Four novel splice variants of sulfonylurea receptor 1

Annette Hambrock2, Regina Preisig-Müller1, Ulrich Russ2, Anke Piehl2, Peter J. Hanley1, John Ray1, Jürgen Daut1, Ulrich Quast2, and Christian Derst1

1 Institute of Physiology, Marburg University, 35037 Marburg; 2 Institute of Pharmacology, Tübingen University, 72074 Tübingen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (SUR1Delta 17), 19 (SUR1Delta 19), or both (SUR1Delta 17Delta 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 SUR1Delta 17 showed high-affinity binding of glibenclamide (Kdapprox 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta  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 (SUR2Delta 17) 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 (SUR1Delta 17) and/or exon 19 (SUR1Delta 17Delta 19 or SUR1Delta 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 SUR1Delta 17 exhibit specific binding of glibenclamide and give rise to ATP-sensitive channels when coexpressed with Kir6.2 subunits.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 lambda 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 SUR1Delta 17 and SUR1Delta 19 splice forms were identified by DNA sequencing. Finally, the Delta 17 and Delta 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 the beta -subunit of F1-ATPase from Neurospora crassa was ligated into the pEGFP-N1 vector) and the endoplasmic reticulum (pEYFP-ER, Clontech). HEK-293 or COS-7 cells were transiently transfected by using Lipofectamine 2000 as transfection reagent and incubated for 24 h at 37°C.

Confocal 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 MOmega . 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.

Experiments using the whole cell configuration were performed as described in Ref. 37. The bath solution contained (in mM) 142 NaCl, 2.8 KCl, 1 MgCl2, 1 CaCl2, 11 D(+)-glucose, and 10 HEPES, titrated to pH 7.4 with NaOH at 37°C. Patch pipettes were filled with (in mM) 132 K-glutamate, 10 NaCl, 2 MgCl2, 10 HEPES, 1 EGTA, 1 Li2GDP, and 0.3 Na2ATP, titrated to pH 7.2 with NaOH. Resistance after filling was 3-5 MOmega . Cells were clamped at -60 mV. Data were recorded with an EPC9 amplifier (HEKA, Lambrecht, Germany) using Pulse software. Signals were filtered at 200 Hz with the four-pole Bessel filter of the EPC9 amplifier and sampled at 1 kHz.

Data analysis and statistics. Inhibition curves from radioligand binding and patch-clamp experiments were analyzed according to the logistic equation
y = 100 − A(1 + 10<SUP><IT>n</IT><SUB>H∗</SUB>(p<IT>x</IT>−pIC<SUB>50</SUB>)</SUP>)<SUP>−1</SUP> (1)
where A denotes the relative extent of inhibition at saturation (A = 100 for complete inhibition of the current by ATP), nH is the Hill coefficient, and pIC50 is the midpoint of the curve with pIC50 = -log IC50; x is the concentration of the compound under study with px = -log x. In binding experiments, two-component analysis with nH = 1 was sometimes required. The dependence of the IC50 value on the concentration of the radioligand, L, was calculated according to the equation (11)
IC<SUB>50</SUB> = <IT>K</IT><SUB>i</SUB>(1 + L/<IT>K</IT><SUB>d</SUB>) (2)
where Ki is the inhibition constant and Kd is the equilibrium dissociation constant of the radioligand.

Fits of the equations to the experimental data were performed according to the method of least squares using the programs FigP (Biosoft, Cambridge, UK) or SigmaPlot (SPSS, Chicago, IL). Assuming that amplitudes and pK values are normally distributed, significance of difference between the parameters was calculated by the two-tailed unpaired Student's t-test or by one-way analysis of variance by using the program GraphPad Prism, version 3.0 (GraphPad Software, San Diego, CA). In the text, the IC50 or K values are followed by the 95% confidence interval in parentheses.

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%).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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, approx 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 gpSUR1Delta 17 (286 bp) as a component of the approx 290-bp PCR product; gpSUR2Delta 17 (252 bp); gpSUR1Delta 19 (224 bp); and gpSUR1Delta 17Delta 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 gpSUR1Delta 17, indicating that the band contained a mixture of two PCR products (291 and 286 bp).


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Fig. 1.   Sequence analysis of the guinea pig sulfonylurea receptor (SUR) sequences around the first nucleotide binding fold (NBF). A: cDNA and amino acid sequence of the amplified PCR products. Primer sequences are underlined; exon 17 of gpSUR1 and gpSUR2 (upper case) and exon 19 of gpSUR1 (lower case) are highlighted. The amino acids of the Walker "A" consensus sequence (GQVGCGKS) are also underlined. B: sequence alignment of exons 16-19 of SUR sequences from different species. gp, Guinea pig; r, rat; m, mouse; h, human; ha, hamster.



