Molecular identification and functional characterization of a mitochondrial sulfonylurea receptor 2 splice variant generated by intraexonic splicing.
BACKGROUND
Cardioprotective pathways may involve a mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel but its composition is not fully understood.


OBJECTIVE
We hypothesized that the mitoK(ATP) channel contains a sulfonylurea receptor (SUR)2 regulatory subunit and aimed to identify the molecular structure.


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 Ends) 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 mitoK(ATP) 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.
Department of Medicine, University of Wisconsin, Madison, WI 53706, USA.
Bin
B
Ye
Stacie L
SL
Kroboth
Jie-Lin
JL
Pu
Jason J
JJ
Sims
Nitin T
NT
Aggarwal
Elizabeth M
EM
McNally
Jonathan C
JC
Makielski
Nian-Qing
NQ
Shi
D018528:ATP-Binding Cassette Transporters / Q000235:genetics* / Q000378:metabolism*; D017398:Alternative Splicing / Q000502:physiology*; D000818:Animals; D002478:Cells, Cultured; D005091:Exons / Q000235:genetics; D005420:Flavoproteins / Q000378:metabolism; D015723:Gene Library; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000502:physiology; D017202:Myocardial Ischemia / Q000235:genetics* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D010084:Oxidation-Reduction; D024661:Potassium Channels, Inwardly Rectifying / Q000235:genetics* / Q000378:metabolism*; D011955:Receptors, Drug / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D064233:Sulfonylurea Receptors
C577510:ABCC9 protein, human; D018528:ATP-Binding Cassette Transporters; D005420:Flavoproteins; D024661:Potassium Channels, Inwardly Rectifying; D011955:Receptors, Drug; D011994:Recombinant Proteins; D064233:Sulfonylurea Receptors