A novel mitochondrial K(ATP) channel assay.
BACKGROUND
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.


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 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.


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.
Wojtovich
Andrew P
AP
Department of Pharmacology, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642, USA.
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
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018687:Antidepressive Agents, Second-Generation / Q000494:pharmacology; D052160:Benzothiazoles; D001681:Biological Assay / Q000379:methods*; D003374:Coumarins; D005456:Fluorescent Dyes; D005473:Fluoxetine / Q000494:pharmacology; D005998:Glycine / Q000031:analogs & derivatives; D066298:In Vitro Techniques; D019157:Ischemic Preconditioning, Myocardial; D007700:Kinetics; D008297:Male; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D008933:Mitochondrial Swelling; D015428:Myocardial Reperfusion Injury / Q000378:metabolism / Q000517:prevention & control; D009995:Osmosis; D010477:Perfusion; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D011188:Potassium / Q000378:metabolism*; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013793:Thallium / Q000378:metabolism*; D014530:Uridine Diphosphate / Q000378:metabolism
D018687:Antidepressive Agents, Second-Generation; D052160:Benzothiazoles; D003374:Coumarins; D005456:Fluorescent Dyes; C116748:N-(3-(2-benzothiazolyl)-6-(2-(2-(bis(carboxymethyl)amino)-5-methylphenoxy)ethoxy)-2-oxo-2H-1-benzopyran-7-yl)-N-(carboxymethyl)glycine; D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; C117780:mitochondrial K(ATP) channel; D005473:Fluoxetine; D014530:Uridine Diphosphate; D000255:Adenosine Triphosphate; D013793:Thallium; D011188:Potassium; D005998:Glycine