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Fig. 2.   Expression pattern of gpSUR splice forms. A: RT-PCR analysis of the first NBF. PCR products of 323, 290, 252, and 224 bp were obtained. Direct sequencing revealed the SUR splice form indicated. Note that the second band is a mixture of two SUR splice forms (gpSUR2 and gpSUR1Delta 17). Asterisk indicates PCR products that were cloned and sequenced. Sequencing revealed that the bands indicated were mixtures of gpSUR2 (>90%) and gpSUR1Delta 17 (<10%). B: RT-PCR analysis of isolated heart cell fractions. Note that (unlike in A) a 191-bp band corresponding to gpSUR1Delta 17Delta 19 was present. C: RT-PCR analysis of the expression of gpSUR1C in different guinea pig tissues. D: cellular expression of gpSUR1C by RT-PCR in capillaries (CAP) and cardiomyocytes (CM). As a control, a 315-bp fragment of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified (data not shown). skel. m., Skeletal muscle.

Figure 1A shows an alignment of the partial cDNA and amino acid sequences of gpSUR1 (GenBank accession no. AY050167) and gpSUR2 (GenBank accession no. AY050168). Sequence comparison showed that the amplified gpSUR1 sequence is highly homologous (>90% identity on the amino acid level) to its human, rat, or hamster orthologs (Fig. 1B). The short exon 17 is less conserved (approx 60% identity), with two additional amino acids in the guinea pig sequence compared with human. The gpSUR2 sequence showed an even higher homology to orthologous sequences (>97% identity).

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 gpSUR1Delta 17Delta 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. gpSUR1Delta 19 was detected at low levels in brain, lung, heart, and pancreas and at even lower amounts in other tissues. The detection of gpSUR1Delta 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 gpSUR1Delta 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. gpSUR2Delta 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 SUR2Delta 18 would correspond to SUR1Delta 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). gpSUR2Delta 17 was also expressed, especially in endothelial cells. In accordance with the analysis of whole heart fraction, low amounts of gpSUR1, gpSUR1Delta 19, and gpSUR1Delta 17 were found in cardiomyocytes. The expression of the splice form lacking both exons, gpSUR1Delta 17Delta 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 lambda 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.

To exclude the possibility that the gpSUR1C clone is an artifact of cDNA synthesis, we confirmed the expression of this short gpSUR1 transcript with 5' RACE experiments. Figure 2C shows the RT-PCR analysis of several guinea pig tissues. gpSUR1C was found to be highly expressed in the atria and ventricles of guinea pig heart. At a lower expression level, gpSUR1C was detected in brain, lung, liver, kidney, and skeletal muscle. Cell-specific expression of gpSUR1C in the heart was studied by using the multicell RT-PCR method described previously (34). gpSUR1C was found to be expressed exclusively in ventricular cardiomyocytes and not in endothelial cells from coronary capillaries, as illustrated in Fig. 2D.

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 rSUR1Delta 17 and rSUR1Delta 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, rSUR1Delta 19, rSUR1Delta 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, rSUR1Delta 19, and rSUR1Delta 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 rSUR1Delta 17 (A-C) and coexpression of Kir6.1-EGFP with rSUR1Delta 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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Fig. 3.   Confocal images of COS-7 cells transfected with localization markers MitoGFP or endoplasmic reticulum (ER)-YFP. MitoGFP was targeted to the mitochondria (MitoGFP) (B), and mitochondria were labeled with the red fluorescent dye MitoTracker red (MitoTRed) (A). The overlay of images (C) reveals identical localization of MitoGFP and MitoTracker red to the mitochondria of MitoGFP transfected cells (yellow). Some cells are not transfected with MitoGFP and therefore show only MitoTracker red labeling. ER-YFP targeted to the lumen of the ER was used to determine the morphology of ER in HEK-293 cells (D) and COS-7 cells (E). Scale bar, 10 µm.



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Fig. 4.   Confocal images of COS-7 cells transfected with rSUR1Delta 17-EGFP (A), rSUR1Delta 19-EGFP (B), or rSUR1-EGFP (C). Mitochondria were labeled with MitoTracker red. In A-C green fluorescence is predominantly localized in the ER. MitoTracker red fluorescence does not overlap. Inset in C shows the same cell without MitoTracker red fluorescence. Note the ER-like distribution of green fluorescence. Scale bar, 10 µm.



