Perovskite-organic tandem solar cells with indium oxide interconnect.

Multijunction solar cells can overcome the fundamental efficiency limits of single-junction devices. The bandgap tunability of metal halide perovskite solar cells renders them attractive for multijunction architectures1. Combinations with silicon and copper indium gallium selenide (CIGS), as well as all-perovskite tandem cells, have been reported2-5. Meanwhile, narrow-gap non-fullerene acceptors have unlocked skyrocketing efficiencies for organic solar cells6,7. Organic and perovskite semiconductors are an attractive combination, sharing similar processing technologies. Currently, perovskite-organic tandems show subpar efficiencies and are limited by the low open-circuit voltage (Voc) of wide-gap perovskite cells8 and losses introduced by the interconnect between the subcells9,10. Here we demonstrate perovskite-organic tandem cells with an efficiency of 24.0 per cent (certified 23.1 per cent) and a high Voc of 2.15 volts. Optimized charge extraction layers afford perovskite subcells with an outstanding combination of high Voc and fill factor. The organic subcells provide a high external quantum efficiency in the near-infrared and, in contrast to paradigmatic concerns about limited photostability of non-fullerene cells11, show an outstanding operational stability if excitons are predominantly generated on the non-fullerene acceptor, which is the case in our tandems. The subcells are connected by an ultrathin (approximately 1.5 nanometres) metal-like indium oxide layer with unprecedented low optical/electrical losses. This work sets a milestone for perovskite-organic tandems, which outperform the best p-i-n perovskite single junctions12 and are on a par with perovskite-CIGS and all-perovskite multijunctions13.

Tumor-induced osteomalacia: experience from a South American academic center.

The majority of tumor-induced osteomalacia cases have been reported in the Northern Hemisphere and Asia. In this first series of South American patients, we show that the clinical presentation and sensitivity of plasmatic fibroblast growth factor 23 and somatostatin analog-based imaging are similar to those described in other populations. INTRODUCTION: Describe the experience of clinical presentation, diagnostic study, and treatment of patients with tumor-induced osteomalacia (TIO) in a South American academic center in comparison to literature. METHODS: Analysis of the records of patients diagnosed with TIO. The clinical presentation, diagnostic studies, and treatment were analyzed. Fibroblast growth factor 23 (FGF23) was measured by ELISA. RESULTS: Six patients were diagnosed with TIO during the studied period. The patients' median age was 53 years (range 22-64). All patients presented with weakness and pain in the extremities. Four experienced fractures during their evolution. The median time to diagnosis was 4.5 years (1-20). Biochemical studies showed hypophosphatemia, median of 1.4 mg/dL (1.2-1.6), with low maximum rates of tubular reabsorption of phosphate adjusted for glomerular filtration rate. FGF23 was elevated in 4/6 patients and inappropriately normal in the other two. In three patients, the location of the tumor was clinically evident and confirmed with anatomical imaging. In the remaining patients, two tumors were located with 68Ga DOTATATE-PET/CT and one with OctreoScan. The causal tumors were located in the lower extremities in five patients and invading the frontal sinus in one patient. In all patients, tumors were successfully removed. Within 14 days, there was normalization of phosphate and FGF23 levels and resolution of clinical symptoms in all patients. In all cases, the histopathology was compatible with a phosphaturic mesenchymal tumor. CONCLUSIONS: The clinical presentation, delay time to diagnosis, FGF23 diagnostic sensitivity and histopathology in this first series of South American patients is similar to those described in other populations. The success of localization by somatostatin analog-based imaging, suggests this may the optimal imaging modality.

3D multicellular models reflect the efficiency of MSC-directed enzyme/prodrug treatment.

Mesenchymal stromal cells (MSC) exhibit beneficial properties to serve as cellular vehicles for enzyme/prodrug cancer gene therapy approaches. We have previously shown that engineered human adipose tissue-derived MSC in combination with non-toxic prodrug mediated substantial cytotoxic and antitumor effect. The aim of this study was to develop advanced 3D cultivation method to serve for modelling of the therapeutic outcome in vitro. We have used engineered MSC expressing fusion transgene cytosine deaminase::uracilphosphoribosyltransferase (CD-MSC) in combination with prodrug 5-fluorocytosine (5FC). This therapeutic regimen designated CD-MSC/5FC was combined with the human melanoma cells A375 or EGFP-A375 in both standard monolayer culture and 3-dimensional (3D) multicellular nodules. The extent of cytotoxicity was evaluated by standard viability assay MTS, ATP-based luminescence assay, fluorimetric test, measurement of Caspase-3/7 activation and DNA laddering. The data have shown that the extent of cytotoxic bystander effect mediated by CD-MSC/5FC is significantly lower in 3D culture conditions. However, these data better recapitulate the therapeutic efficiency as observed previously in vivo. We suggest here to use the 3D multicellular culture conditions for better prediction of the therapeutic outcome in mouse xenograft models.

