1 Institute of Physiology, Marburg University, 35037 Marburg; 2 Institute of Pharmacology, Tübingen University, 72074 Tübingen, Germany
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ABSTRACT |
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ATP-sensitive
K+ (KATP) channels are composed of pore-forming
Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig
SUR1. Three splice forms originate from alternative splicing of the
region coding for NBF1 and lack exons 17 (SUR117), 19 (SUR1
19),
or both (SUR1
17
19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31-39 containing the last two
transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis
showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using
enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide
any evidence for involvement of these splice forms in the
mitochondrial KATP channel. Only SUR1 and SUR1
17 showed high-affinity binding of glibenclamide (Kd
2 nM in the presence of 1 mM ATP) and formed functional KATP
channels upon coexpression with Kir6.2.
ATP-sensitive potassium channel; mitochondria; glibenclamide; meglitinide
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INTRODUCTION |
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ATP-SENSITIVE POTASSIUM
CHANNELS (KATP channels) link the metabolic state of
a cell to excitability: cytosolic ATP inhibits KATP
channels, whereas cytosolic MgADP activates these channels. KATP channels are involved in many physiological processes
including insulin secretion by pancreatic cells, regulation of
vascular tone, and neuronal excitability. The molecular components of
KATP channels have been cloned (1, 22, 23,
25), and the channel has been identified as an octameric
assembly of four channel-forming Kir6.x subunits and four regulatory
sulfonylurea receptor (SUR) subunits (14, 45). The Kir6.x
subunits belong to the family of inwardly rectifying potassium
channels. The SUR is a member of the ATP-binding cassette (ABC)
superfamily and contains 17 transmembrane helices, which are
arranged in three clusters. SUR possesses two intracellular
nucleotide-binding folds (NBFs) as well as binding sites for channel
openers and for blockers of the sulfonylurea type (3).
Binding sites of channel openers and blockers have been localized to
the COOH-terminal transmembrane domain complex of SUR (4, 5, 30,
48), with negative allosteric coupling between these sites
(9, 41). Association of Kir6.x and SUR subunits is a
prerequisite for trafficking of the KATP channel octamer to
the cell membrane. Both subunits contain several anterograde and
retrograde trafficking signal sequences (44, 51) that
determine cell surface expression.
The Kir and the SUR subunits of KATP channels are both encoded by two genes, Kir6.1 and Kir6.2, and SUR1 and SUR2. Alternative splicing of the last exon of SUR2 gives rise to the isoforms SUR2A and B (for review, see Refs. 2 and 3). Channel diversity is created by different combinations of Kir6.x and SURx subunits: the pancreatic KATP channel prototype (Kir6.2/SUR1) is blocked by the sulfonylurea glibenclamide with high potency (IC50 < 1 nM) and shows activation by diazoxide (16, 22). The myocardial KATP channel prototype (Kir6.2/SUR2A) shows a lower affinity for glibenclamide (IC50 = 26 nM; Ref. 38) and, in the absence of MgADP, no activation by diazoxide (23, 32). The vascular KATP channel prototype (Kir6.1/SUR2B) shows a similar sensitivity for glibenclamide (IC50 = 43 nM; Ref. 37), although it is activated by diazoxide (25). However, expression of these isoforms is not restricted to these tissues. KATP channels have also been described in brain (26, 27, 50), kidney (8), and coronary endothelial cells (29). A mitochondrial KATP channel has also been postulated (24), but its molecular composition remains elusive (28, 43).
The SUR2 gene contains 39 exons, and several different splice forms have been found in which the last exon is altered (SUR2A and SUR2B; Refs. 23, 25, and 49) or internal exons are skipped (12, 13, 39). The regions of the NBFs form large cytosolic loops and have attracted particular attention in the search for new splice forms, because alternative splicing may change pharmacological properties of the resulting KATP channels but would not change membrane topology. Changes in the NBFs would be expected to modify the binding of nucleotides and, by allosteric interactions, also the binding of K+ channel openers and sulfonylurea derivatives.
