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A novel mitochondrial K(ATP) channel assay.

ABSTRACT: 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.We sought to develop novel methods to assay mK(ATP) activity and its regulation.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 approximately 20 micromol/L and an ATP IC(50) of approximately 5 micromol/L. (3) The antidepressant fluoxetine (Prozac) inhibited mK(ATP) (IC(50)=2.4 micromol/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.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.

SUBMITTER: Wojtovich AP

PROVIDER: S-EPMC2857559 | biostudies-literature | 2010 Apr

REPOSITORIES: biostudies-literature

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A novel mitochondrial K(ATP) channel assay.

Wojtovich Andrew P AP Williams David M DM Karcz Marcin K MK Lopes Coeli M B CM Gray Daniel A DA Nehrke Keith W KW Brookes Paul S PS

Circulation research 20100225 7

<h4>Rationale</h4>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.<h4>Objective</h4>We sought to develop novel methods to assay mK(ATP) activity and its regulation.<h4>Methods and results</h4>Using a thallium (Tl(+))-sensitive fluorophore, we developed a novel Tl(+) flux based assay ...[more]

PMID: 20185796

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Project description:Background: KATP channels have diverse roles, including regulation of insulin secretion and blood flow, and protection against biological stress responses and are excellent therapeutic targets. Different subclasses of KATP channels exist in various tissue types due to the unique assemblies of specific pore-forming (Kir6.x) and accessory (SURx) subunits. The majority of pharmacological openers and blockers act by binding to SURx and are poorly selective against the various KATP channel subclasses. Methods and Results: We used 3D models of the Kir6.2/SUR homotetramers based on existing cryo-EM structures of channels in both the open and closed states to identify a potential agonist binding pocket in a functionally critical area of the channel. Computational docking screens of this pocket with the Chembridge Core chemical library of 492,000 drug-like compounds yielded 15 top-ranked "hits", which were tested for activity against KATP channels using patch clamping and thallium (Tl+) flux assays with a Kir6.2/SUR2A HEK-293 stable cell line. Several of the compounds increased Tl+ fluxes. One of them (CL-705G) opened Kir6.2/SUR2A channels with a similar potency as pinacidil (EC50 of 9 µM and 11 μM, respectively). Remarkably, compound CL-705G had no or minimal effects on other Kir channels, including Kir6.1/SUR2B, Kir2.1, or Kir3.1/Kir3.4 channels, or Na+ currents of TE671 medulloblastoma cells. CL-705G activated Kir6.2Δ36 in the presence of SUR2A, but not when expressed by itself. CL-705G activated Kir6.2/SUR2A channels even after PIP2 depletion. The compound has cardioprotective effects in a cellular model of pharmacological preconditioning. It also partially rescued activity of the gating-defective Kir6.2-R301C mutant that is associated with congenital hyperinsulinism. Conclusion: CL-705G is a new Kir6.2 opener with little cross-reactivity with other channels tested, including the structurally similar Kir6.1. This, to our knowledge, is the first Kir-specific channel opener.

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A novel mitochondrial K(ATP) channel assay.

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A novel mitochondrial K(ATP) channel assay.

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