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Mitochondrial BKCa channel, mitoBKCa, regulates mitochondria function in the heart but information on its protein partnerships in cardiac mitochondria is missing. A directed proteomic approach discovered the novel interaction of BKCa with Tom22, a component of the mitochondrion outer membrane import system, and the adenine nucleotide translocator (ANT). The expressed protein partners co-immunoprecipitated and co-segregated into mitochondrial fractions in HEK293T cells. The BKCa 50 amino acid splice insert, DEC, facilitated BKCa interaction with ANT. Further, BKCa transmembrane domain was required for the association with both Tom22 and ANT. The results serve as a working framework to understand mitoBKCa import and functional relationships.

Impaired estrogen receptor α (ERα) action promotes obesity and metabolic dysfunction in humans and mice; however, the mechanisms underlying these phenotypes remain unknown. Considering that skeletal muscle is a primary tissue responsible for glucose disposal and oxidative metabolism, we established that reduced ERα expression in muscle is associated with glucose intolerance and adiposity in women and female mice. To test this relationship, we generated muscle-specific ERα knockout (MERKO) mice. Impaired glucose homeostasis and increased adiposity were paralleled by diminished muscle oxidative metabolism and bioactive lipid accumulation in MERKO mice. Aberrant mitochondrial morphology, overproduction of reactive oxygen species, and impairment in basal and stress-induced mitochondrial fission dynamics, driven by imbalanced protein kinase A-regulator of calcineurin 1-calcineurin signaling through dynamin-related protein 1, tracked with reduced oxidative metabolism in MERKO muscle. Although muscle mitochondrial DNA (mtDNA) abundance was similar between the genotypes, ERα deficiency diminished mtDNA turnover by a balanced reduction in mtDNA replication and degradation. Our findings indicate the retention of dysfunctional mitochondria in MERKO muscle and implicate ERα in the preservation of mitochondrial health and insulin sensitivity as a defense against metabolic disease in women.

BackgroundEstrogen pretreatment has been shown to attenuate the development of heart hypertrophy, but it is not known whether estrogen could also rescue heart failure (HF). Furthermore, the heart has all the machinery to locally biosynthesize estrogen via aromatase, but the role of local cardiac estrogen synthesis in HF has not yet been studied. Here we hypothesized that cardiac estrogen is reduced in HF and examined whether exogenous estrogen therapy can rescue HF.Methods and resultsHF was induced by transaortic constriction in mice, and once mice reached an ejection fraction (EF) of ≈35%, they were treated with estrogen for 10 days. Cardiac structure and function, angiogenesis, and fibrosis were assessed, and estrogen was measured in plasma and in heart. Cardiac estrogen concentrations (6.18±1.12 pg/160 mg heart in HF versus 17.79±1.28 pg/mL in control) and aromatase transcripts (0.19±0.04, normalized to control, P

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.

The beta(2) subunit of the large conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca)) modulates a number of channel functions, such as the apparent Ca(2+)/voltage sensitivity, pharmacological and kinetic properties of the channel. In addition, the N terminus of the beta(2) subunit acts as an inactivating particle that produces a relatively fast inactivation of the ionic conductance. Applying voltage clamp fluorometry to fluorescently labeled human BK(Ca) channels (hSlo), we have investigated the mechanisms of operation of the beta(2) subunit. We found that the leftward shift on the voltage axis of channel activation curves (G(V)) produced by coexpression with beta(2) subunits is associated with a shift in the same direction of the fluorescence vs. voltage curves (F(V)), which are reporting the voltage dependence of the main voltage-sensing region of hSlo (S4-transmembrane domain). In addition, we investigated the inactivating mechanism of the beta(2) subunits by comparing its properties with the ones of the typical N-type inactivation process of Shaker channel. While fluorescence recordings from the inactivated Shaker channels revealed the immobilization of the S4 segments in the active conformation, we did not observe a similar feature in BK(Ca) channels coexpressed with the beta(2) subunit. The experimental observations are consistent with the view that the beta(2) subunit of BK(Ca) channels facilitates channel activation by changing the voltage sensor equilibrium and that the beta(2)-induced inactivation process does not follow a typical N-type mechanism.

