Mitochondrial dysfunction is a hallmark of amyloid-beta (A beta)-induced neuronal toxicity in Alzheimer's disease (AD). However, the underlying mechanism of how A beta affects mitochondrial function remains uncertain. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was undertaken to investigate whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive-IbTx-sensitive Ca2+-activated potassium channel (mitoBK(Ca)) is altered in a rat model of A beta neurotoxicity. A beta 1-42 (4 mu g/mu l) was intracerebroventricularly injected in male Wistar rats (220-250 g). Brain A beta accumulation was confirmed two weeks later on the basis of an immunohistochemisty staining assay, and physiological impacts measured in passive avoidance task cognitive performance experiments. Brain mitochondrial inner membranes were then extracted and membrane vesicles prepared for channel incorporation into bilayer lipid. Purity of the cell fraction was confirmed by Western blot using specific markers of mitochondria, plasma membrane, endoplasmic reticulum, and Golgi. Our results first provide evidence for differences in mitoBK(Ca) ion permeation properties with channels coming from A beta vesicle preparations characterized by an inward rectifying I-V curve, in contrast to control mitoBK(Ca) channels which showed a linear I-V relationship under the same ionic conditions (200 mM cis/50 mM trans). More importantly the open probability of channels from A beta vesicles appeared 1.5 to 2.5 smaller compared to controls, the most significant decrease being observed at depolarizing potentials (30 mV to 50 mV). Because BKCa-beta 4 subunit has been documented to shift the BKCa channel voltage dependence curve, a Western blot analysis was undertaken where expression of mitoBK(Ca) alpha and beta 4 subunits was estimated using anti-alpha and beta 4 subunit antibodies. Our results indicated a significant increase in mitoBK(Ca)-beta 4 subunit expression coupled to a decrease in the expression of alpha subunit. Our results thus demonstrate a modification in the mitoBK(Ca) channel gating properties in membrane preparations coming from a rat model of A beta neurotoxicity, an effect potentially linked to a change in mitoBK(Ca)-beta 4 and -alpha subunits expression or increased ROS production due to an enhanced A beta mitochondrial accumulation. Our results may provide new insights into the cellular mechanisms underlying mitochondrial dysfunctions in A beta neurotoxicity. (C) 2015 Elsevier Inc. All rights reserved.

Existing evidence indicates an impairment of mitochondrial functions and alterations in potassium channel activities in diabetes. Because mitochondrial potassium channels have been involved in several mitochondrial functions including cytoprotection, apoptosis and calcium homeostasis, a study was carried out to consider whether the gating behavior of the mitochondrial ATP- and ChTx-insensitive Ca 2+ -activated potassium channel (mitoBK Ca ) is altered in a streptozotocin (STZ) model of diabetes. Using ion channel incorporation of brain mitochondrial inner membrane into the bilayer lipid membrane, we provide in this work evidence for modifications of the mitoBK Ca ion permeation properties with channels from vesicles preparations coming from diabetic rats characterized by a significant decrease in conductance. More importantly, the open probability of channels from diabetic rats was reduced 1.5–2.5 fold compared to control, the most significant decrease being observed at depolarizing potentials. Because BK Ca β4 subunit has been documented to left shift the BK Ca channel voltage dependence curve in high Ca 2+ conditions, a Western blot analysis was undertaken where the expression of mitoBK Ca α and β4 subunits was estimated using of anti-α and β4 subunit antibodies. Our results indicated a significant decrease in mitoBK Ca β4 subunit expression coupled to a decrease in the expression of α subunit, an observation compatible with the observed decrease in Ca 2+ sensitivity. Our results thus demonstrate a modification in the mitoBK Ca channel gating properties in membrane preparations coming from STZ model of diabetic rats, an effect potentially linked to a change in mitoBK Ca β4 and α subunits expression and/or to an increase in reactive oxygen species production in high glucose conditions.

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 (V1/2) of 11.4±1.1mV and an effective gating charge zd 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.