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Fig. 5.   Confocal images of COS-7 cells. Cells cotransfected with Kir6.1-EGFP and rSUR1Delta 17 (A and B) or rSUR1Delta 19 (D and E) showed no localization of EGFP-labeled Kir6.1 to the mitochondria (C and F). EGFP-labeled Kir6.1 was retained in the ER. Scale bar, 10 µm.

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 rSUR1Delta 19, rSUR1Delta 17Delta 19, and gpSUR1C. There was no difference in total binding between the different preparations (at 5 nM [3H]GBC ~150 fmol/mg protein). Addition of 100 nM unlabeled GBC reduced total [3H]GBC binding by ~5%; reduction after addition of 10 µM GBC was 25-30%. Therefore, the binding measured for these splice forms was due to unspecific endogenous GBC-binding to the HEK-293 cells (20, 37). On the other hand, specific binding of [3H]GBC to rSUR1 and rSUR1Delta 17 was detected (radioligand concentration 1 nM). In the absence of MgATP, total binding was 1.3-1.4 pmol/mg protein, and addition of 1 µM unlabeled GBC reduced binding by 1.1-1.3 pmol/mg protein for rSUR1 and rSUR1Delta 17. Because only rSUR1 and rSUR1Delta 17 exhibited high-affinity [3H]GBC binding, the pharmacological properties of these splice forms were compared in binding assays and electrophysiological experiments.

Pharmacological properties of SUR1 and SUR1Delta 17. 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 rSUR1Delta 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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Fig. 6.   Inhibition of [3H]glibenclamide (GBC) binding by GBC (A) and MgATP (B). A: experiments performed with HEK-293 cell membranes containing rSUR1 (n = 4) and rSUR1Delta 17 (n = 6) in the absence of MgATP (0 mM Mg2+, 1 mM EDTA) (open symbols) and in the presence of 1 mM MgATP (2.2 mM Mg2+; 1 mM ATP) (closed symbols) at a [3H]GBC concentration of ~1 nM. The IC50 values were obtained from the fit of Eq. 1 (see MATERIALS AND METHODS). The Ki or Kd values were then calculated from Eq. 2 and are listed in Table 1. Hill coefficients were 1.02 ± 0.31 and 1.10 ± 0.04 in the absence and 0.94 ± 0.04 and 1.0 ± 0.04 in the presence of MgATP. B: effect of MgATP on [3H]GBC binding to HEK-293 cell membranes containing rSUR1 and rSUR1Delta 17 was determined. The data, expressed as percentages of specific binding (Bs), are means from 3-4 experiments. Concentration of [3H]GBC was ~1 nM; 100% Bs corresponded to 1.6 pmol/mg protein (rSUR1) and 1.1 pmol/mg protein (rSUR1Delta 17). The fit of Eq. 1 (see MATERIALS AND METHODS) gave the following parameters (rSUR1/rSUR1Delta 17): amplitudes (A1, %Bs): 34 ± 4%/28 ± 3% and (A2, %Bs): 38 ± 2/47 ± 4%; pIC50/1: 5.41 ± 0.14/5.62 ± 0.06 and pIC50/2: 3.65 ± 0.02/3.79 ± 0.07.


                              
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Table 1.   Properties of [3H]glibenclamide binding to rSUR1 and rSUR1Delta 17

Inhibition of specific [3H]GBC binding by another KATP channel blocker, the benzoic acid derivative meglitinide, which binds to a subpocket of the glibenclamide binding site (4), was also investigated. Similar to the homologous competition by glibenclamide, displacement of specific [3H]GBC binding by meglitinide (Fig. 7A) occurred in a monophasic manner for both splice forms (Hill coefficients very close to 1 in all cases). Again, curves obtained in the absence of MgATP were shifted to the left compared with the curves obtained in the presence of 1 mM MgATP. Results obtained in the absence and in the presence of MgATP were significantly different for both rSUR1 (P < 0.001) and rSUR1Delta 17 (P < 0.0001). On the other hand, there was no significant difference in the parameters obtained for rSUR1 and rSUR1Delta 17 (Table 1).


