Mitochondrial BKCa Channel Research Articles

Coupling of the Electron Transport Chain with the Mitochondrial BKCa Channel in Rat Astrocytes

Potassium channels have been found in the inner mitochondrial membranes of various cells. These channels are regulating the mitochondrial membrane potential, the matrix volume and respiration. In our study, the single-channel activity of a large-conductance Ca2+-regulated potassium (mitoBKCa) channel was measured by a patch-clamping of mitoplasts isolated from rat astrocytes. A potassium-selective current was recorded with a mean conductance of 290 pS. The channel was inhibited by paxilline and iberiotoxin, inhibitors of BKCa channels. Western blot analysis and immunofluorescence demonstrated the presence of the BKCa channel β4 subunit in the inner mitochondrial membranes of the astrocytes. We have shown that substrates of the respiratory chain, such as NADH, succinate, and glutamate/malate are decreasing the activity of the channel at positive voltage. The effect was abolished by rotenone, antimycin and cyanide, inhibitors of the respiratory chain. The interaction of the β4 subunit of mitoBKCa with cytochrome c oxidase was demonstrated by using of Blue Native electrophoresis. Our findings indicate structural and functional coupling of an electron transport chain with the mitoBKCa channel in rat astrocytes.

The inhibitory effect of Ca 2+-activated K + channel activator, BMS on L-type Ca 2+ channels in rat ventricular myocytes

Aims We investigated the effects of BMS-204352 (BMS), a big-conductance calcium-activated potassium (BK Ca) channel activator, on L-type Ca 2+ channels. Main methods Electrophysiological recordings were performed in isolated rat ventricular myocytes. Whole-cell configuration was used. Key findings BMS caused inhibition of the Ca 2+ current in a dose-dependent manner, with K d of 6.00 ± 0.67 μM and a Hill coefficient of 1.33 ± 0.18. BMS did not affect the steady-state activation of L-type Ca 2+ channels. However, for those in steady-state inactivation, BMS shifted the half-maximal potential ( V 1/2) by − 11 mV, but the slope value ( k) was not altered. Iberiotoxin, inhibitor of membrane BK Ca channels and paxilline, inhibitor of mitochondrial BK Ca channel did not affect the inhibitory effect of BMS on L-type Ca 2+ channels. Pretreatment with inhibitors of protein kinase A (PKA), protein kinase C (PKC), and protein kinase G (PKG) did not significantly alter the inhibitory effect of BMS on L-type Ca 2+ current. The presence of a selective β-adrenergic receptor agonist, isoproterenol did not affect the inhibitory effect of BMS on L-type Ca 2+ current. Based on these results, we concluded that the inhibition of L-type Ca 2+ channels by BMS is independent of the inhibition of BK Ca channels or intracellular signaling pathways. Significance It is important to take BMS-204352 (BMS) effects on L-type Ca 2+ channels into consideration when using BMS as a BK Ca channel activator or therapeutic target in ventricular myocytes.

Mitochondrial BKCa channels contribute to protection of cardiomyocytes isolated from chronically hypoxic rats.

Chronic hypoxia protects the heart against injury caused by acute oxygen deprivation, but its salutary mechanism is poorly understood. The aim was to find out whether cardiomyocytes isolated from chronically hypoxic hearts retain the improved resistance to injury and whether the mitochondrial large-conductance Ca2+-activated K+ (BKCa) channels contribute to the protective effect. Adult male rats were adapted to continuous normobaric hypoxia (inspired O2 fraction 0.10) for 3 wk or kept at room air (normoxic controls). Myocytes, isolated separately from the left ventricle (LVM), septum (SEPM), and right ventricle, were exposed to 25-min metabolic inhibition with sodium cyanide, followed by 30-min reenergization (MI/R). Some LVM were treated with either 30 μM NS-1619 (BKCa opener), or 2 μM paxilline (BKCa blocker), starting 25 min before metabolic inhibition. Cell injury was detected by Trypan blue exclusion and lactate dehydrogenase (LDH) release. Chronic hypoxia doubled the number of rod-shaped LVM and SEPM surviving the MI/R insult and reduced LDH release. While NS-1619 protected cells from normoxic rats, it had no additive salutary effect in the hypoxic group. Paxilline attenuated the improved resistance of cells from hypoxic animals without affecting normoxic controls; it also abolished the protective effect of NS-1619 on LDH release in the normoxic group. While chronic hypoxia did not affect protein abundance of the BKCa channel regulatory β1-subunit, it markedly decreased its glycosylation level. It is concluded that ventricular myocytes isolated from chronically hypoxic rats retain the improved resistance against injury caused by MI/R. Activation of the mitochondrial BKCa channel likely contributes to this protective effect.

