]methionine-labeled ROMK proteins were visualized by autoradiography.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of rSUR2B in rat kidney.
A PCR fragment, which showed 99% homology to rat SUR2A, was used to
screen a rat kidney cDNA library. We obtained six positive clones after
screening ~5 × 105 plaques. One of these clones,
named rSUR2B (rat SUR2B), was further analyzed by sequencing, which
revealed a single open reading frame of 4,635 bp encoding a 1,545-amino
acid polypeptide. The rSUR2B polypeptide has been proposed to form 17 membrane-spanning domains based on multisequence alignments of SUR and
the multidrug resistance-associated protein subfamily (1, 35). rSUR2B
contains two potential nucleotide binding folds with Walker A and B
consensus motifs (Fig. 1), like those
observed in other cloned SUR isoforms. Also, potential N-linked
glycosylation sites, protein kinase (PK) A- and PKC-dependent
phosphorylation sites, present in other SUR isoforms are conserved in
the rSUR2B sequence. Alignment of the resulting amino acid sequence
revealed that rSUR2B shared 67% identity with SUR1 (2) and 98% with
SUR2A and SUR2B (17, 19). As shown in Fig. 1, 18 amino acid residues of
rSUR2B were divergent from those of mouse SUR2B (mSUR2B)
(His17, Gln96, Met162,
Pro328, Lys329, Asn331,
Thr333, Arg334, Phe335,
Ser336, Thr618, Asn651,
Val666, Ser771, Asp960,
Asp1022, Ile1133, and Phe1281). In
addition, Phe336 in mSUR2B was absent in rSUR2B.
|
rSUR2B transcript expression in tissues.
Insight into the tissue distribution of rSUR2B transcripts was
obtained by surveying the expression of rSUR2B in rat tissues using
RT-PCR. As shown in Fig. 2, the amplicon
derived from rSUR2B primers (211 bp) was most abundant in brain, heart,
liver, spleen, kidney, and colon, with lesser amounts being detected in
skeletal muscle. This pattern of transcript expression is consistent
with the distribution of SUR2B in mouse tissue (18). SUR2A (387 bp) was
detected in heart and skeletal muscles but was absent in the kidney.
The kidney tubule distribution of rSUR2B is shown in Fig. 3. The products derived from the RT-PCR of
dissected tubules were displayed on a Southern blot following
hybridization with a 32P-labeled rSUR2B-specific probe. The
amplicon showed a discrete distribution, being very abundant in
proximal tubule [proximal convoluted tubule (PCT), proximal
straight tubule (PST)], cortical thick ascending
limb (CTAL), and outer medullary collecting duct (OMCD) segments.
Lesser intensity was seen in the distal convoluted tubule and
connecting tubule segments (Fig. 3). The similarity of the kidney
distributions of ROMK2 (38) and rSUR2B is consistent with the
possibility that renal epithelial secretory KATP channels in distal nephron segments may be comprised of ROMK2 and rSUR2B.
|
|
Heterologous expression of rSUR2B and each isoform of ROMK in X. laevis oocytes.
To assess whether the cloned rSUR2B forms
glibenclamide-sensitive K+ channels with ROMK, we evaluated
Ba2+-sensitive K+ currents in X. laevis
oocytes coinjected with cRNA transcribed from cDNA of rSUR2B and three
ROMK splice isoforms (ROMK1, -2, and -3). Preliminary studies
demonstrated that a molar ratio of 1:3 (coinjection of 5 ng ROMK2 cRNA
and 50 ng rSUR2B cRNA in oocytes) was best for studying the interaction
of ROMK and rSUR2B. Thus this cRNA molar ratio was used for the
functional assays in this study. Figure 4,
A and B, shows the results of these experiments in
which rSUR2B was coinjected with each of three ROMK splice variants.
The coinjected oocytes exhibited significant Ba2+-sensitive
K+ currents (0.5-1 µA; external K+ = 1 mM; Fig. 4A). The inhibitory effect of 0.2 mM glibenclamide on
Ba2+-sensitive K+ currents, however, was
limited to ROMK2; the whole cell K+ currents with ROMK1 and
-3 were unaffected by glibenclamide (Fig. 4, A and B).
