Identification and Properties of a Novel Intracellular (Mitochondrial) ATP-sensitive Potassium Channel in Brain

Robert Bajgar,Subramaniam Seetharaman,Alicia J Kowaltowski,Keith D Garlid,Petr Paucek

doi:10.1074/jbc.m103320200

Robert Bajgar, Subramaniam Seetharaman + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m103320200

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Abstract

Protection of heart against ischemia-reperfusion injury by ischemic preconditioning and K(ATP) channel openers is known to involve the mitochondrial ATP-sensitive K(+) channel (mitoK(ATP)). Brain is also protected by ischemic preconditioning and K(ATP) channel openers, and it has been suggested that mitoK(ATP) may also play a key role in brain protection. However, it is not known whether mitoK(ATP) exists in brain mitochondria, and, if so, whether its properties are similar to or different from those of heart mitoK(ATP). We report partial purification and reconstitution of a new mitoK(ATP) from rat brain mitochondria. We measured K(+) flux in proteoliposomes and found that brain mitoK(ATP) is regulated by the same ligands as those that regulate mitoK(ATP) from heart and liver. We also examined the effects of opening and closing mitoK(ATP) on brain mitochondrial respiration, and we estimated the amount of mitoK(ATP) by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoK(ATP) from brain and liver. Three independent methods indicate that brain mitochondria contain six to seven times more mitoK(ATP) per milligram of mitochondrial protein than liver or heart.

Highlights

The inner membranes of liver and heart mitochondria contain an ATP-sensitive Kϩ channel,1 whose regulation has been studied in both intact mitochondria and liposomes containing reconstituted, purified mitoKATP (1– 6)
We examined the effects of opening and closing mitoKATP on brain mitochondrial respiration, and we estimated the amount of mitoKATP by means of green fluorescence probe BODIPY-FL-glyburide labeling of the sulfonylurea receptor of mitoKATP from brain and liver
We report identification of an ATP-sensitive Kϩ channel in rat brain mitochondria with properties similar to heart and liver mitoKATP (1–3)

Summary

EXPERIMENTAL PROCEDURES

Mitochondrial Isolation—Mitochondria were isolated by differential centrifugation from rat brain cortex (15) and liver (16). The brain mitochondrial preparation utilizes digitonin to disrupt synaptosomal vesicles and is considered to provide a population that is representative of both glial and neuronal tissue (15). Mitochondrial protein was estimated using the Biuret reaction (17). Measurement of Mitochondrial Respiration—Respiration was measured at 25 °C with a Clark-type oxygen electrode in Kϩ- and TEAϩbased media containing 0.5 mg of mitochondrial protein/ml, 2.77 mM CaCl2, 1.38 mM MgCl2, 0.5 mM dithiothreitol, 20 mM imidazole, 2 mM malate, 5 mM pyruvate, 3 mM phosphate, and 10 mM EGTA, pH 7.1 (adjusted by KOH or TEAOH). Measurement of Mitochondrial Volume—Changes in mitochondrial matrix volume, due to net Kϩ salt transport into mitochondria, were monitored by quantitative light scattering, as described previously

Brain Mitochondrial KATP Channel

RESULTS

Glyburide Inhibition

DISCUSSION