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Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium channel (mitoKATP) but its composition is not fully understood.
We hypothesized that mitoKATP contains a SUR2 regulatory subunit and aimed to identify the molecular structure.
Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, two additional short forms (28- and 68-kDa) and a 130-kDa long form. Rapid Amplification of cDNA Ends (RACE) identified a 1.5-Kb transcript, which was generated by a non-conventional intra-exonic splicing (IES) event within the 4th and 29th exons of the SUR2 mRNA. The translated product matched the predicted size of the 55-kDa short form. In a knockout mouse (SUR2KO) where the SUR2 gene was disrupted, the 130-kDa mitoSUR2 was absent but the short forms remained expressed. Diazoxide failed to induce increased fluorescence of flavoprotein oxidation in SUR2KO cells, indicating that the diazoxide-sensitive mitoKATP activity was associated with 130-kDa-based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned WT, suggesting a protective role for the remaining short form-based channels. Heterologous co-expression of the SUR2 IES variant and Kir6.2 in a K+ transport mutant E. coli strain permitted improved cell growth under acidic pH conditions. The SUR2 IES variant was localized to mitochondria, and removal of a predicted mitochondrial targeting sequence allowed surface expression and detection of an ATP-sensitive current when co-expressed with Kir6.2.
We identify a novel SUR2 IES variant in cardiac mitochondria, and provide evidence that the variant-based channel can form an ATP-sensitive conductance and may contribute to cardioprotection.
Alternative splicing generates multiple mRNAs from a single gene, which are subsequently translated into diverse proteins with different structures and functions.1 Up to 60% of mammalian genes are alternatively spliced.2 Eukaryotic ion channel genes are known to have multiple splice variants. The ATP-sensitive potassium channels (KATP) are ubiquitously distributed in many tissue types. Sarcolemmal KATP (sarcKATP) channels consist of a potassium inward-rectifier pore-forming subunit (Kir6.0) and a sulfonylurea receptor (SUR) regulatory subunit.3 Various isoforms and splice variants for the SUR genes have been reported.4,5 The cardiac muscle splice variant (SUR2A) differs from the vascular smooth muscle splice variant (SUR2B) in the alternative use of the SUR2C-terminal exon.6,7 Subtypes of splice variants for SUR2A or SUR2B that lack exon 14 or exon 17 exist in mice7,8 and humans9. Moreover, sarcolemmal SUR short variants are found in heart10 and pancreatic β-cells.11,12 The co-presence of multiple splice variants increases the functional diversity and genetic complexity of KATP channels.
In addition to a sarcolemmal location,13 KATP is present in the inner membrane of mitochondria (mitoKATP).14 Both forms of channels are involved in cardioprotective pathways15 but earlier pharmacological evidence suggests that mitoKATP is more critical in conferring protection.16,17 However, the molecular composition for mitoKATP is uncertain, hampering current efforts in elucidating its role in preconditioning signaling.18 Putative mitoKATP subunits in the sizes of 55-kDa and 63-kDa have been enriched using an ATP-affinity chromatography method, followed by a KATP activity assay in reconstituted lipid bilayers.19,20 The sequences and nature of these proteins, however, remain unresolved.
In this report, we identified transcripts encoding mitochondrial-specific SUR2 (mitoSUR2) 55-kDa A and B forms, with alternative C-termini. Isolation of transcripts encoding the 55-kDa forms revealed a non-conventional intra-exonic splicing (IES) event, which occurred in the SUR2 mRNA to generate such variants. Further investigation suggested that modified channels formed by these IES variants generate a distinct ATP-sensitive current.
Expanded methods are included in an Online supplement.
The accession numbers for SUR2A-55 and SUR2B-55 are DQ991969 and DQ991970. RACE and exonic PCR were performed using a mouse or human heart FirstChoice RACE-Ready cDNA library (Amnion, Austin, TX). Human LV RNA was obtained from Biochain (Hayward, CA). Mouse heart RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA). RT-PCR reactions were carried out using a SuperScript RTIII kit (Invitrogen).
Mouse protocols were performed following the guidelines of NIH at the University of Wisconsin Animal Core Facility. Heterologous SUR2KO mice in the FVB background were interbred and genotyped to obtain homozygous mutants.21 Age-matched homozygous WT and KO littermates were used in this study.
SUR2-specific antibodies were generated as previously described.10 Anti-Na+/K+ ATPase, anti-VDAC1, and anti-COXIV were from Cell Signaling Technology (Danvers, MA). Secondary antibodies were from GE Healthcare (Piscataway, NJ).