Rescue of the protein folding defect in cystic fibrosis

Flecainide has been used to differentiate Kv4.2-based transient-outward K(+)-currents (flecainide-sensitive) from Kv1.4-based (flecainide-insensitive). We found that flecainide also inhibits ultrarapid delayed rectifier (I(Kur)) currents in Xenopus laevis oocytes carried by Kv3.1 subunits (IC(50), 28.3 +/- 1.3 microM) more strongly than Kv1.5 currents corresponding to human I(Kur) (IC(50), 237.1 +/- 6.2 microM). The present study examined molecular motifs underlying differential flecainide sensitivity. An initial chimeric approach pointed to a role for S6 and/or carboxyl-terminal sites in Kv3.1/Kv1.5 sensitivity differences. We then looked for homologous amino acid residues of the two sensitive subunits (Kv4.2 and Kv3.1) different from homologous residues for insensitive subunits (Kv1.4 and Kv1.5). Three candidate sites were identified: two in the S5-S6 linker and one in the S6 segment. Mutation of the proximal S5-S6 linker site failed to alter flecainide sensitivity. Mutation at the more distal site in Kv1.5 (V481L) modestly increased sensitivity, but the reciprocal Kv3.1 mutation (L401V) had no effect. S6 mutants caused marked changes: flecainide sensitivity decreased approximately 8-fold for Kv3.1 L422I (IC(50), 213 +/- 9 microM) and increased approximately 7-fold for Kv1.5 I502L (IC(50), 35.6 +/- 1.9 microM). Corresponding mutations reversed flecainide sensitivity of Kv1.4 and Kv4.2; L392I decreased Kv4.2 sensitivity by approximately 17-fold (IC(50) of 37.4 +/- 6.9 to 628 +/- 36 microM); I547L increased Kv1.4 sensitivity by approximately 15-fold (IC(50) of 706 +/- 37 to 40.9 +/- 7.3 microM). Our observations indicate that the flecainide sensitivity differences among these four voltage-gated K(+)-channels are determined by whether an isoleucine or a leucine is present at a specific amino acid location.

The E462R mutation in the fifth position of the AID (alpha1 subunit interaction domain) region in the I-II linker is known to significantly accelerate voltage-dependent inactivation (VDI) kinetics of the L-type CaV1.2 channel, suggesting that the AID region could participate in a hinged-lid type inactivation mechanism in these channels. The recently solved crystal structures of the AID-CaVbeta regions in L-type CaV1.1 and CaV1.2 channels have shown that in addition to E462, positions occupied by Q458, Q459, E461, K465, L468, D469, and T472 in the rabbit CaV1.2 channel could also potentially contribute to a hinged-lid type mechanism. A mutational analysis of these residues shows that Q458A, Q459A, K465N, L468R, D469A, and T472D did not significantly alter VDI gating. In contrast, mutations of the negatively charged E461, E462, and D463 to neutral or positively charged residues increased VDI gating, suggesting that the cluster of negatively charged residues in the N-terminal end of the AID helix could account for the slower VDI kinetics of CaV1.2. A mutational analysis at position 462 (R, K, A, G, D, N, Q) further confirmed that E462R yielded faster VDI kinetics at +10 mV than any other residue with E462R >> E462K approximately E462A > E462N > wild-type approximately E462Q approximately E462G > E462D (from the fastest to the slowest). E462R was also found to increase the VDI gating of the slow CEEE chimera that includes the I-II linker from CaV1.2 into a CaV2.3 background. The fast VDI kinetics of the CaV1.2 E462R and the CEEE + E462R mutants were abolished by the CaVbeta2a subunit and reinstated when using the nonpalmitoylated form of CaVbeta2a C3S + C4S (CaVbeta2a CS), confirming that CaVbeta2a and E462R modulate VDI through a common pathway, albeit in opposite directions. Altogether, these results highlight the unique role of E461, E462, and D463 in the I-II linker in the VDI gating of high-voltage activated CaV1.2 channels.

The substituted cysteine accessibility method (SCAM) was used to map the external vestibule and the pore region of the ECaC-TRPV5 calcium-selective channel. Cysteine residues were introduced at 44 positions from the end of S5 (Glu515) to the beginning of S6 (Ala560). Covalent modification by positively charged MTSET applied from the external medium significantly inhibited whole cell currents at 15/44 positions. Strongest inhibition was observed in the S5-linker to pore region (L520C, G521C, and E522C) with either MTSET or MTSES suggesting that these residues were accessible from the external medium. In contrast, the pattern of covalent modification by MTSET for residues between Pro527 and Ile541 was compatible with the presence of a alpha-helix. The absence of modification by the negatively charged MTSES in that region suggests that the pore region has been optimized to favor the entrance of positively charged ions. Cysteine mutants at positions -1, 0, +1, +2 around Asp542 (high Ca2+ affinity site) were non-functional. Whole cell currents of cysteine mutants at +4 and +5 positions were however covalently inhibited by external MTSET and MTSES. Altogether, the pattern of covalent modification by MTS reagents globally supports a KcsA homology-based three-dimensional model whereby the external vestibule in ECaC-TRPV5 encompasses three structural domains consisting of a coiled structure (Glu515 to Tyr526) connected to a small helical segment of 15 amino acids (527PTALFSTFELFLT539) followed by two distinct coiled structures Ile540-Pro544 (selectivity filter) and Ala545-Ile557 before the beginning of S6.