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 Ca2+ 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 Ca2+ effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BKCa channel). Furthermore, NS1619 – a BKCa channel opener – induced potassium ion–specific effects on brain mitochondria similar to those induced by Ca2+. 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-β4 subunit (of the BKCa channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of β4 subunit in neuronal mitochondria. We hypothesize that the mitochondrial BKCa channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.

Delayed preconditioning with NS1619 protects cultured cortical neurons against both apoptotic and necrotic cell deaths

NS1619, a potent activator of the large conductance Ca2+ activated potassium (BKCa) channel, has been demonstrated to induce preconditioning in the heart. The aim of our study was to test the delayed preconditioning effect of NS1619 in rat cortical neuronal cultures against oxygen‐glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. In addition, we tested the activation of the phosphoinositide 3‐kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase‐3/7. NS1619 dose‐dependently protected the cells against the toxic insults, and the protection was blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BKCa channel inhibitors. Furthermore, NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. Finally, NS1619 inhibited the activation of capase‐3/7. In summary, NS1619 is a potent inducer of delayed neuronal preconditioning. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BKCa channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation. Supported by the NIH Grants HL‐030260, HL‐065380, and HL‐077731.

Differential distribution of Ca 2+-activated potassium channel β4 subunit in rat brain: Immunolocalization in neuronal mitochondria

Large conductance Ca 2+-activated potassium channels (BK Ca channels) are expressed in the plasma membrane of various cell types. Interestingly, recent studies provided evidence for the existence of BK Ca channels also in mitochondria. However, the molecular composition of these channels as well as their cellular and tissue distribution is still unknown. The goal of the present study was to find a candidate for the regulatory component of the mitochondrial large conductance calcium activated potassium (mitoBK Ca) channel in neurons. A combined approach of Western blot analysis, high-resolution immunofluorescence and immunoelectron microscopy with the use of antibodies directed against four distinct β subunits demonstrated the presence of the BK Ca channel β4 subunit (KCNMB4) in the inner membrane of neuronal mitochondria in the rat brain and cultured neurons. Within the cell, the expression of β4 subunit was restricted to a subpopulation of mitochondria. The analysis of β4 subunit distribution throughout the brain revealed that the highest expression levels occur in the thalamus and the brainstem. Our results suggest that β4 subunit is a regulatory component of mitochondrial BK Ca channels in neurons. These findings may support the perspectives for the neuroprotective role of mitochondrial BK Ca channel in specific brain structures.

Delayed neuronal preconditioning by NS1619 is independent of calcium activated potassium channels

