Published online before print October 1, 2009, doi: 10.1161/CIRCRESAHA.109.195040
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Submitted on January 31, 2009
Revised on August 28, 2009
Accepted on September 23, 2009
Molecular Identification and Functional Characterization of a Mitochondrial Sulfonylurea Receptor 2 Splice Variant Generated by Intraexonic Splicing
Bin Ye ;
From the Department of Medicine (B.Y., S.K., J.-L.P., N.A., J.M., N.-Q.S.) and School of Pharmacy (J.S.), University of Wisconsin, Madison; and Department of Medicine (E.M.), University of Chicago, Ill.
* To whom correspondence should be addressed. E-mail: nqs{at}medicine.wisc.edu.
Rationale: Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoKATP) channel but its composition is not fully understood.
Objective: We hypothesized that the mitoKATP channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure.
Methods and Results: Western blot analysis in cardiac mitochondria detected a 55-kDa mitochondrial SUR2 (mitoSUR2) short form, 2 additional short forms (28 and 68 kDa), and a 130-kDa long form. RACE (rapid amplification of cDNA end products) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic 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), in which 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 channel activity was associated with 130-kDa–based channels. However, SUR2KO mice displayed similar infarct sizes to preconditioned wild type, suggesting a protective role for the remaining short form-based channels. Heterologous coexpression of the SUR2 IES variant and Kir6.2 in a K+ transport mutant Escherichia 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 coexpressed with Kir6.2.
Conclusions: 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.
Key words: KATP channel • SUR2 • ischemia • intraexonic splicing • mitochondria