A SUR2 splice form lacking exon 17 (SUR217) was found by RT-PCR in
several mouse tissues, including heart, skeletal muscle, bladder, small
intestine, and prostate (13, 15). Heterologous coexpression of this splice form with Kir6.2 shows a small decrease in
ATP-sensitivity (13). Another SUR2 splice form identified in cardiac tissue (12) lacks exon 14 but could not be
functionally expressed. In the case of SUR1, a splice form lacking exon
33 has been described that shows unique electrophysiological properties (39). The lack of exon 33 introduces a frame shift that
leads to a deletion of the second nucleotide binding domain (NBF2). A
second SUR1 splice variant lacking exon 31 has not been functionally characterized (6). Here we describe four novel splice
forms of SUR1. Three of them lack exon 17 (SUR1
17) and/or exon 19 (SUR1
17
19 or SUR1
19), which are both located in the coding
region of the first NBF. The fourth splice form is a truncated
COOH-terminal SUR1 fragment (SUR1C). Functional analysis showed that
only SUR1 containing all the exons (referred to as SUR1 in the
following text) and SUR1
17 exhibit specific binding of glibenclamide
and give rise to ATP-sensitive channels when coexpressed with Kir6.2 subunits.
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MATERIALS AND METHODS |
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RT-PCR of splice forms. RNA from different guinea pig tissues was prepared using a modified single-step method (10). RNA (1 µg) was reverse transcribed with Superscript reverse transcriptase (Life Technologies). As a positive control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was tested by RT-PCR (29). RNA from ventricular cardiomyocytes and capillary endothelial cells was prepared and reverse transcribed as described previously (34). The purity of cell fractions was verified by RT-PCR experiments by using the cell-specific markers troponin T and endothelin-1 (34). Two degenerated primers were used to amplify both SUR1 and SUR2 from RT samples, with the forward primer located on exons 15 and 16 (5'-AGGYCAGCTRACYATGATYGTRGG-3') and the reverse primer located on exon 21 (5'-ATGTCTGGCTGMAGRGAGCAGGC-3'). The SUR1C fragment was amplified with primers located in the 5'-noncoding region of the SUR1C cDNA (see below) (forward: 5'-GAAGGGTGGCTATGATCACTGATGGG-3') and in exon 31 (reverse: 5'-CACAGGCACCGATGTACTCCATTCG-3'). PCR products of different sizes, depending on the splice form (see RESULTS), were amplified by using AmpliTaq Gold DNA polymerase (Applied Biosystems) for 35 cycles at 94°C for 1 min, 55°C for 30 s, and 73°C for 30 s (with an initial polymerase activation at 94°C for 6 min and a final elongation at 73°C for 5 min). Finally, PCR products were visualized on a 4% Nusieve agarose gel and directly sequenced. In addition, PCR products were cloned into pBluescript SK+ vector (Life Technologies) and sequenced to identify minor SUR1 or SUR2 splice forms.
Isolation of SUR1C from a guinea pig cardiac cDNA library.
A cardiac ventricle cDNA library was constructed, ligated into the
TriplEx vector (Clontech, Palo Alto, CA), and screened with a
COOH-terminal gpSUR1 fragment as described previously
(29). Total sequence information was obtained from the
converted pTriplEx. For 5' and 3' RACE (rapid amplification of cDNA
ends), a Marathon cDNA was synthesized from 5 µg of
poly(A)+ RNA as described previously (36).
Construction of SUR1 expression vectors.
To construct a SUR1 expression vector of the different splice forms, an
ApaI/XhoI cassette was amplified from rat heart,
kidney, and brain cDNA by using rat SUR1 specific primers (forward:
5'-CGAGAAGAAGTCCGGGACCTCT-3', reverse:
5'-CTGTCCTCTTGTCATCCCGGAG-3'). The PCR products were digested with
ApaI and XhoI and cloned into pBluescript SK+
vector. Rat SUR117 and SUR1
19 splice forms were identified by DNA
sequencing. Finally, the
17 and
19 cassettes were subcloned into
rat SUR1/pBF1 (kindly provided by Dr. B. Fakler, Tübingen,
Germany). For heterologous expression in mammalian cells, the ratSUR1
splice forms were subcloned into pcDNA3.1 (Invitrogen, Karlsruhe,
Germany) or pEGFP-C1 (NH2-terminal fusion of the enhanced
green fluorescent protein; Clontech) vectors. For expression of the
SUR1C splice form, the guinea pig clone was used.
