Effectors of large-conductance calcium-activated potassium channel modulate glutamate excitotoxicity in organotypic hippocampal slice cultures.

Mitochondria have been suggested as a potential target for cytoprotective strategies. It has been shown that increased K+ uptake mediate by mitochondrial ATP-regulated potassium channels (mitoKATP channel) or large-conductance Ca2+-activated potassium channels (mitoBKCa channel) may provide protection in different models of cell death. Since recent findings demonstrated the presence of BKCa channels in neuronal mitochondria, the goal of the present study was to test the potential neuroprotective effects of BKCa channel modulators. Using organotypic hippocampal slice cultures exposed to glutamate, we demonstrated that preincubation of the slices with the BKCa channel opener NS1619 resulted in decreased neuronal cell death measured as reduced uptake of propidium iodide. This neuroprotective effect was reversed by preincubation with the BKCa channel inhibitors paxilline and Iberiotoxin (IbTx). Moreover, mitochondrial respiration measurements revealed that NS1619 induced an IbTx-sensitive increase in state 2 respiration of isolated brain mitochondria. In addition, electrophysiological patch-clamp studies confirmed the presence of BKCa channels in mitoplasts isolated from embryonic hippocampal cells. Taken together, our results confirm presence of BKCa channel in rat hippocampal neurons mitochondria and suggest putative role for mitoBKCa in neuroprotection.

Calcium ions regulate K⁺ uptake into brain mitochondria: the evidence for a novel potassium channel.

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca(2+) additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca(2+) effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK(Ca) channel). Furthermore, NS1619 - a BK(Ca) channel opener - induced potassium ion-specific effects on brain mitochondria similar to those induced by Ca(2+). These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-beta(4) subunit (of the BK(Ca) channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of beta(4) subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK(Ca) channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.

[Mitochondrial neuroprotection]. / Mitochondrialna neuroprotekcja.

Mitochondria play a key function in cellular metabolism. Additionally to ATP synthesis, mitochondria may buffor cytosolic calcium ions and generate reactive oxygen species. Due to these processes, mitochondria are involved in complex cytoprotective phenomena. Neuroprotection is very often based on changes in the integrity of mitochondrial membranes. In this report potential neuroprotective role of mitochondrial ion channels is discussed.

A novel potassium channel in skeletal muscle mitochondria.

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.