There was no apparent voltage dependence of the glibenclamide block. We
next assessed the concentration dependence of glibenclamide-mediated inhibition Ba2+-sensitive K+ currents in ROMK2
and rSUR2B coinjected oocytes. As shown in Fig.
5, K+ currents compared with
controls without glibenclamide (I/Io) were
reduced to 0.73 ± 0.095 (n = 8), 0.54 ± 0.064 (n = 15), and 0.38 ± 0.044 (n = 7) of control with exposure to
0.1, 0.2, and 0.4 mM glibenclamide, respectively. The EC50
for glibenclamide-mediated inhibition of K+ currents in
ROMK2 + rSUR2B coinjected oocytes was 185 µM. Glibenclamide had no
significant effect on whole cell K+ currents in oocytes
injected with ROMK2 alone (I/Io = 0.96 ± 0.08 with 0.2 mM glibenclamide).
|
|
Coimmunoprecipitation of ROMK and rSUR2B. A direct interaction between SUR proteins and Kir6 channels is required to form KATP channels in neuronal tissues, and this association provides for glibenclamide sensitivity via the SUR subunit (15, 16). To assess whether a direct interaction between ROMK and rSUR2B is needed for the glibenclamide sensitivity of K+ currents in oocytes, we used an in vitro translation assay to examine the ability of the two proteins to associate. Because our anti-ROMK antibody (38) is weak at immunoprecipitation, influenza virus HA-tagged ROMK constructs (ROMK1-HA and ROMK2-HA) were utilized for this study. We had previously shown that these constructs formed functional channels (39). In addition, coexpression of ROMK2-HA and rSUR2B in oocytes yielded a similar sensitivity to glibenclamide [I/Io = 0.55 ± 0.06 (n = 5) with 0.2 mM glibenclamide] as that for the untagged ROMK2 construct (Fig. 4).
Figure 6 shows the results of these in vitro translation assay experiments performed in the presence of microsomes. Similar results were obtained in the absence of microsomal membranes (data not shown). In vitro translation of ROMK2-HA (Fig. 6, lane 2) or rSUR2B (Fig. 6, lane 3) yielded the expected 43-kDa (38) and 174-kDa (molecular mass is predicted by amino acid sequence) core proteins, respectively. No significant bands were seen when either ROMK2 or rSUR2B was absent (H2O control; Fig. 6, lane 1). Fainter bands representing glycosylation products of these two proteins are visible just above each of the dense-core bands. With cotranslation of ROMK2-HA and rSUR2B, both the 43- and 174-kDa bands are detected (Fig. 6, lane 4). When ROMK2-HA and rSUR2B were translated separately, the anti-HA antibody precipitated only ROMK2-HA (Fig. 6, lane 5) but not wild-type rSUR2B (Fig. 6, lane 6). In contrast, when ROMK2-HA and rSUR2B are cotranslated, the anti-HA antibody precipitated a complex of ROMK2-HA and rSUR2B (Fig. 6, lane 7). This finding demonstrates that when cotranslated in vitro, ROMK2-HA and rSUR2B are physically associated. Finally, to determine the relationship between the direct interaction of ROMK with rSUR2B and glibenclamide sensitivity, we assessed the ability of ROMK1-HA to coimmunoprecipitate rSUR2B (Fig. 6, lanes 8 and 9). As expected, the ROMK1-HA protein was slightly larger (~45 kDa; Fig. 6, lanes 8 and 9) than ROMK2-HA (~43 kDa; Fig. 6, lane 2). When ROMK1-HA and rSUR2B were cotranslated, the anti-HA antibody precipitated only ROMK1-HA, but not the rSUR2B protein (Fig. 6, lane 9). Thus association of ROMK and rSUR2B proteins was observed only with ROMK2-HA, the channel isoform that exhibits sensitivity to glibenclamide (Fig. 4).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report here the cloning of a rat homolog of SUR2B, rSUR2B. The predicted amino acid sequence for rSUR2B is 98.7% and 96.6% identical to mouse and human SUR2B, respectively. The proposed Walker A and B consensus sites and several additional motifs (PKA and PKC phosphorylation sites, glycosylation sites) identified in other SUR isoforms are conserved in rSUR2B. The wide expression of rSUR2B transcripts in all of the tested tissues is also in agreement with the expression pattern determined for mouse SUR2B, confirming the cloning of rat member of the SUR family.