Cell culture and transfections. Human embryonic kidney (HEK)-293 cells were cultured as described previously (19, 20) in minimum essential medium containing glutamine, supplemented with 10% fetal bovine serum and 20 µg/ml gentamycin. COS-7 cells were grown in DMEM (Life Technologies) supplemented with 10% fetal bovine serum and 10 µg/ml streptomycin-ampicillin mixture (Biochrom, Berlin, Germany). For binding studies and electrophysiological measurements, cells were transfected with mammalian expression vector pcDNA3.1 (Invitrogen) containing the coding sequence of rat SUR1 (rSUR1; GenBank accession no. X97279; Ref. 1), the different splice forms of SUR1 mentioned above, or pcDNA3.1 vector alone with Lipofectamine and OptiMem (Invitrogen) (19). For electrophysiological studies, SUR1 splice forms were cotransfected transiently with Kir6.2 (GenBank accession no. D50581; Ref. 22) at a molar plasmid ratio of 1:1. Here, pEGFP-C1 vector (Clontech), encoding for green fluorescent protein, was added for easier identification of transfected cells. Two to four days after transfection, cells were used for binding studies and electrophysiological experiments. Cell lines stably transfected with SUR1 wild-type or SUR1 splice forms were isolated in the presence of 700 µg geneticin/ml in the first 3 wk and 300 µg geneticin/ml medium; thereafter, 1 wk before experiments, the antibiotic was withdrawn.
For confocal microscopy, rSUR1 splice forms and guinea pig Kir6.1 cDNAs (GenBank accession nos. AF183918 and AF183919; Ref. 29) were fused to the EGFP gene by cloning into the expression vector pEGFP-C1 or -N1 (Clontech). Subcellular localization vectors were used to label mitochondria (mitoGFP: targeting sequence of theConfocal microscopy. Confocal images were produced using a FV300 confocal laser scanning microscope (Olympus, Hamburg, Germany). Images of 600 × 800 pixels were obtained via a ×60 oil-immersion objective lens (numerical aperture 1.4). Laser intensity was set at 6%, and the pinhole was optimized for the ×60 objective. COS-7 cells were grown on coverslips, and mitochondria were labeled by incubation with 50 nM MitoTracker red (Molecular Probes, Leiden, The Netherlands) for 7-8 min at ~30°C. MitoTracker red was excited with the 568-nm line of a Kr-Ar ion laser, and red fluorescence was detected at >585 nm. We avoided repeatedly scanning MitoTracker red-labeled cells because we found that repeated excitation induced a change in mitochondrial morphology, probably secondary to the phototoxic effects of this indicator as previously described (31). Simultaneous to MitoTracker red excitation, EGFP was excited at 488 nm, and green fluorescence was detected at 510-540 nm. Areas of overlap between the red and green signals were seen as yellow.
Equilibrium-binding experiments.
