Reduced activity of inward rectifier potassium channels contributes to an increased risk of arrhythmia in heart failure (Pogwizd et al., 2001). Understanding the regulation of these channels may therefore provide a basis for the development of novel antiarrhythmic agents. Mitochondria are increasingly recognized as a source of important signaling molecules, and we recently reported evidence for an inhibitor of inward rectifier potassium channels that is generated by the action of the mitochondrial uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (Collins et al., 2005). To gain further support for the concept of an endogenous inhibitor, we prepared extracts from homogenized Xenopus oocytes and tested them for inhibitory activity. Oocytes were homogenized in 4-6.5 ml/g of (mM) 40 KCl, 46 K-gluconate, 5 KF, 0.1 NaVO3, 10 K-pyrophosphate, 1 EGTA, 10 EDTA, 0.1 spermine, 20 HEPES, pH 7.4 with KOH (150 mM final [K+]) (FVPPE) with a Polytron-type or Dounce homogenizer. Homogenates were centrifuged twice at 1000xg, 4 degrees C and the supernatant was retained each time. The supernatant was centrifuged again at 16,000xg, 4 degrees C for 20 minutes, and this supernatant was further centrifuged at 200,000xg, 4 degrees C for 1 hour to pellet the membrane fraction. Alternatively, oocytes were fractionated directly by centrifugation at 16,800xg, 4 degrees C, and the cytoplasmic fraction was diluted into 0.8 ml/g (original oocyte weight) FVPPE. This sample was centrifuged at 200,000xg, 4 degrees C for 1 hour, and the supernatant was further diluted by 8-fold into FVPPE. Complementary RNA for the inward rectifier potassium channel Kir2.2 was transcribed in vitro and injected into isolated Xenopus oocytes. Two to five days later, inward rectifier currents were recorded in giant membrane patches (Hilgemann, 1995). The pipettes and recording chamber contained FVPPE. Inside-out patches were perfused with FVPPE and with oocyte extracts. Kir2.2 current was inhibited by the 1000xg (3 patches), 16,000xg (4 patches) and 200,000xg (8 patches) supernatants of the Polytron-type or Dounce homogenates, but not by the supernatant from the centrifugation-fractionated oocytes (3 patches). The 200,000xg homogenate supernatant was filtered through Centricon columns with molecular weight cutoffs of 100, 30 and 10kD and the filtrates were tested for their ability to inhibit Kir2.2 current in inside-out patches. All three filtrates inhibited the current. Based on these data, we propose that Xenopus oocytes have an endogenous inhibitor of inward rectifier potassium channels that is released from an intracellular compartment by homogenization but not by centrifugation-fractionation, and has a molecular weight of less than 10kD.