The present study provides strong evidence that the renal
KATP channel responsible for K+ secretion in
TAL cells and the principal cells of the CCD is comprised of ROMK2 and
rSUR2B. First, the expression of ROMK2 (7, 38) and rSUR2B (Fig. 3; Ref.
6) transcripts and protein overlap in the CTAL and CCD. Second,
coexpression of ROMK2 and rSUR2B in X. laevis oocytes generates
whole cell K+ currents that are inhibited by glibenclamide
(Figs. 4 and 5) with an EC50 of 185 µM (Fig. 5). A
comparison of the glibenclamide sensitivities of ROMK coexpressed with
SUR2B (from Fig. 5) or CFTR (from Refs. 25, 31) and the native
KATP channels in rat TAL (36, 37) and principal (36) cells
is shown in Fig. 7. The striking
concordance in the sensitivities to glibenclamide of whole cell
K+ currents in ROMK2 + rSUR2B coinjected oocytes and that
of native renal KATP channels measured by inside-out
patches [EC50 = 150 µM (36); Fig. 7, native
TAL] strongly supports the hypothesis that ROMK2 and SUR2B encode
the renal secretory KATP channel. Third, the in vitro
binding assays (Fig. 6) show direct physical association of ROMK and
rSUR2b and that this association of subunits is required for
glibenclamide sensitivity. This is consistent with the direct
association of Kir + SUR in forming KATP channels in other
tissues (22).
|
The ROMK2-rSUR2B complex can be coimmunoprecipitated in the presence (Fig. 6) and absence (data not shown) of microsomal membranes, suggesting that the glycosylated forms of ROMK2 or rSUR2B are not required for the heteromultimeric complex formation. The results of the coimmunoprecipitation studies in the in vitro translation assay also imply that an intermediate protein(s) is not necessary for the ROMK2 + rSUR2B channel subunit assembly. In contrast to ROMK2, whole cell K+ currents in ROMK1 + rSUR2B or ROMK3 + rSUR2B coinjected oocytes exhibited no significant sensitivity to glibenclamide (Fig. 5), indicating that among the three known ROMK isoforms in rat kidney only ROMK2 can coassemble with rSUR2B. Interestingly, ROMK2 is the most widely expressed of the channel isoforms along the distal nephron and collecting duct, being absent only in the outer medullary collecting duct (7). Because ROMK1 and ROMK3 differ from ROMK2 only in the NH2 terminus by a 19- or 26-amino acid extension (7), respectively, the results shown in Fig. 6 suggest that the direct physical interaction between ROMK and rSUR2B is interrupted by the NH2-terminal extensions of ROMK1 and ROMK3. In support of this hypothesis, Reimann et al. (30) showed that NH2-terminal deletions of Kir6.2 abolish high-affinity sulfonylurea block, implying that the NH2 terminus of Kir6.2 is involved in coupling to the SUR subunit. Several other studies have also demonstrated that the NH2-terminal domain of Kir6.2 determines the gating properties and biochemical interaction between the two subunits (5, 8, 11, 12, 19) of this KATP channel. We are currently examining this issue for ROMK2 + rSUR2B interactions.
Several recent studies (2, 15-18) and reviews (1, 43) have suggested that the different SUR isoforms can explain the varying glibenclamide sensitivity found in KATP channel from a variety of tissues. For example, Kir6.2 coexpressed with either SUR1 or SUR2A in mammalian cells exhibit EC50 values for glibenclamide of 9 and 350 nM, respectively. The present results indicate, however, that the associated Kir subunit can also affect glibenclamide sensitivity. When SUR2B is coexpressed with Kir6.1, 3 µM glibenclamide completely abolishes channel activity (40), whereas this concentration would have negligible effect on ROMK2 + rSUR2B (Fig. 5). Thus both the SUR isoforms and the associated Kir subunits of KATP channels determine the glibenclamide sensitivity, a finding also consistent with direct interaction of SUR with the Kir subunit.