Membranes were prepared from HEK-293 cells at 80% confluence (16 million cells/dish) (19). Cells were easily detached by repeated rinsing with medium in the absence of trypsin and centrifuged at 37°C for 6 min at 500 g. The pelleted cells were lysed
by addition of 5 ml/dish of ice-cold hypotonic buffer containing 10 mM
HEPES and 1 mM EGTA (pH 7.4), and the lysate was centrifuged (100,000 g) for 60 min at 4°C. The resulting membrane pellet was
resuspended in a buffer containing 5 mM HEPES, 5 mM KCl, and 139 mM
NaCl (pH 7.4, 4°C) at a protein concentration of 0.7 mg/ml and frozen
at 80°C. In the binding assay, membranes (final protein
concentration 0.03-0.4 mg/ml) were added to the incubation buffer
(139 mM NaCl, 5 mM KCl, 5 mM HEPES, and 2.2 mM MgCl2)
supplemented with the radioligand [1-6 nM
[3H]glibenclamide (GBC)], the competitor of interest,
and either 2.2 mM MgCl2 + 1 mM Na2 ATP
(for binding experiments in the presence of MgATP) or 0 mM
MgCl2 + 0 mM Na2 ATP + 1 mM EDTA (for
experiments in the absence of MgATP). After 15 min of incubation at
37°C, the reaction was stopped by diluting 0.3-ml aliquots in
triplicate into 8 ml of ice-cold quench solution [50 mM
tris(hydroxymethyl)aminomethane, 154 mM NaCl, pH 7.4] and rapid
filtration under vacuum over Whatman GF/B filters. Filters were washed
twice with 8 ml of ice-cold quench solution and counted for
3H in the presence of 6 ml of scintillant (Ultima Gold;
Packard, Meriden, CT). Nonspecific binding of [3H]GBC
(BNS) was determined in the presence of 1 µM GBC and was about 5-10% of total binding.
Patch-clamp experiments.
Whole cell or inside-out patch-clamp recordings (21) were
carried out in HEK-293 cells cotransfected with KATP
channel subunits and green fluorescent protein (GFP). Patch pipettes
were drawn from borosilicate glass capillaries (GC 150T; Harvard
Apparatus, Edenbridge, UK) and heat polished using a horizontal
microelectrode puller (Zeitz, Augsburg, Germany). For inside-out
patches, bath and pipette were filled with a high-K+ Ringer
solution containing (in mM) 142 KCl, 2.8 NaCl, 1 MgCl2, 1 CaCl2, 11 D(+)-glucose, and 10 HEPES, titrated to pH 7.4 with NaOH at 22°C. After being filled with buffer, pipettes had a
resistance of 1-1.5 M. After the patch was excised, the pipette
was moved in front of a superfusion cannula filled with high-
K+ EGTA Ringer solution containing 143 mM KCl, 0.85 mM
MgCl2, 1 mM CaCl2, 5 mM EGTA, 11 mM
D(+)-glucose, and 10 mM HEPES (pH 7.2, 22°C). ATP was added to the
solution, keeping free Mg2+ constant (0.7 mM), and KCl was
reduced to maintain osmolarity. The solution with low free
Mg2+ contained 142 mM KCl, 0.5 mM CaCl2, 0.5 mM
MgCl2, 5 mM EDTA, 10 mM HEPES, and 11 mM
D-glucose (pH 7.2, 22°C). The free Ca2+ and
Mg2+ concentrations were calculated to be 5.4 and 270 nM,
respectively (19). ATP was added in appropriate amounts
and had only minor effects on free Ca2+ or
Mg2+. After addition of 1 mM ATP, the concentration of
MgATP was 2.3 µM. Excised patches were clamped at a transmembrane
potential of
50 mV.
Data analysis and statistics.
Inhibition curves from radioligand binding and patch-clamp experiments
were analyzed according to the logistic equation
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(1) |
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(2) |
Materials. [3H]GBC [specific activity 1.85 TBq (50 Ci)/mmol] was purchased from Perkin Elmer Life Science Products (Bad Homburg, Germany). The reagents and media used for cell culture and transfection were from Invitrogen. Na2ATP and Li2GDP were obtained from Roche Molecular Biochemicals (Mannheim, Germany) and glibenclamide from Sigma (Deisenhofen, Germany). The following drugs were kind gifts of the pharmaceutical companies indicated: diazoxide (Essex Pharma, Munich, Germany), meglitinide (Aventis, Frankfurt, Germany), and l-pinacidil (Leo Pharmaceuticals, Ballerup, Denmark). KATP channel modulators were dissolved in dimethyl sulfoxide/ethanol [50/50 (vol/vol)] and further diluted with the same solvent or with incubation buffer (final solvent concentration < 0.3%).
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RESULTS |
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Identification of guinea pig SUR1 and SUR2 splice forms.