Noncanonical Role of Telomerase in Regulation of Microvascular Redox Environment With Implications for Coronary Artery Disease.

Telomerase reverse transcriptase (TERT) (catalytic subunit of telomerase) is linked to the development of coronary artery disease (CAD); however, whether the role of nuclear vs. mitchondrial actions of TERT is involved is not determined. Dominant-negative TERT splice variants contribute to decreased mitochondrial integrity and promote elevated reactive oxygen species production. We hypothesize that a decrease in mitochondrial TERT would increase mtDNA damage, promoting a pro-oxidative redox environment. The goal of this study is to define whether mitochondrial TERT is sufficient to maintain nitric oxide as the underlying mechanism of flow-mediated dilation by preserving mtDNA integrity.Immunoblots and quantitative polymerase chain reaction were used to show elevated levels of splice variants α- and ß-deletion TERT tissue from subjects with and without CAD. Genetic, pharmacological, and molecular tools were used to manipulate TERT localization. Isolated vessel preparations and fluorescence-based quantification of mtH2O2 and NO showed that reduction of TERT in the nucleus increased flow induced NO and decreased mtH2O2 levels, while prevention of mitochondrial import of TERT augmented pathological effects. Further elevated mtDNA damage was observed in tissue from subjects with CAD and initiation of mtDNA repair mechanisms was sufficient to restore NO-mediated dilation in vessels from patients with CAD. The work presented is the first evidence that catalytically active mitochondrial TERT, independent of its nuclear functions, plays a critical physiological role in preserving NO-mediated vasodilation and the balance of mitochondrial to nuclear TERT is fundamentally altered in states of human disease that are driven by increased expression of dominant negative splice variants.

Comparing Articaine brands: A randomized non-inferiority controlled trial.

INTRODUCTION: The substitution of reference drugs for similar, new or existing drugs in the market is a dilemma present in daily dental procedures. In order to decide whether or not to adopt a new drug in relation to the reference, a clinical trial can provide the necessary evidence. METHODS: A total of 179 healthy volunteers (18-25 years) requiring extraction of mandibular third molars completed the study. Subjects were randomized into 4 groups: right, left, Arteek-SP and Septanest. Allocations consisted of 4% Articaine hydrochloride with 1:10000 epinephrine. The primary aim of this randomized controlled trial was to determine whether the test drug Arteek-SP was noninferior by a margin of 10% in the proportion of patients with total absence of pain, compared to the reference drug Septanest in the extraction of mandibular third molars using inferior alveolar nerve blocks. Secondary outcomes included, hemodynamic parameters, volume (mL), pain scores assessed during one visit and reported postoperative discomfort assessed during 8 days. Noninferiority was declared if total absence of pain for both groups was equal to 0.9, with a significance level of 5% (P < 0.05) and power at 90% (ß = 0.1) at 95% confidence level. RESULTS: Arteek-SP was applied during the first surgery to 90 volunteers (50.28%) and Septanest was applied to 89 subjects (49.72%). Less than 10% difference was identified, in the proportion of patients with total absence of tooth pain P < 0.05 and in the gum P < 0.1, at 95% CI, when Arteek-SP was applied first in comparison with Septanest, establishing noninferiority. CONCLUSION: The clinical performance of the test drug Arteek-SP is noninferior to the reference drug Septanest. They can be considered interchangeable in terms of cost or convenience. REGISTRATION: ClinicalTrials.org, number NCT4166890.

Ca2+- and thromboxane-dependent distribution of MaxiK channels in cultured astrocytes: from microtubules to the plasma membrane.