Ventricular myocytes were isolated25 from 10-wks-old mice for fluorescence measurement of flavoprotein oxidation.26 Diazoxide, 5-hydroxydecanoate, dinitrophenol or pinacidil was added at 100 μM, 500 μM, 100 μM or 100 μM. ATP-sensitive potassium current (IKATP) was recorded as previously described.10
MG1655 was provided by the University of Wisconsin E. coli Sequencing Project.27 ME6106 (kdpABC5, trkA) was obtained from The Japanese National BioResource Project.28 Each SUR2 IES vairant or a full-length SUR2A cDNA was sub-cloned into pcDNA3 (Invitrogen) as pNQ55, pNQ64 and pSUR2A. A mouse Kir6.2 plasmid was obtained as previously described.10
Each SUR2 IES variant was sub-cloned into a bi-cistronic pGFPIRS vector (a kind gift from David Johns, Johns Hopkins University) as pNQ66 or pNQ67 for mitochondrial localization. A GFP that was fused to a known mitochondrial targeting sequence (MTS) in a eukaryotic expression vector (Clonetech, Valencia, CA) was used as a control. Each plasmid was transfected into COS1 cells and the transfected cells were stained with MitoTracker (Invitrogen) 24 h post transfection. The cells were then scanned with a confocal microscope (Bio-Rad, Hercules, CA) and analyzed subsequently. The putative MTS for the SUR2 IES variants was removed by mutagenesis. A MTS-less SUR2B-55 or the full-length SUR2A was subcloned into pGFPIRS as pNQ62 or pNQ69. Each plasmid was transfected in a Kir6.2 stable COS1 line.10 A vector control was also transfected. IKATP or ATP sensitivity (100 μM) was recorded as previously described.10
Data are reported as mean±SEM in the ischemia experiments and as mean±SD in the cell growth experiments. A student t test or two-way ANOVA was performed where appropriate with P<0.05 considered significant.
The presence of SUR2 variants in heart and brain mitochondria were assessed using SUR2-specific antibodies (Figure 1A). BNJ-2 detected a 130-kDa long form in mouse heart samples (Figure 1B). BNJ-39 (SUR2A-specific) detected three short forms in the sizes of 68-kDa, 55-kDa and 28-kDa while BNJ-40 (SUR2B-specific) detected a 55-kDa band. Based on terminal exon usage and protein size, the detected bands were designated as SUR2-130, SUR2A-68, SUR2A-55, SUR2A-28, and SUR2B-55. The mitoSUR2 short forms were also found in dog heart (Figure 1C). In the mouse brain samples, mitoSUR2 short forms were also found except for SUR2A-68 (Figure 1D).
DNA sequences encoding the mitoSUR2 short forms were sought in a mouse heart cDNA library using 5′ or 3′ RACEs, and the variants were amplified subsequently by nested exonic PCR (Figure 2A&2B). A 1.5-Kb band was obtained in addition to a 4.6-Kb SUR2 band (Figure 2C). Cloning and sequencing of the 1.5-Kb transcripts revealed that they resulted from a non-conventional intra-exonic splicing event (IES), which occurred within exons 4 and 29 of the SUR2 mRNA (Figure 2B). Each IES variant contained 13 exons encoding a 55-kDa protein. PCR with Primer P4, which spans the IES junction of exons 4 and 29 of SUR2, detected an expected 1.1-Kb band (Figure 2C), confirming the existence of an IES junction that was not present in SUR2 conventional splice variants. To ensure that IES did occur in isolated SUR2 mRNA, Primer P5, located just before the IES junction, was designed. RT-PCR with P5 using isolated WT mouse heart mRNA detected a 4.2-Kb band, suggesting that P5 annealed to SUR2 conventional variants as templates. A 1.1-Kb band was also obtained indicating that P5 could anneal to the SUR2 IES variants as templates. The results suggested that the IES variants co-existed with SUR2 conventional splice variants.
Because significant homologies between the human and mouse SUR2 conventional splice variants at the nucleic acid level have been reported,8,9 the same approach was used to amplify the 1.5-Kb IES variants from a human heart cDNA library (Figure 2D). PCR reactions using P4/h2 or P4/h3 detected an expected 1.1-Kb band. Moreover, an expected 1.1-Kb RT-PCR band using primers P4/h2 or P4/h3 was detected in mRNA isolated from human heart tissues. Since it was relatively easier to characterize functions of the IES variants using SUR2KO mice, we focused on the mouse variants in subsequent studies.