Neutralization of the aspartate near the selectivity filter in the GYGD pore sequence (D292N) of the voltage- and Ca(2+)-activated K+ channel (MaxiK, BKCa) does not prevent conduction like the corresponding mutation in Shaker channel, but profoundly affects major biophysical properties of the channel (Haug, T., D. Sigg, S. Ciani, L. Toro, E. Stefani, and R. Olcese. 2004. J. Gen. Physiol. 124:173-184). Upon depolarizations, the D292N mutant elicited mostly gating current, followed by small or no ionic current, at voltages where the wild-type hSlo channel displayed robust ionic current. In fact, while the voltage dependence of the gating current was not significantly affected by the mutation, the overall activation curve was shifted by approximately 20 mV toward more depolarized potentials. Several lines of evidence suggest that the mutation prevents population of certain open states that in the wild type lead to high open probability. The activation curves of WT and D292N can both be fitted to the sum of two Boltzmann distributions with identical slope factors and half activation potentials, just by changing their relative amplitudes. The steeper and more negative component of the activation curve was drastically reduced by the D292N mutation (from 0.65 to 0.30), suggesting that the population of open states that occurs early in the activation pathway is reduced. Furthermore, the slow component of the gating current, which has been suggested to reflect transitions from closed to open states, was greatly reduced in D292N channels. The D292N mutation also affected the limiting open probability: at 0 mV, the limiting open probability dropped from approximately 0.5 for the wild-type channel to 0.06 in D292N (in 1 mM [Ca2+]i). In addition to these effects on gating charge and open probability, as already described in Part I, the D292N mutation introduces a approximately 40% reduction of outward single channel conductance, as well as a strong outward rectification.

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as "charge immobilization" (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567-590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at -90 mV return potential changed from a single fast component to at least two components, the slower requiring approximately 200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at -120 and -90 mV. In contrast, at higher potentials (-70 and -50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of "parallel" inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH2-terminus region.

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Delta(6-46) K+ channel and in the nonconducting mutant (Shaker H4-Delta(6-46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547-556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to -90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non-slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011-1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

Am J Physiol Heart Circ Physiol 298: H16-H23, 2010. First published October 30, 2009; doi: 10.1152/ajpheart.00588.2009.--Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardio-protection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% [O.sub.2] and 5% C[O.sub.2]) Krebs Henseleit buffer (control), with G1 (1 [micro]M), and G1 (1 [micro]M) together with [Ca.sup.2+]acellular signal-regulated kinase (Erk) inhibitor PD-98059 (5 [micro]M). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37[degrees]C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the [Ca.sup.2+] load required to induce mitochondria permeability transition pore (mPTP) opening. Gl-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 [+ or ] 264 vs. 2,640 [+ or ] 334 beats x mm[Hg.sup.-1] x [min.sup.-1,] P < 0.05). The infarct size decreased significantly in Gl-treated hearts (21 [+ or ] 2 vs. 46 [+ or ] 3%, P < 0.001), and the [Ca.sup.2+] load required to induce mPTP opening increased (2.4 [+ or ] 0.06 vs. 1.6 [+ or ] 0.11 [micro]M/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 [+ or ] 46 beats x mm[Hg.sup.-1] x [min.sup.-1], infarct size: 53 [+ or ] 2%, and [Ca.sup.2+] retention capacity: 1.4 [+ or ] 0.11 [micro]M/mg mitochondrial protein (P < 0.05)]. These results suggest that GPER activation provides a cardioprotectire effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway. G protein-coupled receptor 30; mitochondrial permeability transition pore; cardioprotection; infarct size; heart function

Sepiapterin reductase (SPR) catalyzes the final step of tetrahydrobiopterin ([H.sub.4]B) biosynthesis and the first step of [H.sub.4]B regeneration from an exogenous precursor sepiapterin. Despite the potential significance of SPR in regulating [H.sub.4]B-dependent nitric oxide (N[O.sup.*]) production, the endothelium-specific sequence and functions of SPR remain elusive. We first cloned endothelial SPR cDNA from bovine aortic endothelial cells (Genebank: DQ978331). In cells transiently transfected with SPR gene, SPR activity (HPLC) was dramatically increased by 19-fold, corresponding to a significant increase in endothelial [H.sub.4]B content (HPLC) and N[O.sup.*] production (electron spin resonance). In vivo delivery of SPR gene significantly increased vascular SPR protein expression (mouse vs. bovine antibodies to differentiate endogenous vs. exogenous), activity, [H.sub.4]B content, and N[O.sup.*] production, as well as N[O.sup.*]-dependent vasorelaxation. In endothelial cells transfected with small interfering RNA specific for SPR, ~87% of mRNA were attenuated (real-time quantitative RT-PCR), corresponding to a significant reduction in SPR protein expression and activity, which was associated with decreases in both intracellular [H.sub.4]B content and N[O.sup.*] level. Exogenous administration of sepiapterin to endothelial cells significantly upregulated [H.sub.4]B and N[O.sup.*] levels, which were attenuated by SPR RNA interference (RNAi). [H.sub.4]B-stimulated increase in NO* production, however, was SPR RNAi independent. GTP cyclohydrolase 1 expression and activity, as well as dihydrofolate reductase expression, were not affected by SPR RNAi, whereas dihydrofolate reductase activity was significantly downregulated. These data represent the first to study endothelial SPR functionally and clearly demonstrate an important role of endothelial SPR in modulating [H.sub.4]B and N[O.sup.*] bioavailability. endothelial nitric oxide synthase; aortic endothelial cells; guanosine 5'-triphosphate cyclohydrolase 1; dihydrofolate reductase