Although CFTR is also expressed at the apical regions of distal nephron segments (9) and forms glibenclamide-sensitive K+ channels when expressed with either ROMK1 (31) or ROMK2 (24, 25) in oocytes, two observations suggest that CFTR may not form the renal epithelial KATP channel in CTAL and CCD. First, the glibenclamide sensitivity of K+ channels in ROMK + CFTR coinjected X. laevis oocytes [ROMK1 + CFTR, EC50 = 33 µM (31); ROMK2 + CFTR, EC50 = 2.0 µM (24); Fig. 7] is significantly higher than for the native KATP channels in renal cells [EC50 = 150 µM (36)]. Second, CFTR transcripts appear to be predominantly expressed in intercalated cells in the CCD (33), whereas ROMK is expressed only in the principal cells (38). This does not exclude the real possibility that CFTR regulates the function of ROMK in CTAL in some other fashion as it does with other channels (13, 14). Interestingly, we did not find rSUR2B in the rat MTAL (Fig. 3), whereas CFTR [or its truncated isoform; TNR-CFTR (27)] is expressed in this nephron segment. Because neither SUR1 (6) nor SUR2A (Fig. 2; Ref. 6) is expressed in the kidney, the low-conductance KATP channel in the MTAL is either formed by an unidentified SUR or by CFTR. The latter would suggest a relatively higher (low µM) glibenclamide sensitivity of the MTAL KATP compared with that in the CTAL (36).
Finally, KATP channels, in parallel with Na+-K+-ATPase, have been suggested to play an important role in K+ recycling across basolateral membranes of rabbit (34, 26) and amphibian proximal tubule (28). The expression of SUR2B in rat proximal tubules raises the possibility that this SUR in association with an unidentified Kir subunit forms the basolateral KATP channel in this nephron segment.
In conclusion, the present study provides strong evidence that the small-conductance, secretory KATP channel in renal CTAL and principal cells of the CCD may be comprised of ROMK2 and SUR2B, consistent with the proposed heteromultimeric formation of KATP channels in nonrenal cells.
![]() |
ACKNOWLEDGEMENTS |
---|
The nucleotide sequence reported in this manuscript has been submitted to the GenBank with accession number AF019628.
![]() |
FOOTNOTES |
---|
This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-37605 (to S. C. Hebert)
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. C. Hebert, Div. of Nephrology, Vanderbilt University Medical Center, 21st Ave. South and Garland, Rm. 53223, Medical Center North, Nashville, TN 37232-2372 (E-mail: steven.hebert{at}mcmail.vanderrbilt.edu).
Received 10 September 1999; accepted in final form 11 November 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aguilar-Bryan, L,
Clement JP, IV,
Gonzalez G,
Kunjilwar K,
Babenko A,
and
Bryan J.
Toward understanding the assembly and structure of KATP channels.
Physiol Rev
78:
227-245,
1998
2.
Aguilar-Bryan, L,
Nicholas CG,
Wechesler SW,
Clement JP, IV,
Boyd AE, III,
Gonzalez G,
Herrera-Sosa H,
Nauy K,
Bryan J,
and
Nelson DA.
Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion.
Science
268:
423-426,
1995[ISI][Medline].
3.
Ashcroft, FM.
Mechanisms of the glycaemic effects of sulfonylureas.
Horm Metab Res
28:
456-463,
1996[ISI][Medline].
4.
Ashcroft, SJH,
and
Ashcroft FM.
Properties and function of ATP-sensitive K-channels.
Cell Signal
2:
197-214,
1990[ISI][Medline].
5.
Babenko, AP,
Gonzalez G,
and
Bryan J.
The N-terminus of Kir6.2 limits spontaneous bursting and modulates the ATP-inhibition of KATP channels.
Biochem Biophys Res Commun
255:
231-238,
1999[ISI][Medline].
6.
Beesley, AH,
Qureshi IZ,
Giesberts AN,
Parker AJ,
and
White SJ.
Expression of sulfonylurea receptor protein in mouse kidney.
Pflügers Arch
438:
1-7,
1999[ISI][Medline].
7.
Boim, MA,
Ho K,
Shuck ME,
Bienkowski MJ,
Block JH,
Slightom JL,
Yang Y,
Brenner BM,
and
Hebert SC.
ROMK inwardly rectifying ATP-sensitive K+ channel. II. Cloning and distribution of alternative forms.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1132-F1140,
1995
8.
Clement, JP, IV,
Kunjilwar K,
Gonzalez G,
Schwanstecher M,
Panten U,
Aguilar-Bryan L,
and
Bryan J.
Association and stoichiometry of KATP channel subunits.
Neuron
18:
827-838,
1997[ISI][Medline].