When cloning the transcripts encoding the SUR2 IES variants, we found both in-frame and “out-of-frame” products in WT mouse hearts. The “out-of-frame” product lacked a “G” at the IES junction region between exons 4 and 29 (Figure 3A). The extra “G” abolished the IES event, resulting in a 510-bp short transcript encoding a 20-kDa protein (Figure 3B). However, the G was present in the in-frame 1464-bp transcript, which encodes a 55-kDa protein. Our observations suggested that IES splicing could be controlled in cardiac cells.
Expression levels of both IES variants were compared in WT mice at 3-wks, 10-wks or 50-wks of age by a quantitative RT-PCR (Figure 3C). IES variant expression in the 3-wk-old group was significantly higher than that in the 10-wk-old or 50-wk-old group, suggesting that SUR2 IES variant expression decreased with aging.
SUR2KO mice were previously generated by inserting a disruption cassette between exons 12-16 of the SUR2 cDNA (Figure 4A).32 RT-PCR reactions were carried out using mRNA isolated from the SUR2KO hearts and detected presence of SUR2 IES variants (Figure 4B&4C). The 1.1-Kb bands encoding the 55-kDa forms were detected, cloned and confirmed by sequencing. SUR2-specific antibodies were used to cross-react with SUR2KO cardiac mitochondrial samples (Figure 4D). Neither T1 nor BNJ-2 detected any signal in the mutant, suggesting that the 130-kDa mitoSUR2 long form was disrupted. However, BNJ-39 and BNJ-40 still detected the mitoSUR2 short forms. The results indicated that mitoSUR2 short forms remained expressed in SUR2KO cardiac mitochondria. We reasoned that the transcripts encoding SUR2 IES variants were unaffected in the mutant because IES occurred within exons 4 and 29, bypassing the targeted disruption site.
Flavoprotein oxidation26 was used to characterize whether the diazoxide-induced mitoKATP activity was intact in SUR2KO cells (Figure 5A&5B). In the presence of diazoxide, fluorescence resulted from flavoprotein oxidation was significantly enhanced in WT cells. By contrast, diazoxide failed to elicit increased fluorescence in SUR2KO cells, suggesting that the diazoxide-sensitive mitoKATP activity was absent. To ensure that diazoxide acted at the level of mitoKATP but not sarcKATP, IKATP was recorded from the isolated cells in parallel (Figure 5C&5D). Diazoxide did not activate IKATP in WT or SUR2KO cells while pinacidil-induced IKATP was recorded from WT cells. Because the 130-kDa mitoSUR2 (SUR2-130) was disrupted in SUR2KO mice, these results are consistent with the presence of a mitoKATP and that the diazoxide-sensitive mitoKATP activity may be associated with SUR2-130.
Mitochondria and bacteria possess similar protein synthesizing machinery, and bacteria can not recognize a mitochondrial targeting sequence so they are frequently used to express mitochondrial proteins. Potassium transport is a fundamental requirement for maintaining pH homeostasis in bacteria and three potassium uptake systems exist in bacteria. An E. coli mutant ME6106 that lacks both kdpABC and trkA systems relies on the remaining kup system for growth.33 The kup system specifically supports cell growth under low pH conditions. When grown at pH5.5 in the presence of glucose, this mutant extrudes H+ in exchange for K+.34 We hypothesized that an introduced potassium transport machinery could improve cell growth in ME6106. It is known that acidic pH conditions activate KATP channels.35 Modulation of KATP activities by pH is mainly through Kir6.236 but recent evidence suggests that SUR2 is involved in regulating Kir6.2 under low pH conditions.37 We tested whether co-expression of each IES variant with Kir6.2 could enhance cell growth of ME6106 at pH5.5 (Figure 6A). ME6106 was transformed with Kir6.2 plus SUR2-55A, SUR2-55B, SUR2A or the empty vector. All positive transformants, the untransformed ME6106, and an untransformed WT E. coli K12 strain, MG1655, were tested for growth at pH5.5. MG1655 (blue) stopped growing as expected when pH in the medium dropped to 2.2. The untransformed ME6106 (orange) grew relatively slower than MG1655 at the beginning as reported but it continued to grow during the testing period. ME6106 containing Kir6.2/vector (green) showed a significantly 10% higher growth than the untransformed ME6106, suggesting that the expressed Kir6.2 could improve growth in ME6106. ME6106 containing Kir6.2/SUR2-55A (pink), Kir6.2/SUR2-55B (cyan) or Kir6.2/SUR2A (red) displayed a significantly 18%, 19% or 27% higher cell growth than ME6106. Our results supported a regulatory role for the SUR2 IES variants to Kir6.2 but the full-length SUR2A had a stronger effect relative to the SUR2 IES variants.