To investigate alternative splicing of the short exons of the first NBF
of guinea pig SUR subunits, we designed primers for amplification of
exons 16-20 (SUR1) or 16-19 (SUR2) from both gpSUR1 and
gpSUR2 in one PCR reaction (Fig. 1).
After the PCR products were separated on a 4% Nusieve agarose gel,
bands of 323, 290, 252, 224, and 191 bp were identified in several
tissues and cell fractions (Fig. 2).
Direct sequencing or cloning of PCR products revealed that these bands
correspond to the following gpSUR splice forms: gpSUR1 (323 bp); gpSUR2
(291 bp); and gpSUR1
17 (286 bp) as a component of the
290-bp PCR
product; gpSUR2
17 (252 bp); gpSUR1
19 (224 bp); and
gpSUR1
17
19 (191 bp). Direct sequencing of the second PCR band
shown in Fig. 2A revealed only the sequence of gpSUR2.
However, cloning and sequencing of isolated clones showed that a minor
portion of this band (<10%) represented gpSUR1
17, indicating that
the band contained a mixture of two PCR products (291 and 286 bp).
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Tissue distribution of SUR splice forms.
Figure 2A shows the RT-PCR analysis of several guinea pig
tissues. The different gpSUR splice forms were detected in significant amounts in all tissues tested (Fig. 2A), except for
gpSUR117
19, which was found only in the cardiomyocyte fraction
(see SUR expression in heart cell
fraction). The relative amounts of the PCR products varied
from organ to organ. gpSUR1 was detected in all tissues tested,
although the band observed in heart was very faint. gpSUR1
19 was
detected at low levels in brain, lung, heart, and pancreas and at even
lower amounts in other tissues. The detection of gpSUR1
17 was
difficult because the size of the PCR product was the same as for
gpSUR2. However, from sequencing of cloned PCR products, it became
clear that the number of gpSUR1
17 clones were always <10% of the
total clones tested, indicating that this isoform is a minor splice
variant. gpSUR2 was particularly abundant in brain, heart, kidney, and
liver. gpSUR2
17 was found at high RNA levels in stomach and, in
agreement with previous work (13), in skeletal muscle.
Note that exon 18 of SUR2 is equivalent to exon 19 of SUR1 (Fig.
1B); thus SUR2
18 would correspond to SUR1
19. However,
we have not detected a gpSUR2 splice form without exon 18.
SUR expression in heart cell fraction.
Cell-specific RT-PCR analysis was carried out with isolated
cardiomyocytes and with coronary capillaries, which consist only of
microvascular endothelial cells. In both cell types, gpSUR2 was the
dominant PCR signal (Fig. 2B). gpSUR217 was also
expressed, especially in endothelial cells. In accordance with the
analysis of whole heart fraction, low amounts of gpSUR1, gpSUR1
19,
and gpSUR1
17 were found in cardiomyocytes. The expression of the splice form lacking both exons, gpSUR1
17
19, appeared to be
restricted to cardiomyocytes because it was not observed in other
tissues or in coronary capillaries. In endothelial cells, only gpSUR1 was detected but not (or only very weakly) the other gpSUR1 splice forms.
Identification of a short splice variant of SUR1.
To isolate SUR1 from a guinea pig heart, we constructed a cDNA library
and screened with a COOH-terminal guinea pig-specific SUR1 probe
(29). Several positive clones were identified and isolated. After the TriplEx DNA was converted from an isolated clone
to pTriplEx, sequencing revealed a gpSUR1 cDNA 1,663 bp in length
(GenBank accession no. AF183921). The 5' noncoding region (nucleotides
1-556) of this clone showed an as yet unknown sequence from bp 1 to 205 and continued with the known sequence of gpSUR1 starting at exon
28. The putative coding region of the cDNA (557-1552 bp)
represents exons 31 (with a putative Start-Codon) to 39 of gpSUR1 and
encodes a truncated gpSUR1 protein of 332 amino acids, which we named
gpSUR1C. The deduced amino acid sequence contains only the last two
putative transmembrane regions and the COOH-terminal domain of gpSUR1.
