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 (). 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 (). The SUR2 IES variants can regulate Kir6.2 activity and support better growth under acidic pH conditions (). 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.