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

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Abstract
Activation of the mitochondrial ATP-sensitive K+channel (mitoKATP) 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 mitoKATPchannel are unknown. Furthermore, there is no evidence that cytosolic PKC can directly regulate the mitoKATPchannel 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 mitoKATPchannel 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 mitoKATPchannels 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 mitoKATPchannel 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 mitoKATPchannel previously inhibited by ATP ( P < 0.05), but its inactive analog 4α-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-δ in the IMM. These data provide the first characterization of the human cardiac mitoKATPchannel 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.

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