In the assay, 0.3 mg BTC-AM loaded mitochondria were added to a rapidly stirred cuvet containing 2 ml of chloride-free Tl+assay buffer at 37C. The antidepressant fluoxetine (Prozac) inhibited mKATP(IC502.4 mol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mKATPindependent stimulus. The related antidepressant zimelidine was without effect on either mKATPor IPC. == Conclusions == The Tl+flux mKATPassay was validated by correlation with a classical mKATPchannel osmotic swelling assay (R20.855). The pharmacologic profile of mKATP(response to ATP, UDP, PIP2, and fluoxetine) is consistent with that of an inward rectifying K+channel (KIR) and is somewhat closer to that of the KIR6.2 than the KIR6.1 isoform. The effect of fluoxetine on mKATP-dependent cardioprotection has implications for the growing use of antidepressants in patients who may benefit from preconditioning. Keywords:mitochondrial ATP sensitive potassium channel, ischemia, reperfusion, ischemic preconditioning, fluoxetine == INTRODUCTION == The mitochondrial ATP DDR1-IN-1 sensitive potassium channel (mKATP) is thought to be essential for cardioprotection recruited by ischemic preconditioning (IPC),1,2but despite intense research the DDR1-IN-1 molecular identity of this channel remains unclear. The simplest thesis is that mKATPchannels are derivative of surface KATPchannels, and thus composed of inward rectifying K+channels (KIR) and sulfonylurea receptors (SUR). The cardiomyocyte IFN-alphaA surface KATPchannel is comprised of KIR6.2 and SUR2A isoforms,3but efforts to conclusively assign these proteins to the cardiac mKATPhave been unsuccessful to date. Neither Kir6 nor SUR genes contain mitochondrial target sequences, and Kir6/SUR proteins are not found in mitochondrial proteome databases or prediction engines.4,5Furthermore, immune-based methods to detect KIR/SUR subunits in mitochondria are plagued by issues of antibody specificity6and mitochondrial purity/contamination. Several of the key pharmacologic reagents used to study mKATPchannels (e.g. the agonist diazoxide (DZX) and antagonist 5-hydroxydecanoate (5-HD)) are also known to exhibit off-target effects.7,8Targeted gene deletion in mice to identify the mKATPchannel involved in IPC has proven futile, due to the confounding cardiovascular effects of knocking out KIR6 and SUR genes (Kcnj8,Kcnj11,Abcc8, andAbcc9) on surface KATPchannel function. In general, KIRand SUR knockouts exhibit profound defects in glucose/insulin handling,912which impacts the response to IPC.13 A recent study using custom-made antibodies and SUR knockout mice identified shortform splice variants of SUR2 in mitochondria.14Furthermore, recent pharmacological evidence suggests that complex II of the respiratory chain (succinate dehydrogenase) may be a regulatory component of the mKATPchannel.15,16However, both these findings leave the identity of the K+channel-forming subunit of mKATPunknown. In this regard, mKATPis similar to other mitochondrial ion channels which exist at a phenomenological level but have not been molecularly identified (e.g., the mitochondrial Ca2+uniporter). A major obstacle in studying the mKATPchannel has been the availability of a reliable assay. Most studies to date have utilized an DDR1-IN-1 isolated mitochondrial rapid swelling assay, in which K+uptake into mitochondria is followed by osmotically-obliged water, leading to mild DDR1-IN-1 swelling that is assayed as light scattering in a spectrophotometer.17,18This assay has been criticized as irreproducible by some laboratories,19with the precise timing of mitochondrial isolation appearing to be a critical factor.20 Studying the literature on surface KATPchannels, two key biochemical properties that appeared to have been overlooked in the mKATPchannel field were the permeability of surface KATPchannels for the heavy metal thallium (Tl+),21and the modulation of channel run-down by phospholipids such as phosphatidylinositol-4,5-bisphosphate (PIP2).22,23Herein, we developed a novel Tl+fluorescence based assay for mKATPchannel activity, and used this assay to show that the channel is subject to run-down that is reversed by PIP2. It is anticipated that both these discoveries will advance the study of this channel. Furthermore, the antidepressant fluoxetine (FLX), which is known to modulate KIRchannels,24,25was found herein to block the mKATPchannel and to block IPC, but FLX did not block mKATP-independent cardioprotection. The implications of these data for clinical use of FLX in cardiovascular disease patients are discussed. == METHODS == Full experimental details are in theonline supplement. Cardiac mitochondria were rapidly isolated from male Sprague-Dawley rat hearts by differential centrifugation in sucrose-based buffer as previously described.20Protein was determined by the Folin-phenol method.26Within 1.5 hr of mitochondrial isolation the activity of mKATPwas monitored by the osmotic swelling assay as previously described.20 A novel fluorescence-based Tl+flux assay for mKATPactivity was also developed. The ionic radii of Tl+(0.154 nm) and K+(0.144 nm) are similar,27and thus Tl+is widely used as an analog to study membrane K+transport.21,2730The assay made use of the fluorescent indicator BTC-AM, which is better.