We further tested whether heterologously expressed SUR2 IES variants could be targeted to mitochondria. Each SUR2 IES variant-GFP fusion or a mitoGFP control was transiently transfected into COS1 cells and imaged by confocal scanning. Green fluorescence distribution of each expressed target, MitoTracker Red labeling and the overlay images were recorded (Figure 6B). Like the positive control mitoGFP, green signals in the SUR2 IES variant-GFP fusions overlapped with MitoTracker staining, suggesting that both variants were localized to mitochondria.
Protein import into mitochondria generally requires a N-terminal cleavable MTS that normally contains 20-30 amino acids but the length can be longer in some cases.38 Deleting MTS is expected to allow a mitochondrial protein to be expressed on cell surface for further functional study such as voltage clamping.39 However, some MTS motifs are non-cleavable, therefore, they remain intact as part of the mature proteins.38 Although consensus for each type of MTS has been reported, a functional test is ultimately required to either demonstrate mitochondrial expression by fusing the putative motif to a known non-mitochondrial protein or to show non-mitochondrial expression after removing the putative MTS. We hypothesized that a MTS is present in the N-terminus of SUR2 IES variants and removal of it could lead to non-mitochondrial expression. This putative MTS, which contains the first 30 amino acids of SUR2 IES variants, was deleted by mutagenesis. Each MTS-less SUR2 IES variant-GFP fusion was transiently transfected into COS1 cells for confocal imaging (Figure 7A). The detected green signal and MitoTracker staining did not overlap in the modified SUR2 IES variants, indicating that the mitochondrial expression was altered. We also observed some aggregated MTS-less SUR2 IES variant signal (i.e. the green panel for MTS-less SUR2A-55), suggesting that some proteins may not traffic properly to cell surface as previously reported.39
To confirm that cell surface expression was achieved in the MTS-less SUR2 IES variants, K+ currents were recorded from a modified SUR2B-55 IES variant-based channel by expressing the mutant variant into a Kir6.2 stable COS1 line. Currents were recorded from the recombinant channel while no currents were detected from the vector control in the absence of glibenclamide (Figure 7B). Unlike the glibenclamide-sensitive current detected from the SUR2A/Kir6.2 channel, the MTS-less variant-based channel had a smaller amplitude and displayed insensitivity to glibenclamide (Figure 7C). The current had kinetics and conductance typical of IKATP and was inhibited by 100 μM ATP (Figure 7D). Our data demonstrated that the recombinant channel conferred an ATP-sensitive IKATP activity.
We evaluated ischemic protection in SUR2KO mice, in which the mitoSUR2 short forms remained expressed. In the ischemia-reperfusion experiment, mice were subjected to 30-min ischemia and 24-h reperfusion. The average infarct sizes of preconditioned hearts in WT mice (27%) were significantly reduced relative to the ischemic hearts (45%), suggesting that our protocols were effective (Figure 8A). As observed previously,40 SUR2KO mice (24%) displayed significantly smaller infarcts than WT (45%) in ischemia, suggesting that they were less susceptible to ischemic/reperfusion injuries. IPC significantly limited infarcts in WT mice (27%) but it did not allow significant further improvement in SUR2KO mice (21%). The average infarct sizes recorded from the ischemic SUR2KO hearts were comparable to in the preconditioned WT hearts, suggesting that the SUR2KO mice were “constitutively” engaged in a protected state without the need of being preconditioned after losing the 130-kDa mitoSUR2.