SUR1 splice forms from rat.
Because we have not cloned the entire SUR1 from guinea pig, we
reconstituted the splice forms lacking exons 17 and 19 with the rat
SUR1 cDNA (see MATERIALS AND METHODS). By amplifying a 690-bp fragment (ApaI-XhoI cassette) from
different rat tissues, we were able to detect the rSUR117 and
rSUR1
19 splice forms in this species as well (data not shown).
However, we have not performed a detailed expression analysis in rat
tissues. Although there are significant sequence differences between
guinea pig and rat exons 17-18, including several amino acid
substitutions and one insertion in exon 17 (see Fig. 1B),
the overall sequence identity (21 of 27 residues) and the conservation
of alternative splicing are consistent with conserved functional
properties. For further functional analysis, these rSUR1 splice forms
(except for gpSUR1C) were used.
The novel SUR1 splice forms are not part of the mitochondrial
KATP channel.
In recent years much work has been done to elucidate the
molecular nature of the mitochondrial KATP channel. To
investigate the possibility that the novel SUR1 splice forms may be
targeted to the mitochondria, we constructed several
EGFP-fusion proteins of rSUR1, rSUR119, rSUR1
17, and gpSUR1C
as well as Kir6.1. Figure 3 shows COS-7
cells labeled with the red fluorescent dye MitoTracker red
(A) or transfected only with the subcellular localization markers MitoGFP but not with SUR or Kir6.1 subunits (B).
MitoTracker red and MitoGFP were used as positive controls for
targeting the mitochondria. It can be seen that the fluorescence of
these two markers overlapped (Fig. 3C), thus providing a
reliable picture of the subcellular localization of mitochondria (Fig.
3, A-C). To determine the morphology of the endoplasmic
reticulum (ER), a YFP-tagged ER-marker was used (Fig. 3, D
and E). Figure 4 shows the
fluorescence of COS-7 cells transfected with EGFP-tagged rSUR1, rSUR1
19, and rSUR1
17. No overlap with MitoTracker red
fluorescence was seen for these EGFP-tagged splice forms. Recently, it
has been suggested that the Kir6.1 subunit may be part of the
mitochondrial KATP channel (46). Coexpression
of the SUR1 splice forms with NH2- or COOH-terminally
EGFP-tagged Kir6.1, however, never resulted in a mitochondrial
targeting of the Kir6.1 subunits, as illustrated in Fig.
5. Figure 5 shows coexpression of
Kir6.1-EGFP with rSUR1
17 (A-C) and coexpression of
Kir6.1-EGFP with rSUR1
19 (D-F). Similar results were
obtained for rSUR1 and gpSUR1C (not shown). In summary, our results
suggest that SUR1 splice forms and Kir6.1 are not part of the
mitochondrial KATP channel. The sometimes diffusely labeled
cytosol may indicate that EGFP-labeled KATP channel
subunits may not only be retained in the ER but may also be found in
other vesicular structures yet to be characterized.
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Binding of [3H]GBC to the different SUR1 splice forms. After transfection of HEK-293 cells with different SUR1 splice forms, the binding of glibenclamide to membrane preparations of these cells was investigated. First, [3H]GBC binding was assayed using high concentrations of radioligand (5 nM) and protein (0.2-0.4 mg/ml). These conditions were chosen to allow us to differentiate between low-affinity specific and endogenous [3H]GBC binding to control cells transfected with pcDNA3.1 vector alone. Experiments were performed with 0 mM Mg2+ and 1 mM EDTA to avoid inhibition of specific [3H]GBC binding by MgATP (40).
We found no significant difference between membranes from cells transfected with pcDNA3.1 vector alone (control) and with rSUR1Pharmacological properties of SUR1 and SUR117.
