Electro-pharmacological profile of a mitochondrial inner membrane big-potassium channel from rat brain

Abstract

Recent studies have indicated a calcium-activated large conductance potassium channel in rat brain mitochondrial inner membrane (mitoBK channel). Accordingly, we have characterized the functional and pharmacological profile of a BK channel from rat brain mitochondria in the present study. Brain mitochondrial inner membrane preparations were subjected to SDS-PAGE analysis and channel protein reconstitution into planar lipid bilayers. Western blotting and antibodies directed against various cellular proteins revealed that mitochondrial inner membrane fractions did not contain specific proteins of the other subcellular compartments except a very small fraction of endoplasmic reticulum. Channel incorporation into planar lipid bilayers revealed a voltage dependent 211 pS potassium channel with a voltage for half activation (V(1/2)) of 11.4±1.1mV and an effective gating charge z(d) of 4.7±0.9. Gating and conducting behaviors of this channel were unaffected by the addition of 2.5mM ATP, and 500 nM charybdotoxin (ChTx), but the channel appeared sensitive to 100 nM iberiotoxin (IbTx). Adding 10mM TEA at positive potentials and 10mM 4-AP at negative or positive voltages inhibited the channel activities. These results demonstrate that the mitoBK channel, present in brain mitochondrial inner membrane, displays different pharmacological properties than those classically described for plasma membrane, especially in regard to its sensitivity to iberiotoxin and charybdotoxin sensitivity.

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

“…The activity of the putative mitochondrial BK Ca channel (mitoBK Ca ), thought to be stimulated by Ca 2+ and blocked by charybdotoxin, was initially detected in mitoplasts from a glioma cell line (Siemen et al 1999). Later, similar single-channel recordings were performed for the inner membranes of heart, skeletal muscle, brain and endothelium mitochondria (Xu et al 2002, Skalska et al 2008, Douglas et al 2006, Fahnik-Babaei et al 2011, Bednarczyk et al 2013a). The BK Ca channel opener NS1619 was found to increase K + influx of intact heart mitochondria in a way that was sensitive to BK Ca channel blockers, charybdotoxin and Iberiotoxin (Xu et al 2002).…”

Brain mitochondrial ATP-insensitive large conductance Ca+2-activated K+ channel properties are altered in a rat model of amyloid-β neurotoxicity

“…3D. As can be seen P o estimates for the microinjected-Aβ model reached values close to~0.4 ± 0.01 (n = 5) at + 50 mV with values~0.2 ± 0.02 (n = 5) at − 40 mV, while the control mitoBK Ca channel P o increased from 0.5 ± 0.01 (n = 5) at −40 mV to a maximum of 0.9 ± 0.01 (n = 5) at +40 mV (Fahanik-Babaei et al, 2011a). Curve fitting the observed P o voltage dependence to a Boltzmann constant led to a voltage for half-maximal activation (V 1/2 ) of 5.60 ± 0.9 mV (n = 5) for Aβ group compared to 11.4 ± 1.1 (n = 5) in control rats with an equivalent gating charge z of − 1.33 ± 0.32 (n = 5) and 4.7 ± 0.9 (n = 5) respectively.…”

“…We have reported in a previous work two different kinds of brain mitoBK Ca channels namely an ATP-insensitive mitoBK Ca channel blocked by IbTx but not ChTx (Fahanik-Babaei et al, 2011a), and an ATP-sensitive mitoBK Ca channel blocked by IbTx and ChTx (FahanikBabaei et al, 2011b). Experiments were thus performed to determine the nature of the mitoBK Ca channels observed from microinjected-Aβ brain rat preparations.…”

“…Single potassium channel recordings were obtained following fusion of vesicles prepared from brain mitochondrial inner membranes of microinjected-Aβ rats onto black (bilayer) lipid membranes (BLMs) as described elsewhere (Fahanik-Babaei et al, 2011a). Single channel currents were measured with a BC-525D amplifier (Warner Instrument), and the cis chamber was voltage-clamped relative to the trans chamber, which was grounded.…”

“…A BK Ca channel is composed of four similar α-subunits that co-assemble around a central axis to form a single K + -selective conduction pore, plus β auxiliary subunits that co-associated with each α-subunits in a 1:1 ratio To date, four β subunits (β1-4) have been described with β1 being the principal form found in vascular smooth muscle cells, while β2 (chromaffin cells, brain, lung), β3 (testis, pancreas, spleen) and β4 (neuronal, lung) are thought to be restricted to other tissues (Orio et al, 2002;Salkoff et al, 2006;Wang et al, 2006). We have shown in a previous work the presence in control animals of two mitoBK Ca channels, a ChTxand ATP-insensitive-IbTx-sensitive mitoBK Ca channel (Fahanik-Babaei et al, 2011a) and a mitoBK Ca channel sensitive to ChTx-and ATP (Fahanik-Babaei et al, 2011b) Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was undertaken to investigate if the gating behavior of the ChTx-and ATP-insensitive-IbTx-sensitive mitoBK Ca channel was altered in a model of Aβ toxicity. Our results show that significant abnormalities in mitoBK Ca channel permeation and gating properties, added to an increase in β4 and a decrease in mitoBK Ca α subunit expression respectively.…”