Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria.

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.

Reconstitution of the mitochondrial ATP-dependent potassium channel into bilayer lipid membrane.

Electrical properties and regulation of the mitochondrial ATP-dependent potassium channel were studied. The channel protein was solubilized from the mitochondrial membrane using an ethanol/water mixture. Reconstituted into a bilayer lipid membrane BLM), the protein formed a slightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl. Often, several channels worked simultaneously (clusters) when many channels were incorporated into the BLM. The elementary channel and the clusters were both highly potassium selective. At concentrations of 1 to 10 microM, ATP favors channel opening, while channels become closed at 1-3 mM ATP. GDP (0.5 mM) reactivated the ATP-closed channels without affecting the untreated channels. The sulfhydryl-reducing agent ditiothreitol increased the open probability at concentrations of 1 to 3 mM, but damaged the selectivity of the channel.

ATP-dependent potassium channel from rat liver mitochondria: inhibitory analysis, channel clusterization.

An inhibitor of potassium mitochondrial channels quinidine (0.1 mM) closed the ATP-sensitive potassium channels isolated from mitochondria and reconstituted into a bilayer lipid membrane. Inhibitors of cytoplasm membrane K-channels (ouabain, tetraethyl ammonium, and cesium ions) produced no effect on the ATP-sensitive mitochondria K-channels; 0.5-2 microM glybenclamide did not affect the reconstituted channels, either. The multiplicity of jumps of conductivity at small concentration of the protein and the maintenance of ion selectivity at switching various levels of conductivity lead us to assume that the large channels are clusters of elementary channels. The average frequency of channel-cluster switchovers between various levels of conductivity is much higher than at the elementary channel.