Characterization of human cardiac mitochondrial ATP-sensitive potassium channel and its regulation by phorbol ester in vitro

Departments of 1 Anesthesiology, 2 Physiology, 3 Surgery, and 4 Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin Submitted 13 October 2005 ; accepted in final form 15 December 2005 Activation of the mitochondrial ATP-sensitive K + channel (mitoK ATP ) and its regulation by PKC are critical events in preconditioning induced by ischemia or pharmaceutical agents in animals and humans. The properties of the human cardiac mitoK ATP channel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoK ATP channel located in the inner mitochondrial membrane (IMM) due to the physical barrier of the outer mitochondrial membrane. In the present study, we characterized the human cardiac mitoK ATP channel and its potential regulation by PKC associated with the IMM. IMM fractions isolated from human left ventricles were fused into lipid bilayers in symmetrical potassium glutamate (150 mM). The conductance of native mitoK ATP channels was usually below 80 pS ( 70%), which was reduced by ATP and 5-hydroxydecanoic acid (5-HD) in a dose- and time-dependent manner. The native mitoK ATP channel is activated by diazoxide and inhibited by ATP and 5-HD. The PKC activator phorbol 12-myristate 13-acetate (2 µM) increased the cumulative open probability of the mitoK ATP channel previously inhibited by ATP ( P < 0.05), but its inactive analog 4 -phorbol 12,13-didecanoate had no effect. Western blot analysis detected an inward rectifying K + channel (Kir6.2) immunoreactive protein at 56 kDa and PKC- in the IMM. These data provide the first characterization of the human cardiac mitoK ATP channel and its regulation by PKC(s) in IMM. This local PKC control mechanism may represent an alternative pathway to that proposed previously for cytosolic PKC during ischemic/pharmacological preconditioning. bilayers; mitochondria; protein kinase C; ischemia; preconditioning Address for reprint requests and other correspondence: M. T. Jiang, Dept. of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (e-mail: mtjiang{at}mcw.edu )