In homologous competition experiments, [3H]GBC binding to
rSUR1 was displaced by unlabeled GBC with Kd
values of 0.2 nM in the absence and 1.6 nM in the presence of 1 mM
MgATP (Fig. 6A, Table
1). The results obtained with rSUR1
and rSUR1
17 were not significantly different, with
Kd values of 0.2 nM in the absence of MgATP and
2.0 nM in the presence of 1 mM MgATP. Hill coefficients were close to 1 for all curves, indicating the presence of a single class of
glibenclamide binding sites in each splice form. Specific binding of
[3H]GBC was inhibited to maximally 30% by MgATP in a
concentration-dependent manner, giving very similar curves for both
splice forms (Fig. 6B). Both inhibition curves displayed two
components with nearly the same amplitudes and IC50 values
of ~3 and 200 µM (see Fig. 6B for precise parameters).
Control experiments in the presence of 1 mM EDTA or 1 mM EDTA + 1 mM ATP showed that neither free magnesium nor free ATP affected
(specific or nonspecific) [3H]GBC binding (data not
shown).
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Electrophysiological characterization.
The ability of the splice forms to produce functional plasmalemmal
K+ channels was tested in the whole cell configuration of
the patch-clamp technique. Whereas 4 of 4 green fluorescent cells
examined after transfection with Kir6.2 and rSUR1 developed a prominent
inward current, no such currents were detected in 23 cells from two
transfections with Kir6.2/gpSUR1C or in 18 cells transfected with
Kir6.2/rSUR119.
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DISCUSSION |
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Alternative splicing of SUR is a functionally important mechanism
for creating KATP channel diversity. Most prominently, the differential usage of the two last SUR2 exons (38A and 38B) gives rise
to channels with very different pharmacological properties (23,
25). The SUR1 genes consist of 39 exons, only some of which can
be spliced out without generating a frame shift. Several "spliceable" SUR exons are found within the coding region of the first NBF. Previous reports demonstrated skipping of exons 14, 17, and
18 of SUR2 (2, 13, 15). In the present work we describe
three novel splice variants of SUR1 in which the reading frames are
preserved: SUR117, in which 36 bp are spliced out; SUR1
19, in
which 99 bp are spliced out; and SUR
17
19.
The SUR1 splice forms lacking exons 17 and/or 19 were detected in various guinea pig and rat tissues, although in smaller amounts than SUR1 containing both exons 17 and 19. Different cell-specific expression patterns were found in the heart, indicating different regulation of alternative splicing in cardiac muscle cells and microvascular endothelial cells. The other novel splice variant, gpSUR1C, consisting only of exons 31-39 of SUR1, was found to be strongly expressed in cardiac atria and ventricles. Its expression appears to be restricted to cardiomyocytes because no PCR signal was obtained in the capillary fractions. This short splice variant has a predicted molecular mass of 36 kDa.
Because recent biochemical work has demonstrated that the molecular
mass of the mitochondrial SUR is much smaller (29 kDa, Ref.
47, or 63 kDa, Ref. 18) than
that of SUR1 and SUR2 (177 and 174 kDa), we considered gpSUR1C as a
potential candidate for the mitochondrial KATP channel.
However, the restricted expression pattern of gpSUR1C and the
subcellular localization of the EGFP-tagged KATP channel
subunits make it very unlikely that gpSUR1C represents a subunit of the
mitochondrial KATP channel. Thus the functional role of
this truncated splice form remains unclear. Our experiments with
EGFP-tagged proteins suggest that none of the other novel SUR1 splice
forms, rSUR117 and rSUR1
19, is targeted to the mitochondria, either alone or in combination with Kir6.1. By using immunogold labeling, Suzuki et al. (46) speculated that Kir6.1 may be
the Kir6 subunit of the mitochondrial KATP channel. In
agreement with other work (43), our results using
NH2- or COOH-terminally tagged Kir6.1 showed no
mitochondrial targeting.
The radioligand studies showed no specific [3H]GBC
binding to rSUR119, rSUR1
17
19, or gpSUR1C. On the other hand,
specific binding of [3H]GBC was detected with rSUR1 and
rSUR1
17. Both splice forms also showed great similarity in the other
pharmacological properties tested: specific binding of
[3H]GBC was inhibited by MgATP in a
concentration-dependent manner, consistent with the shift in
[3H]GBC/glibenclamide and
[3H]GBC/meglitinide inhibition curves determined in the
absence and the presence of MgATP. Heterologous displacement of
[3H]GBC binding by l-pinacidil and
diazoxide was only detectable in the presence of MgATP, because
high-affinity binding of these openers requires MgATP (19, 33,
35, 41, 42). The amplitudes of [3H]GBC
displacement by l-pinacidil (~80%) and diazoxide
(~50%) differed from the results of Schwanstecher et al.
