Abstract 1294: Molecular Identification of the Sequence Encoding a 55-kDa SUR2 Protein in Mouse Heart Mitochondria
The cell surface cardiac ATP-sensitive potassium channel (cellKATP) is formed by a KIR6.x pore and a SUR2 (~174 kDa) regulatory subunit. SUR2 is a member of the ABC transporter super-family and it contains 3 transmembrane domains (TMD0 –2) and 2 nucleotide-binding folds (NBFs). The mitochondrial form of the KATP channel (mitoKATP) is thought to confer cardioprotection during ischemia but its molecular composition remains unknown. We designed and raised a panel of 10 antibodies, which cover the critical regions of the SUR2 protein, to investigate the nature of the mitochondrial SUR2 regulatory subunit. Previously, our C-terminal antibodies that are raised against the ends of SUR2 splice variants identified a 55-kDa short form SUR2 in purified mouse heart mitochondria. In this report, we attempted to address the molecular nature of the 55-kDa protein. We hypothesized that the 55-kDa SUR2 is a gene product resulted from post-transcriptional modification. Initial RACE and nested PCR experiments identified a 1.5-kb band from a RACE-ready mouse heart cDNA library. We were able to amplify the full-length 4.6-kb SUR2 gene from the same library indicating that the quality of the cDNA library was satisfactory. Cloning and sequencing of the 1.5-kb band revealed an alternatively spliced, in-frame “mini” SUR2 gene encoding a functional protein containing TMD0 and TMD2 only. RT-PCR in isolated mouse heart cDNA confirmed that the 1.5-kb gene product exists and it can encode a SUR2A or SUR2B short form in the size of 55-kDa when translated. Western blot analysis using the SUR2A or SUR2B specific antibody detected the presence of a 55-kDa SUR2A or SUR2B protein in purified mitochondria isolated from ventricular myocytes but this band is unlikely present in the plasma membrane-enriched fractions. The 55-kDa SUR2, which lacks TMD1 and NBF1, is considered insensitive to those KATP channel drugs acting on those regions but its SUR2B form is diaxozide-sensitive. With the presence of TMD0 and TMD2, the 55-kDa SUR2 should be interact with a proper KIR to form a channel complex. We proposed that the 55-kDa SUR2 is part of the mitoKATP complex in mouse heart.