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Lack of manifestations of diazoxide/5‐hydroxydecanoate‐sensitive K ATP channel in rat brain nonsynaptosomal mitochondria
Author(s) -
Brustovetsky Tatiana,
Shalbuyeva Natalia,
Brustovetsky Nickolay
Publication year - 2005
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2005.091199
Subject(s) - diazoxide , mitochondrion , potassium channel , biophysics , chemistry , inner mitochondrial membrane , membrane potential , microbiology and biotechnology , biochemistry , biology , pharmacology , endocrinology , insulin
Pharmacological modulation of the mitochondrial ATP‐sensitive K + channel (mitoK ATP ) sensitive to diazoxide and 5‐hydroxydecanoate (5‐HD) represents an attractive strategy to protect cells against ischaemia/reperfusion‐ and stroke‐related injury. To re‐evaluate a functional role for the mitoK ATP in brain, we used Percoll‐gradient‐purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (ΔΨ) and depolarization‐induced K + efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K + inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP‐sensitive K + channel of the plasma membrane (cellK ATP ), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5‐hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoK ATP were applied to show the role of the channel in regulating matrix volume, respiration, and ΔΨ and K + fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoK ATP activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoK ATP , similar to the cellK ATP , in brain mitochondria. On the other hand, the depolarization‐evoked release of K + suppressed by ATP could be re‐activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K + efflux similarly to ATP. These observations implicate the ANT in ATP‐sensitive K + transport in brain mitochondria.