Fibrates are peroxisome proliferator-activated receptor-[alpha] (PPAR[alpha]) ligands in widespread clinical use to lower plasma triglyceride levels. We investigated the effect of fenofibrate and clofibrate on ion transport in mouse intestine and in human T84 colonic adenocarcinoma cells through the use of short-circuit current ([I.sub.sc]) and ion flux analysis. In mice, oral administration of fenofibrate produced a persistent inhibition of cAMP-stimulated electrogenic [Cl.sup.-] secretion by isolated jejunum and colon without affecting electroneutral fluxes of [sup.22][Na.sup.+] or [sup.86][Rb.sup.+] ([K.sup.+]) across unstimulated colonic mucosa. When applied acutely to isolated mouse intestinal mucosa, 100 [micro]M fenofibrate inhibited cAMP-stimulated [I.sub.sc] within 5 min. In T84 cells, fenofibrate rapidly inhibited ~80% the [Cl.sup.-] secretory responses to forskolin (cAMP) and to heat stable enterotoxin STa (cGMP) without affecting the response to carbachol ([Ca.sup.2+]). Both fenofibrate and clofibrate inhibited cAMP-stimulated [I.sub.sc] with an [IC.sub.50] ~1 [micro]M, whereas other PPAR[alpha] activators (gemfibrozil and Wy-14,643) were without effect. Membrane permeabilization experiments on T84 cells indicated that fenofibrate inhibits basolateral camp-stimulated [K.sup.+] channels (putatively KCNQ1/KCNE3) without affecting [Ca.sup.2+]-stimulated [K.sup.+] channel activity, whereas clofibrate inhibits both [K.sup.+] pathways. Fenofibrate had no effect on apical cAMP-stimulated [Cl.sup.-] channel activity. Patch-clamp analysis of HEK-293T cells confirmed that 100 [micro]M fenofibrate rapidly inhibits [K.sup.+] currents associated with ectopic expression of human KCNQ1 with or without the KCNE3 [beta]-subunit. We conclude that fenofibrate inhibits intestinal cAMP-stimulated [Cl.sup.-] secretion through a nongenomic mechanism that involves a selective inhibition of basolateral KCNQ1/KCNE3 channel complexes. Our findings raise the prospect of fenofibrate as a safe and effective antidiarrheal agent. clofibrate; peroxisome proliferator-activated receptor-[alpha]; [K.sub.v]7.1; [K.sub.v]LQT1; KCNE3; MiRP2; secretory diarrhea; mouse intestine; human T84 cells; antidiarrheal agents

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory ([beta]) subunits. The most remarkable effects of [beta]1 and [beta]2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by [alpha] and [beta]1 or [beta]2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of [beta]1 but not [beta]2. Here we reveal the molecular regions in these [beta] subunits that determine their differential functional coupling with the pore-forming [alpha]-subunit. We made chimeric constructs between [beta]1 and [beta]2 subunits, and BK channels formed by [alpha] and chimeric [beta] subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the [beta]1 and [beta]2 subunits demonstrates that the N[H.sub.3] and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these [beta] subunits. Moreover, the intracellular domains of [beta]1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the [alpha]-subunit to be the target of the modulation by the [beta]1-subunit.

Voltage-dependent and calcium-sensitive [K.sup.+] (MaxiK) channels are key regulators of neuronal excitability, secretion, and vascular tone because of their ability to sense transmembrane voltage and intracellular [Ca.sup.2+]. In most tissues, their stimulation results in a noninactivating hyperpolarizing [K.sup.+] current that reduces excitability. In addition to noninactivating MaxiK currents, an inactivating MaxiK channel phenotype is found in cells like chromaffin cells and hippocampal neurons. The molecular determinants underlying inactivating MaxiK channels remain unknown. Herein, we report a transmembrane [Beta] subunit ([Beta]2) that yields inactivating MaxiK currents on coexpression with the pore-forming [Alpha] subunit of MaxiK channels. Intracellular application of trypsin as well as deletion of 19 N-terminal amino acids of the [Beta]2 subunit abolished inactivation of the [Alpha] subunit. Conversely, fusion of these N-terminal amino acids to the noninactivating smooth muscle [Beta]1 subunit leads to an inactivating phenotype of MaxiK channels. Furthermore, addition of a synthetic N-terminal peptide of the [Beta]2 subunit causes inactivation of the MaxiK channel [Alpha] subunit by occluding its [K.sup.+]-conducting pore resembling the inactivation caused by the 'ball' peptide in voltage-dependent [K.sup.+] channels. Thus, the inactivating phenotype of MaxiK channels in native tissues can result from the association with different [Beta] subunits.

Copyright of Biomédica: Revista del Instituto Nacional de Salud is the property of Instituto Nacional de Salud of Colombia and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Revista Biomedica is the property of Centro de Investigaciones Regionales Dr. Hideyo Noguchi; Facultad de Medicina, UADY and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)