We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance δ of 0.49 ± 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that δ should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of δ measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect δ for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.

The topological model proposed for the Kir2.1 inward rectifier predicts that seven of the channel 13 cysteine residues are distributed along the N- and C-terminus regions, with some of the residues comprised within highly conserved domains involved in channel gating. To determine if cytosolic cysteine residues contribute to the gating properties of Kir2.1, each of the N- and C-terminus cysteines was mutated into either a polar (S, D, N), an aliphatic (A,V, L), or an aromatic (W) residue. Our patch-clamp measurements show that with the exception of C76 and C311, the mutation of individual cytosolic cysteine to serine (S) did not significantly affect the single-channel conductance nor the channel open probability. However, mutating C76 to a charged or polar residue resulted either in an absence of channel activity or a decrease in open probability. In turn, the mutations C311S (polar), C311R (charged), and to a lesser degree C311A (aliphatic) led to an increase of the channel mean closed time due to the appearance of long closed time intervals (Tc ≥ 500 ms) and to a reduction of the reactivation by ATP of rundown Kir2.1 channels. These changes could be correlated with a weakening of the interaction between Kir2.1 and PIP2, with C311R and C311S being more potent at modulating the Kir2.1-PIP2 interaction than C311A. The present work supports, therefore, molecular models whereby the gating properties of Kir2.1 depend on the presence of nonpolar or neutral residues at positions 76 and 311, with C311 modulating the interaction between Kir2.1 and PIP2.

The effect of melatonin on the Ca2+ signaling process in bovine aortic endothelial cells (BAE) and in primary cultured vascular endothelial cells from normotensive Sprague Dawley (SDR) and genetically hypertensive (SHR) rats was investigated using the Ca2+ indicator Fura‐2. Acute applications of melatonin failed to initiate a Ca2+ response in the three cell types considered. However, preincubating SHR aortic endothelial cells with exposure to melatonin increased the internal Ca2+ release triggered by bradykinin (BK) and ATP while stimulating the related agonist‐evoked Ca2+ entry. This effect appeared specific for SHR cells, as a similar incubation period failed to alter the Ca2+ responses in BAE and SDR cells. Because of the known overproduction of free radicals in SHR cells, the effect of melatonin on Ca2+ signaling was also tested in SDR and BAE cells exposed to the superoxide anion radical. Melatonin reversed the deleterious action of free radicals on Ca2+ signaling in both cases, suggesting that its stimulatory effect in SHR was linked to its antioxidative properties. Finally, experiments where melatonin was applied between successive BK stimulation periods showed an enhancement of the agonist‐evoked Ca2+ entry in BAE and SDR cells. This effect appeared to be independent of the production of second messengers as no specific binding sites for melatonin, including MT1, MT2 and MT3 receptors, could be detected in BAE cells. We conclude that melatonin improves Ca2+ signaling in dysfunctional endothelial cells characterized by an overproduction of free radicals while stimulating the agonist‐evoked Ca2+ entry in normal endothelial cells through a mechanism not related to its antioxidative properties.

Cysteine-scanning mutagenesis (SCAM) and computer-based modeling were used to investigate key structural features of the S6 transmembrane segment of the calcium-activated K+ channel of intermediate conductance IKCa. Our SCAM results show that the interaction of [2-(trimethylammonium) ethyl] methanethiosulfonate bromide (MTSET) with cysteines engineered at positions 275, 278, and 282 leads to current inhibition. This effect was state dependent as MTSET appeared less effective at inhibiting IKCa in the closed (zero Ca2+ conditions) than open state configuration. Our results also indicate that the last four residues in S6, from A283 to A286, are entirely exposed to water in open IKCa channels, whereas MTSET can still reach the 283C and 286C residues with IKCa maintained in a closed state configuration. Notably, the internal application of MTSET or sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) caused a strong Ca2+-dependent stimulation of the A283C, V285C, and A286C currents. However, in contrast to the wild-type IKCa, the MTSET stimulated A283C and A286C currents appeared to be TEA insensitive, indicating that the MTSET binding at positions 283 and 286 impaired the access of TEA to the channel pore. Three-dimensional structural data were next generated through homology modeling using the KcsA structure as template. In accordance with the SCAM results, the three-dimensional models predict that the V275, T278, and V282 residues should be lining the channel pore. However, the pore dimensions derived for the A283-A286 region cannot account for the MTSET effect on the closed A283C and A286 mutants. Our results suggest that the S6 domain extending from V275 to V282 possesses features corresponding to the inner cavity region of KcsA, and that the COOH terminus end of S6, from A283 to A286, is more flexible than predicted on the basis of the closed KcsA crystallographic structure alone. According to this model, closure by the gate should occur at a point located between the T278 and V282 residues.