A 55-kDa band was previously purified from inner mitochondrial membranes using ATP-affinity chromatography followed by a KATP-drug based activity assay in reconstituted lipid bilayers.41 When this band was separated on a 2-D gel, at least five distinct spots were detected. No protein sequence was reported from those candidates, suggesting that the approach may have flaws in identifying mitoKATP subunits. This approach, however, does not assume that the mitoKATP structure resembles sarcKATP, which may allow detection of other non-KATP proteins. A modified approach intends to use KATP subunit antibodies to immunoprecipitate purified cardiac mitochondrial fractions, and then separate the detected band by 2-D gels for mass spectrometry analysis.42 This is a significant improvement compared to the earlier approach if the target proteins are enriched enough to meet the detection and sensitivity requirements of the selected mass spectrometry methods. However, the specificity of a commercial Kir6.1 antibody compromised the detection, leading to identification of other proteins.42 In this report, we used a combined approach of RACEs, SUR2-specific antibodies and a SUR2KO mutant mouse to identify two 55-kDa proteins and other mitoSUR2 forms. It is possible that we resolved the nature of two 55-kDa species reported previously.41
Transcripts encoding the 55-kDa mitoSUR2 short forms were found to be products of a non-conventional IES event,43 which occurred within the 4th and the 29th exons of the SUR2 mRNA. Based on the sequence information, we noticed that the SUR2 IES variants are 100% identical to the SUR2 conventional splice variants at the protein level except the truncated transmembrane helix5-16. Therefore, attempts using SUR2-specific antibodies to immunoprecipitate a purified mitochondrial fraction for mass spectrometry might not identify these variants because the resultant hits could be considered as partial sequences of those SUR2 conventional splice variants. The co-presence of various types of SUR2 splice variants may have hindered the progress of other protein-based efforts in the past.
In contrast to conventional splicing that occurs between an exon and an intron, IES takes place within two exons, leading to shorter but in-frame variants.43 To date, about 10 cases of IES events have been reported in mammalian genes, mostly under disease conditions.44 This is the first report of finding an IES event in an ABC transporter and a mammalian ion channel. Due to the rarity of IES, mechanisms that generate these IES variants are not fully understood. It is thought that recognition of the IES junction is influenced by two major factors, assembly of a splicesome complex and quantitative scanning of the target sequence from a transcriptional/splicing machinery complex primed by upstream splicing signals. Previous results have shown that a typical 5′ IES donor site has a nAG/GTnnnn consensus while a 3′ IES acceptor site has a frequent CAG/G motif.45 We found nnG/GTnnnn as the 5′ donor site and nAG/G as the 3′ acceptor site for SUR2 IES variants, which matched the consensus motifs. In this work, we also found that IES splicing can be controlled and regulated in cardiac cells.
SURs belong to the ABC transporter super-family.46 Traditional ABC transporters have two transmembrane domains (TMD) and two symmetric nucleotide binding domains (NBD). SURs do not play transport functions and differ from other ABC transporters by possessing an additional TMD and two asymmetric NBDs. Hemi-ABC transporters have been described to use their single TMD to receive regulatory signals from the only NBD,47 and they are found in mitochondria.48 The identified SUR2 IES variants lack TMD1 and NBD1 but they have an intact NBD2 and a new hybrid TMD (Figure 8B). The SUR2 IES variant-based channels are expected to be relatively insensitive to most sulfonylureas based on the deduced topology. We provided evidence that the modified SUR2B-55 IES variant and Kir6.2 form an ATP-sensitive potassium channels (Figures 7B-D). The SUR2 IES variants can regulate Kir6.2 activity and support better growth under acidic pH conditions (Figure 6A). These results suggest that the SUR2 IES variants may function as hemi-ABC transporters. It is unclear how the SUR2A-68 and SUR2A-28 variants remain expressed in SUR2KO mice yet but we demonstrated that the 55-kDa variants were unaffected because of the IES event. IES may serve as a mechanism to retain certain level of mitoKATP activity in cardiac cells.
Results from the flavoprotein oxidation experiment suggest that SUR2KO mice lacked the diazoxide-sensitive mitoKATP activity found in WT. This finding differs from a previously reported Kir6.2 KO, where the diazoxide-sensitive mitoKATP activity is comparable to the WT controls.49 Our observation provides evidence that the diazoxide-sensitive florescence could be associated with a distinct SUR2-130-based mitoKATP channel. IPC study showed that the recorded infarcts in SUR2KO mice, where the mitoSUR2 short forms remained expressed, is similar to preconditioned WT, suggesting that the mutant mice were protected. The SUR2-130 based channels may not be required for conferring protection itself as the mutant mice lacking the long form are “constitutively” protected in ischemia. Future pharmacological study in the mitoKATP conductance formed by both the long and short mitoSUR2 forms will be evaluated to interpret their roles in preconditioning.
In summary, we have characterized the sequence and function of SUR2 IES variants in cardiac mitochondria, and provided evidence that it forms a KATP channel when the MTS is removed. Future studies in the pharmacology and pathophysiology of channels constituted by these forms may provide new insights into the cardioprotective pathway.
SOURCES OF FUNDING
This study was supported by The American Heart Association National Center (0630268N to N.Q.S) and (0435030N to B.Y), and by NIH (R01HL-57414 to J.C.M. and R01 HL078926 to E.M.M).