(42) who found 100% displacement in both cases. To
explain this discrepancy, it has to be taken into account that
experimental conditions (expression system, incubation time, and
temperature) in these studies were different.
The characteristics of ATP inhibition of Kir6.2/rSUR1 reported in the
present study are in excellent agreement with a previous report
(22). Surprisingly, we observed no difference in ATP block
of Kir6.2/rSUR1 and Kir6.2/rSUR117 in either the presence or absence
of free Mg2+. For the
17 splice variants of SUR2A and
SUR2B, however, Chutkow et al. (13) measured a reduced ATP
sensitivity for channels containing Kir6.2 and SUR2x
17. Therefore,
SUR1/SUR1
17 clearly differ from SUR2/SUR2
17 in this respect.
Because of the prominent differences of SUR1 and SUR2 in exon 17 itself
and the neighboring region (Fig. 1), a divergent behavior of the two
proteins is plausible.
Our electrophysiological characterization of ATP and Mg2+
sensitivity and our radioligand binding studies suggest that the lack of exon 17 does not affect functional expression of rSUR1 in the plasma
membrane and its pharmacological properties. In contrast, exon 19 of
rSUR1 appears to be essential for glibenclamide binding and functional
channel expression because no specific glibenclamide binding was
detected in the splice variants rSUR119 and rSUR1
17
19 and no
KATP currents were observed upon coexpression of Kir6.2 with rSUR1
17
19. Previous studies (4, 30) have shown
that the region between transmembrane helix 14 and CL8 (the cytosolic loop linking helices 15 and 16) contribute to glibenclamide binding by
interacting with the cyclohexyl ring of the glibenclamide molecule (4); in addition, CL3 (the cytosolic loop linking
transmembrane helices 5 and 6) is important for high-affinity
glibenclamide binding to SUR1 (30). On the basis of the
existing data, it cannot be decided whether the region encoded by exon
19 represents an essential part of the glibenclamide binding site
(e.g., for the benzamido or the sulfonylurea part of the molecule) or
whether the absence of exon 19 affects glibenclamide binding
indirectly, for example, by introducing a conformational change in the
SUR1 molecule.
In conclusion, we have found three splice variants of SUR1 originating
from alternative splicing of the region encoding NBF1 and a short
splice variant containing only exons 31-39. The splice variants
SUR117 and SUR1
19 were expressed in all tissues tested, albeit to
a lesser amount than the SUR1 containing all exons. Only one of these
splice forms, SUR1
17, showed glibenclamide binding and functional
expression in combination with the pore-forming subunit Kir6.2. The
short splice variant was found to be strongly expressed in cardiac
muscle cells and displayed no high affinity for glibenclamide. Because
none of the four novel splice variants was targeted to the
mitochondria, they do not appear to participate in the formation of
mitochondrial KATP channels.
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ACKNOWLEDGEMENTS |
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We thank C. Löffler-Walz, Günter Schlichthörl, and Anette Hennighausen for excellent technical assistance.
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FOOTNOTES |
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This study was supported by the Deutsche Forschungsgemeinschaft (grants Da177/7-3 to J. Daut and Qu100/2-4 to A. Hambrock and U. Quast) and by the Kempkes-Stiftung (R. Preisig-Müller and C. Derst).
Address for reprint requests and other correspondence: J. Daut, Institute of Physiology, Marburg Univ., Deutschhausstrasse 2, 35037 Marburg, Germany (E-mail: daut{at}mailer.uni-marburg.de).
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.
April 18, 2002;10.1152/ajpcell.00083.2002
Received 9 April 2002; accepted in final form 11 April 2002.
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