9.
Crawford, I,
Maloney PC,
Zeitlin PL,
Guggino WB,
Hyde SC,
Turley H,
Harris A,
and
Higgins CF.
Immunocytochemical localization of the cystic fibrosis gene product CFTR.
Proc Natl Acad Sci USA
88:
9262-9266,
1991[Abstract].
10.
Edwards, G,
and
Weston AH.
The pharmacology of ATP-sensitive potassium channels.
Annu Rev Pharmacol Toxicol
33:
597-637,
1993[ISI][Medline].
11.
Fink, M,
Duprat F,
Heurteaux C,
Lesage Romey F,
Barhanin G,
and
Lazdunski J.
Dominant negative chimeras provide evidence for homo and heteromultimeric assembly of inward rectifier K+ channel proteins via their N-terminal end.
FEBS Lett
378:
64-68,
1996[ISI][Medline].
12.
Giblin, JP,
Leaney JL,
and
Tinker A.
The molecular assembly of ATP-sensitive potassium channels.
J Biol Chem
274:
22652-22659,
1999
13.
Higgins, CF.
The ABC of channel regulation.
Cell
82:
693-696,
1995[ISI][Medline].
14.
Ho, K,
Nichols CG,
Lederer WJ,
Lytton J,
Vassilev PM,
Kanazirska MV,
and
Hebert SC.
Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.
Nature
362:
31-38,
1993[ISI][Medline].
15.
Inagaki, N,
Gonoi T,
Clement JP, IV,
Namba N,
Inazawa J,
Gonzalez G,
Aguilar-Bryan L,
Seino S,
and
Bryan J.
Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor.
Science
270:
1166-1170,
1995[Abstract].
16.
Inagaki, N,
Gonoi T,
Clement JP, IV,
Wang C-Z,
Aguilar-Bryan L,
Bryan J,
and
Seino S.
A family of sulfonylurea receptors determines the pharmacological properties of ATP-sensitive K+ channels.
Neuron
16:
1011-1017,
1996[ISI][Medline].
17.
Inagaki, N,
Tsuura Y,
Namba N,
Masuda K,
Gonoi T,
Seino Y,
Mizula M,
and
Seino S.
Cloning and functional characterization of a novel ATP-sensitive potassium channel ubiquitously expressed in rat tissues, including pancreatic islets, pituitary, skeletal muscle, and heart.
J Biol Chem
270:
5691-5694,
1995
18.
Isomoto, S,
Kondo C,
Yamada M,
Matsumoto S,
Higashiguchi O,
Horio Y,
Maysuzawa Y,
and
Kurachi Y.
A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP-sensitive K+ channel.
J Biol Chem
271:
24321-24324,
1996
19.
Koster, JC,
Bentle KA,
Nichols CG,
and
Ho K.
Assembly of ROMK1 (Kir1.1a) inward rectifier K+ channel subunits involves multiple interaction sites.
Biophys J
74:
1821-1829,
1998
20.
Lee, WS,
and
Hebert SC.
ROMK inwardly rectifying ATP-sensitive K+ channel. I. Expression in rat distal nephron segments.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F1124-F1131,
1995
21.
Liman, ER,
Tytgat J,
and
Hess P.
Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs.
Neuron
9:
861-871,
1992[ISI][Medline].
22.
Lorenz, E,
Alekseev AE,
Krapivinsky GB,
Carrasco AJ,
Clapham DE,
and
Terzic A.
Evidence for direct physical association between a K+ channel (Kir6.2) and an ATP-binding cassette protein (SUR1) which affects cellular distribution and kinetic behavior of an ATP-sensitive K+ channel.
Mol Cell Biol
18:
1652-1659,
1998
23.
Mauerer, UR,
Boulpaep EL,
and
Segal AS.
Regulation of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule.
J Gen Physiol
111:
161-180,
1998
24.
McNicholas, CM,
Guggino WB,
Schwiebert EM,
Hebert SC,
Giebisch G,
and
Egan ME.
Sensitivity of a renal K+ channel (ROMK2) to the inhibitory sulfonylurea compound glibenclamide is enhanced by coexpression with the ATP-binding cassette transporter cystic fibrosis transmembrane regulator.
Proc Natl Acad Sci USA
93:
8083-8088,
1996
25.
