A Novel Mitochondrial KATP Channel Assay

Rationale: The mitochondrial ATP sensitive potassium channel (mKATP) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mKATP activity is disputed. Objective: We sought to develop novel methods to assay mKATP activity and its regulation. Methods and Results: Using a thallium (Tl+)-sensitive fluorophore, we developed a novel Tl+ flux based assay for mKATP activity, and used this assay probe several aspects of mKATP function. The following key observations were made. (1) Time-dependent run down of mKATP activity was reversed by phosphatidylinositol-4,5-bisphosphate (PIP2). (2) Dose responses of mKATP to nucleotides revealed a UDP EC50 of ≈20 μmol/L and an ATP IC50 of ≈5 μmol/L. (3) The antidepressant fluoxetine (Prozac) inhibited mKATP (IC50=2.4 μmol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mKATP-independent stimulus. The related antidepressant zimelidine was without effect on either mKATP or IPC. Conclusions: The Tl+ flux mKATP assay was validated by correlation with a classical mKATP channel osmotic swelling assay ( R 2=0.855). The pharmacological 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.