1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one (NS1619), a potent activator of the large conductance Ca2+ activated potassium (BK(Ca)) channel, has been demonstrated to induce preconditioning (PC) in the heart. The aim of our study was to test the delayed PC effect of NS1619 in rat cortical neuronal cultures against oxygen-glucose deprivation, H2O2, or glutamate excitotoxicity. We also investigated its actions on reactive oxygen species (ROS) generation, and on mitochondrial and plasma membrane potentials. Furthermore, we tested the activation of the phosphoinositide 3-kinase (PI3K) signaling pathway, and the effect of NS1619 on caspase-3/7. NS1619 dose-dependently protected the cells against the toxic insults, and the protection was completely blocked by a superoxide dismutase mimetic and a PI3K antagonist, but not by BK(Ca) channel inhibitors. Application of NS1619 increased ROS generation, depolarized isolated mitochondria, hyperpolarized the neuronal cell membrane, and activated the PI3K signaling cascade. However, only the effect on the cell membrane potential was antagonized by BK(Ca) channel blockers. NS1619 inhibited the activation of capase-3/7. In summary, NS1619 is a potent inducer of delayed neuronal PC. However, the neuroprotective effect seems to be independent of cell membrane and mitochondrial BK(Ca) channels. Rather it is the consequence of ROS generation, activation of the PI3K pathway, and inhibition of caspase activation.

Mitochondrial Ca2+-activated K+channels more efficiently reduce mitochondrial Ca2+overload in rat ventricular myocytes

We investigated the role of the mitochondrial ATP-sensitive K(+) (K(ATP)) channel, the mitochondrial big-conductance Ca(2+)-activated K(+) (BK(Ca)) channel, and the mitochondrial permeability transition pore (MPTP) in the ouabain-induced increase of mitochondrial Ca(2+) in native rat ventricular myocytes by loading cells with rhod 2-AM. To overload mitochondrial Ca(2+), we pretreated cells with ouabain before applying mitochondrial K(ATP) or BK(Ca) channel and/or MPTP opener. Ouabain (1 mM) increased the rhod 2-sensitive fluorescence intensity (160 +/- 5.0% of control), which was dramatically decreased to the control level on application of diazoxide and NS-1619 in a dose-dependent manner (half-inhibition concentrations of 78.3 and 7.78 muM for diazoxide and NS-1619, respectively). This effect was reversed by selective inhibition of the mitochondrial K(ATP) channel by 5-hydroxydecanoate, the mitochondrial BK(Ca) channel by paxilline, and the MPTP by cyclosporin A. Although diazoxide did not efficiently reduce mitochondrial Ca(2+) during prolonged exposure to ouabain, NS-1619 reduced mitochondrial Ca(2+). These results suggest that although mitochondrial BK(Ca) and K(ATP) channels contribute to reduction of ouabain-induced mitochondrial Ca(2+) overload, activation of the mitochondrial BK(Ca) channel more efficiently reduces ouabain-induced mitochondrial Ca(2+) overload in our experimental model.

Modulatory effects of endogenous nitric oxide on the bioenergetics of BK Ca channels in guinea pig isolated cardiac mitochondria

Plasmalemmal studies have shown that exogenous nitric oxide (NO) promotes big-conductance Ca2+-sensitive K+(BKCa) channel opening, but it is unknown if NO interacts with putative BKCa channels in the mitochondrial inner membrane. The presence of a mitochondrial NO synthase (NOS) is established, and studies support involvement of NO in regulating respiration by competing with the O2 binding site on cytochrome oxidase. We examined the effects of 500 μM L-NIO, a NOS inhibitor, and 5 μM paxilline (PAX), a BKCa channel inhibitor, in mitochondria respiring on succinate at 37°C. Mitochondria respired in state II until O2 fell from about 222 to 67 μM before addition of 10 μM NS1619, a BKCa channel opener, and then 500 μM ADP (onset of state III). L-NIO and NS1619 independently increased state III respiration rate by 8.0±1.0% and 8.4±1.8%, respectively, and together by 16.1±1.5%. State II respiration was also increased by 5.2±1.1% with NS1619, but was unaffected by L-NIO. PAX abolished the effects of NS1619 on respiration, but did not change the effect of L-NIO. Our study indicates that L-NIO and NS1619 enhance respiration by independent mechanisms, i.e., removal of the inhibitory effect of NO on cytochrome oxidase, and K+/H+ exchange with matrix K+ influx. It remains unclear from these results alone if interaction of NO occurs at the level of the mitochondrial BKCa channel as suggested at the plasmalemmal level. (NIH, AHA)