McNicholas, CM,
Nason MW,
Guggino WB,
Schwiebert EM,
Hebert SC,
Giebisch G,
and
Egan ME.
A functional CFTR-NBF1 is required for ROMK2-CFTR interaction.
Am J Physiol Renal Physiol
273:
F843-F848,
1997
26.
Misler, S,
and
Giebisch G.
ATP-sensitive potassium channels in physiology, pathophysiology, and pharmacology.
Curr Opin Nephrol Hypertens
1:
21-33,
1992[Medline].
27.
Morales, MM,
Carroll TP,
Morita T,
Schwiebert EM,
Devuyst O,
Wilson PD,
Lopes AG,
Stanton BA,
Dietz HC,
Cutting GR,
and
Guggino WB.
Both the wild type and a functional isoform of CFTR are expressed in kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F1038-F1048,
1996
28.
Noulin, J-F,
Brochiero E,
Lapointe J-Y,
and
Laprade R.
Two-types of K+ channels at the basolateral membrane of proximal tubule: inhibitory effect of taurine.
Am J Physiol Renal Physiol
277:
F290-F297,
1999
29.
Quast, U.
ATP-sensitive K+ channels in the kidney.
Naunyn-Schimiedebergs Arch Pharmacol
354:
213-222,
1996.
30.
Reimann, F,
Tucker SJ,
Proks P,
and
Ashcroft FM.
Involvement of the N-terminus of Kir6.2 in coupling to the sulfonylurea receptor.
J Physiol (Lond)
518:
325-336,
1999
31.
Rukundin, A,
Schulze DH,
Sullivan SK,
Lederer WJ,
and
Welling PA.
Novel subunit composition of a renal epithelial KATP channel.
J Biol Chem
273:
14165-14171,
1998
32.
Tinker, A,
Jan YN,
and
Jan LY.
Regions responsible for the assembly of inwardly rectifying potassium channels.
Cell
87:
857-868,
1996[ISI][Medline].
33.
Todd-Turla, KM,
Rusval E,
Náray-Fejes-Tóth A,
and
Fejes-Tóth G.
CFTR expression in cortical collecting duct cells.
Am J Physiol Renal Fluid Electrolyte Physiol
270:
F237-F244,
1996
34.
Tsuchiya, K,
Welling PA,
and
Giebisch G.
Pharmacological evidence for an ATP-sensitive K+ conductance in the rabbit proximal tubule basolateral membrane.
J Am Soc Nephrol
1:
693,
1990.
35.
Tusnády, GE,
and
Bakos
É, Váradi A, and Sarkadi B. Membrane topology distinguishes a subfamily of the ATP-binding cassette (ABC) transporters.
FEBS Lett
402:
1-3,
1997[ISI][Medline].
36.
Wang, T,
Wang WH,
Klein-Robbenhaar G,
and
Giebisch G.
Effects of glyburide on renal tubule transport and potassium-channel activity.
Renal Physiol Biochem
18:
169-182,
1995[ISI][Medline].
37.
Wang, WH.
Two types of K+ channel in thick ascending limb of rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
267:
F599-F605,
1994
38.
Xu, JZ,
Hall AE,
Peterson LN,
Bienkowski MJ,
Eessalu TE,
and
Hebert SC.
Localization of the ROMK protein on apical membranes of rat kidney nephron segments.
Am J Physiol Renal Physiol
273:
F739-F748,
1997[ISI][Medline].
39.
Xu, ZC,
Yang Y,
and
Hebert SC.
Phosphorylation of the ATP-sensitive, inwardly rectifying K+ channel, ROMK, by cyclic AMP-dependent protein kinase.
J Biol Chem
271:
9313-9319,
1996
40.
Yamada, M,
Isomoto S,
Matsumoto S,
Kondo C,
Shindo T,
Horio Y,
and
Kurachi Y.
Sulfonylurea receptor 2B and Kir6.1 form a sulfonylurea-sensitive but ATP-insensitive K+ channel.
J Physiol (Lond)
499:
715-720,
1997[Abstract].
41.
Yokoshiki, H,
Sunagawa M,
Seki T,
and
Sperelakis N.
ATP-sensitive K+ channels in pancreatic, cardiac, and vascular smooth muscle cells.
Am J Physiol Cell Physiol
274:
C25-C37,
1998