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Fig. 7.   Inhibition of [3H]GBC binding by meglitinide (A) or by l-pinacidil or diazoxide (B) in HEK-293 membranes containing rSUR1 and rSUR1Delta 17. A: inhibition of [3H]GBC binding by meglitinide in the absence and presence of 1 mM MgATP (n = 3-4). [3H]GBC concentration was 1 nM. Hill coefficients (nH) were 0.90 ± 0.07 and 0.95 ± 0.03 (without MgATP) and 1.00 ± 0.11 and 0.90 ± 0.1 (with MgATP). B: inhibition of [3H]GBC binding by l-pinacidil or diazoxide in HEK-293 membranes containing rSUR1 and rSUR1Delta 17 (n = 2-7). [3H]GBC concentration was 1 nM. Hill coefficients were set to 1 for l-pinacidil and were 1.35 ± 0.18 (rSUR1) and 1.02 ± 0.07 (rSUR1Delta 17) for diazoxide.

To test the binding properties of selected openers, inhibition of [3H]GBC binding by unlabeled l-pinacidil and diazoxide was analyzed at 0 mM Mg2+ and 1 mM EDTA and at 2.2 mM Mg2+ and 1 mM ATP. Displacement of [3H]GBC by both openers was found only in the presence of MgATP (Fig. 7B), as has been shown previously (33, 41). The curves obtained for both openers were very similar for rSUR1 and rSUR1Delta 17.

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/rSUR1Delta 19.

Chutkow et al. (13) described differences in the potency of ATP block between channels containing SUR2 or SUR2Delta 17. Therefore, the ATP sensitivities of Kir6.2/rSUR1 and Kir6.2/rSUR1Delta 17 channels were compared in the inside-out patch-clamp configuration (Fig. 8). As shown in Fig. 8A, rundown and refreshment of the channel in the presence of 0.7 mM free Mg2+ led to some minor differences in ATP-block efficiency during repetitive application and washout of ATP. Therefore, the mean value of two successive runs was determined for each cell, and cells transfected with rSUR1 and with rSUR1Delta 17 were subjected to exactly the same protocol. Figure 8B shows that there was no difference in ATP sensitivity between the two splice forms at low (270 nM) and physiological (0.7 mM) free Mg2+ concentrations.


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Fig. 8.   Concentration-dependent ATP block of ATP-sensitive potassium (KKATP) current (I) from channels containing rSUR1 or rSUR1Delta 17. A: sample trace from an inside-out patch with Kir6.2/rSUR1Delta 17 channels. The current was inhibited by MgATP concentrations of 3, 10, 30, 100, 300 and 1,000 µM, indicated by the thickness of the horizontal bar. B: concentration dependence of the inhibition of KATP channels composed of Kir6.2 and rSUR1 (solid line) or rSUR1Delta 17 (dashed line) by ATP. The currents were recorded in the presence of low (270 nM) (open symbols) or standard (0.7 mM) (closed symbols) concentrations of Mg2+. Fit of Eq. 1 to the data gave the following values for IC50 (95% confidence interval in brackets) and nH: at 270 nM Mg<UP><SUB>free</SUB><SUP>2+</SUP></UP>, 6.0 (5.6, 6.3) µM [nH = 1.19 ± 0.04] for rSUR1 and 6.7 (6.1, 7.3) µM [nH = 1.30 ± 0.07] for rSUR1Delta 17; at 0.7 mM Mg<UP><SUB>free</SUB><SUP>2+</SUP></UP>, 14.0 (13, 15) µM [nH = 1.09 ± 0.05] for rSUR1 and 12.0 (11, 13) µM [nH = 0.96 ± 0.05] for rSUR1Delta 17.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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: SUR1Delta 17, in which 36 bp are spliced out; SUR1Delta 19, in which 99 bp are spliced out; and SURDelta 17Delta 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, rSUR1Delta 17 and rSUR1Delta 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 rSUR1Delta 19, rSUR1Delta 17Delta 19, or gpSUR1C. On the other hand, specific binding of [3H]GBC was detected with rSUR1 and rSUR1Delta 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/rSUR1Delta 17 in either the presence or absence of free Mg2+. For the Delta 17 splice variants of SUR2A and SUR2B, however, Chutkow et al. (13) measured a reduced ATP sensitivity for channels containing Kir6.2 and SUR2xDelta 17. Therefore, SUR1/SUR1Delta 17 clearly differ from SUR2/SUR2Delta 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 rSUR1Delta 19 and rSUR1Delta 17Delta 19 and no KATP currents were observed upon coexpression of Kir6.2 with rSUR1Delta 17Delta 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 SUR1Delta 17 and SUR1Delta 19 were expressed in all tissues tested, albeit to a lesser amount than the SUR1 containing all exons. Only one of these splice forms, SUR1Delta 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.


    ACKNOWLEDGEMENTS

We thank C. Löffler-Walz, Günter Schlichthörl, and Anette Hennighausen for excellent technical assistance.


    FOOTNOTES

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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DISCUSSION
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