A Novel Mitochondrial K ATP Channel Assay

Abstract

Rationale : The mitochondrial ATP sensitive potassium channel (mK ATP ) is implicated in cardioprotection by ischemic preconditioning (IPC), but the molecular identity of the channel remains controversial. The validity of current methods to assay mK ATP activity is disputed. Objective : We sought to develop novel methods to assay mK ATP activity and its regulation. Methods and Results : Using a thallium (Tl + )-sensitive fluorophore, we developed a novel Tl + flux based assay for mK ATP activity, and used this assay probe several aspects of mK ATP function. The following key observations were made. (1) Time-dependent run down of mK ATP activity was reversed by phosphatidylinositol-4,5-bisphosphate (PIP 2 ). (2) Dose responses of mK ATP to nucleotides revealed a UDP EC 50 of ≈20 μmol/L and an ATP IC 50 of ≈5 μmol/L. (3) The antidepressant fluoxetine (Prozac) inhibited mK ATP (IC 50 =2.4 μmol/L). Fluoxetine also blocked cardioprotection triggered by IPC, but did not block protection triggered by a mK ATP -independent stimulus. The related antidepressant zimelidine was without effect on either mK ATP or IPC. Conclusions : The Tl + flux mK ATP assay was validated by correlation with a classical mK ATP channel osmotic swelling assay ( R 2 =0.855). The pharmacological profile of mK ATP (response to ATP, UDP, PIP 2 , and fluoxetine) is consistent with that of an inward rectifying K + channel (K IR ) and is somewhat closer to that of the K IR 6.2 than the K IR 6.1 isoform. The effect of fluoxetine on mK ATP -dependent cardioprotection has implications for the growing use of antidepressants in patients who may benefit from preconditioning.