Reconstitution and partial purification of the glibenclamide-sensitive, ATP-dependent K+ channel from rat liver and beef heart mitochondria.

P Paucek,G Mironova,F Mahdi,A.D Beavis,G Woldegiorgis,K.D Garlid

doi:10.1016/s0021-9258(18)35717-x

P Paucek, G Mironova + Show 4 more

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https://doi.org/10.1016/s0021-9258(18)35717-x

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Abstract

The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.

Highlights

Partial Purification of the Mitochondrial K+Channel-The detergent-solubilized extract of rat liver mitochondria was fractionated on aDEAE-cellulose column and assayed for K+ uniport activity following reconstitution, as described under “Experimental Procedures.”
To probe for other similarities, we examined the effects of the sulfonylurea, glibenclamide,which is a potent and classical inhibitor of plasmalemma1 ATP
We found no K+ uniport activity in the fraction isolated from mitochondria that had medium, supplemented as described in the legend to Fig

Summary

Two protocols were used for measurement of the electrophoretic

K’uptake as follows. ( a )Vesicles (0.4 mg of lipid/ml) were added to a medium containing 150mM KCI, 25mM TEA-HEPES, pH8.5, and 0.5 mM EDTA. ( a )Vesicles (0.4 mg of lipid/ml) were added to a medium containing 150mM KCI, 25mM TEA-HEPES, pH8.5, and 0.5 mM EDTA. (b) Vesicles (0.4 mgof lipid/ ml) were added to a medium containing 150mM KSCN, 25 mM TEA-. Internal medium contained 0.10 mM KSCN, 0.5 mM EDTA, 25 mM TEA-HEPES, pH 7.4, 100 mM. ElectricalMeasurements in Lipid Bilayer Membranes-Lipids were isolated from bovine brain [18], solubilized in n-decane (20 mg/ml), and used to form a lipid bilayer membrane across a circular hole (0.975 X cm2)ina Teflon partitionseparating two cuvettes [19] Medium on both sides of the membrane contained 1 M KC1 and 10 mM Tris-HC1, pH 7.2. Aliquots of the eluate were added to the “cis)) side of the membrane. Gel patterns were visualized with Coomassie Brilliant Blue R-250 and silver staining [21]

RESULTS

This is entirely consistent with competition between ATP

TIME lminl

DISCUSSION