Redox Regulation of ATP Sensitive Potassium Channels

New York University School Of Medicine, New York NY

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Abstract

DESCRIPTION (provided by applicant): The cardiovascular system circulates oxygen supply in the body, and is also one of the most vulnerable systems to oxidative damage. Oxidative stress has been shown to be involved in various cardiac disorders, including myocardial ischemia-reperfusion injury and heart failure. Thioredoxins are small proteins that act as antioxidants by reducing disulfide bonds via a thiol-disulfide exchange reaction. The major functions of thioredoxins are to maintain normal protein functions against oxidative modification. In our preliminary studies, using bioinformatic approaches, we identified novel putative protein-protein interaction domains, the coiled-coil (CC) domain, in the nucleotide binding fold (NBF) of SUR subunits of ATP-sensitive K+ (KATP) channel. Using the SUR1 CC domain as the bait in a two-hybrid screen against a rat cardiac cDNA library, we identified thioredoxin-2 as a putative KATP channel interacting protein. Interaction of thioredoxins with the SUR CC- domain was subsequently confirmed using GST pull-down assays. Moreover, co-immunoprecipitation assays demonstrated that thioredoxin-2 and the cytosolic isoform thioredoxin 1 also direct interact with full-length SUR1 or SUR2 subunit in a heterologous expression system. The presumed proximity of thioredoxins to SUR subunits suggests that thioredoxins are crucial to protect KATP channels against redox modification. Experiments proposed here will 1) use co-immunoprecipitation assays and molecular techniques to characterize interaction of thioredoxins and KATP channel subunits in the heterologous expression system and isolated myocytes 2) use patch-clamp technique at inside-out configuration and pharmacological studies to examine whether thioredoxin binding is functionally relevant to KATP channel activity and to the redox-induced channel modification. It is known that opening of KATP channels protect myocytes under various pathological conditions. This project will provide novel insights in the redox modification of KATP channel functions in cardiac myocytes under physiological conditions as well as the involvement of this modification in pathological states. Elucidation of the role of thioredoxins in regulating KATP channel functions during pathological conditions might provide valuable information to facilitate therapeutic development. PUBLIC HEALTH RELEVANCE: The KATP channel activity responds to alterations in the redox state that occurs in physiological and pathological conditions. Our preliminary study demonstrates that thioredoxins, members of reducing proteins, interact with KATP channels. In this study, we will 1) characterize the interaction between thioredoxins and KATP channels, and 2) explore the functional relevance of this interaction in redox regulation of KATP channel activity under physiological and pathological conditions.