The Ca(2+) affinity and permeation of the epithelial Ca(2+) channel (ECaC1) were investigated after expression in Xenopus oocytes. ECaC1 displayed anomalous mole-fraction effects. Extracellular Ca(2+) and Mg(2+) reversibly inhibited ECaC1 whole cell Li(+) currents: IC(50) = 2.2 +/- 0.4 microM (n = 9) and 235 +/- 35 microM (n = 10), respectively. These values compare well with the Ca(2+) affinity of the L-type voltage-gated Ca(2+) (Ca(V)1.2) channel measured under the same conditions, suggesting that high-affinity Ca(2+) binding is a well-conserved feature of epithelial and voltage-gated Ca(2+) channels. Neutralization of D550 and E535 in the pore region had no significant effect on Ca(2+) and Mg(2+) affinities. In contrast, neutralization of D542 significantly decreased Ca(2+) affinity (IC(50) = 1.1 +/- 0.2 mM, n = 6) and Mg(2+) affinity (IC(50) > 25 +/- 3 mM, n = 4). Despite a 1,000-fold decrease in Ca(2+) affinity in D542N, Ca(2+) permeation properties and the Ca(2+)-to-Ba(2+) conductance ratio remained comparable to values for wild-type ECaC1. Together, our observations suggest that D542 plays a critical role in Ca(2+) affinity but not in Ca(2+) permeation in ECaC1.

Objective To test the hypothesis that the Ca2+ signal transduction process in endothelial cells from genetically hypertensive rats (SHR) is affected by an overproduction of free radicals. Methods The Ca2+ response to the inositol 1,4,5-triphosphate (IP3) mobilizing agonist, ATP, was measured using the fluorescent probe, fura-2, in endothelial cells from Sprague–Dawley rats, and in young and age-matched genetically hypertensive rats (SHR). The effect of free radicals and reducing agents on the intracellular release of Ca2+ and IP3production was determined in resting and ATP-stimulated cells. Experiments were also performed to compare the level of expression and enzymatic activity of catalase and superoxide dismutase (SOD ) in endothelial cells from SHR and Sprague–Dawley rats. Results The exposure of aortic endothelial cells from Sprague–Dawley rats to the free-radical generating system, hypoxanthine + xanthine oxidase (HX/XO), caused a time- and concentration-dependent inhibition of the ATP-induced Ca2+ response. A similar HX/XO-dependent inhibition was also observed in Sprague–Dawley cells stimulated with the endoplasmic reticulum Ca2+-ATPase inhibitor, thapsigargin. Incubation with the antioxidative enzymes, catalase and SOD, had no effect on the ATP-induced Ca2+ release in Sprague–Dawley cells, but led to a strong increase in the internal release of Ca2+ in cells from adult (12 weeks old) or young (3 weeks old) SHR. The effect of antioxidants was not related either to an enhancement of the ATP-induced production of IP3, or to a lower expression and activity of SOD and catalase. Conclusion The present work provides evidence that the Ca2+ signalling process in SHR endothelial cells is affected by an overproduction of free radicals, resulting in a depletion of releasable Ca2+ from IP3-sensitive and -insensitive Ca2+ pools. These results point towards a beneficial action of antioxidants on Ca2+ signalling in endothelial cells from models of hypertension.

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified CaV1.2 (α1C) and CaV2.3 (α1E) channels. Mibefradil inhibition of peak Ba2+ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Cav1.2 currents (IC 50) and from IC 50= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for CaV2.3 currents. In the presence of mibefradil, CaV1.2 and CaV2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered CaV1.2 and CaV2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I–II linker from CaV1.2 in the CaV2.3 host channel), in EC(AID)EEE (part of the I–II linker from CaV1.2 in the CaV2.3 host channel) as well as in CaV1.2 E462R, and CaV2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC 50= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC 50= 41 ± 5 μm (n= 4) and IC 50= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and CaV2.3, whereas intermediate-inactivating channel kinetics (CEEE, CaV1.2 E462R, and CaV1.2 E462K) were inhibited by similar concentrations of mibefradil with IC 50≈ 55–75 μm. The slower CaV1.2 wild-type and CaV1.2 Q473K channels responded to higher doses of mibefradil with IC 50≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (CaV1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (CaV2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca2+ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a ``receptor guarded mechanism'' where fast inactivation kinetics efficiently trap mibefradil into the channel.

Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca2+ concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 μM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.