Abstract 5210: Redox Regulation of ATP-sensitive Potassium Channels: A Possible Role for Thioredoxins

Abstract

Cardiac ATP-sensitive potassium (K ATP ) channels are gated by intracellular nucleotides, which link energy metabolism to membrane excitability and consequent contractility. However, there is an increasing realization that the K ATP channel is also under the control of other associated proteins belonging to channel megadalton complex. Using bioinformatic approaches we identified a possible coiled-coil (CC) domain in SUR subunits. To identify proteins that associate with SUR subunits, we used this CC-domain as the bait in a two-hybrid screen against a rat cardiac cDNA library and identified thioredoxin 2 (Trx2) as a putative candidate. We confirmed interaction between Trx2 and the SUR CC-domain using GST pull-down assays. In a heterologous expression system we further demonstrated using co-immunoprecipitation assays that both forms of thioredoxin (Trx1 and Trx2) interact with full-length SUR1 or SUR2. Immunoprecipitation of SUR1 from MIN6 cells was followed by mass spectrometry, which confirmed that Trx1 natively associates with SUR1 subunits. Given that thioredoxins act as antioxidants to reduce proteins by cysteine thiol-disulfide exchange, our data suggest that K ATP channel activity is likely regulated by redox state. To examine this hypothesis, we assessed the effects of oxidation with hydrogen peroxide (H 2 O 2 ; 10 μ M to 1.5 mM) on the activity of K ATP channels (Kir6.2/SUR1) expressed in CHO cells using patch clamp techniques. H 2 O 2 had no effect on K ATP channels in the absence of ATP. When channels were partially blocked by sub-maximal levels of ATP (10 μ M to 1 mM), H 2 O 2 dose-dependently increased channel activity. Interestingly, low H 2 O 2 concentrations (10 to 30 μ M) transiently activated K ATP channels, whereas higher concentrations (>300 μ M) caused a sustained activation. H 2 O 2 decreased the channel sensitivity to ATP (IC 50 8.9±0.26 vs. 21.7±0.15 uM; n = 9 –10) and had no effect on MgADP sensitivity. Our data demonstrate that oxidation (which occurs with ischemia/reperfusion) activates K ATP channels and suggest that thioredoxins, as components of the K ATP channel macromolecular complex, may regulate its response to cellular redox state.