Molecular Identification and Functional Characterization of a Mitochondrial Sulfonylurea Receptor 2 Splice Variant Generated by Intraexonic Splicing

Abstract

Rationale: 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. 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 Ends) identified a 1.5-Kb transcript, which was generated by a nonconventional intraexonic splicing ( Key Words: K ATP channel Ⅲ SUR2 Ⅲ ischemia Ⅲ intraexonic splicing Ⅲ mitochondria A lternative 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 (K ATP ) channels are ubiquitously distributed in many tissue types. Sarcolemmal K ATP (sarcK ATP ) 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 SUR2 C-terminal exon. 6,7 Subtypes of splice variants for SUR2A or SUR2B that lack exon 14 or exon 17 exist in mice 7,8 and humans. 9 Moreover, sarcolemmal SUR short variants are found in heart 10 and pancreatic ␤ cells. 11,12 The copresence of multiple splice variants increases the functional diversity and genetic complexity of K ATP channels.In addition to a sarcolemmal location, 13 the K ATP channel is present in the inner membrane of mitochondria (mitoK ATP ). 14 Both forms of channels are involved in cardioprotective pathways, 15 but earlier pharmacological evidence suggests that the mitoK ATP channel is more critical in conferring protection. 16,17 However, the molecular composition of the mitoK ATP channel is uncertain, hampering present efforts in elucidating its role in preconditioning signaling. 18 Putative mitoK ATP channel subunits in the sizes of 55 and 63

Kir6.2 is not the mitochondrial KATP channel but is required for cardioprotection by ischemic preconditioning

“…Such a pattern is common in the cardioprotection field; for example, ablation of STAT3 abolishes protection by IPC while having no impact on baseline I/R injury (29,40). It is also notable that genetic ablation of SUR2 increases resistance to I/R injury (30), but this may be due to expression of short-form SUR2 splice variants in the SUR2 Ϫ/Ϫ knockout mouse, which are thought to independently mediate cardioprotection (25,41).…”

KATPChannels in the Cardiovascular System

“…Indeed, low-molecular-weight SUR2 isoforms have been identified that may be components of the mitochondrial K ATP channel. When heterologously expressed, a 55 kDa SUR2 splice variant colocalizes with mitoTracker dyes in COS1 cells (901). However, when coexpressed with Kir6.x, patch-clamp methods demonstrate the presence of plasmalemmal K ATP channels that are blocked by millimolar ATP levels but are practically insensitive to pinacidil, diazoxide, or glibenclamide (3).…”

Vasodilation induced by oxygen/glucose deprivation is attenuated in cerebral arteries of SUR2 null mice

“…Murine cardiac myocytes express both full-length (130 kDa) and short (28,55, and 68 kDa) SUR2 forms (32,45). Fulllength SUR2 is absent, whereas all three short forms are present, in SUR2 nl mouse cardiac myocytes (32).…”