Large-conductance, voltage- and Ca2+-activated K+ channels (MaxiK) are broadly expressed ion channels minimally assembled by four pore-forming alpha-subunits (MaxiKalpha) and typically observed as plasma membrane proteins in various cell types. In murine astrocyte primary cultures, we show that MaxiKalpha is predominantly confined to the microtubule network. Distinct microtubule distribution of MaxiKalpha was visualized by three independent labeling approaches: (1) MaxiKalpha-specific antibodies, (2) expressed EGFP-labeled MaxiKalpha, and (3) fluorophore-conjugated iberiotoxin, a specific MaxiK pore-blocker. This MaxiKalpha association with microtubules was further confirmed by in vitro His-tag pulldown, co-immunoprecipitation from brain lysates, and microtubule depolymerization experiments. Changes in intracellular Ca2+ elicited by general pharmacological agents, caffeine or thapsigargin, resulted in increased MaxiKalpha labeling at the plasma membrane. More notably, U46619, an analog of thromboxane A2 (TXA2), which triggers Ca2+-release pathways and whose levels increase during cerebral hemorrhage/trauma, also elicits a similar increase in MaxiKalpha surface labeling. Whole-cell patch clamp recordings of U46619-stimulated cells develop a approximately 3-fold increase in current amplitude indicating that TXA2 stimulation results in the recruitment of additional, functional MaxiK channels to the surface membrane. While microtubules are largely absent in mature astrocytes, immunohistochemistry results in brain slices show that cortical astrocytes in the newborn mouse (P1) exhibit a robust expression of microtubules that significantly colocalize with MaxiK. The results of this study provide the novel insight that suggests that Ca2+ released from intracellular stores may play a key role in regulating the traffic of intracellular, microtubule-associated MaxiK stores to the plasma membrane of developing murine astrocytes.

Decreased event-related theta power and phase-synchrony in young binge drinkers during target detection: An anatomically-constrained MEG approach.

BACKGROUND: The prevalence of binge drinking has risen in recent years. It is associated with a range of neurocognitive deficits among adolescents and young emerging adults who are especially vulnerable to alcohol use. Attention is an essential dimension of executive functioning and attentional disturbances may be associated with hazardous drinking. The aim of the study was to examine the oscillatory neural dynamics of attentional control during visual target detection in emerging young adults as a function of binge drinking. METHOD: In total, 51 first-year university students (18 ± 0.6 years) were assigned to light drinking ( n = 26), and binge drinking ( n = 25) groups based on their alcohol consumption patterns. A high-density magnetoencephalography signal was combined with structural magnetic resonance imaging in an anatomically constrained magnetoencephalography model to estimate event-related source power in a theta (4-7 Hz) frequency band. Phase-locked co-oscillations were further estimated between the principally activated regions during task performance. RESULTS: Overall, the greatest event-related theta power was elicited by targets in the right inferior frontal cortex and it correlated with performance accuracy and selective attention scores. Binge drinkers exhibited lower theta power and dysregulated oscillatory synchrony to targets in the right inferior frontal cortex, which correlated with higher levels of alcohol consumption. CONCLUSIONS: These results confirm that a highly interactive network in the right inferior frontal cortex subserves attentional control, revealing the importance of theta oscillations and neural synchrony for attentional capture and contextual maintenance. Attenuation of theta power and synchronous interactions in binge drinkers may indicate early stages of suboptimal integrative processing in young, highly functioning binge drinkers.

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.

Chemical treatment of olive pomace: effect on acid-basic properties and metal biosorption capacity.

In this study, olive pomace, an agricultural waste that is very abundant in Mediterranean area, was modified by two chemical treatments in order to improve its biosorption capacity. Potentiometric titrations and IR analyses were used to characterise untreated olive pomace (OP), olive pomace treated by phosphoric acid (PAOP) and treated by hydrogen peroxide (HPOP). Acid-base properties of all investigated biosorbents were characterised by two main kinds of active sites, whose nature and concentration were determined by a mechanistic model assuming continuous distribution for the proton affinity constants. Titration modelling denoted that all investigated biosorbents (OP, PAOP and HPOP) were characterised by the same kinds of active sites (carboxylic and phenolic), but with different total concentrations with PAOP richer than OP and HPOP. Single metal equilibrium studies in batch reactors were carried out to determine the capacity of these sorbents for copper and cadmium ions at constant pH. Experimental data were analysed and compared using the Langmuir isotherm. The order of maximum uptake capacity of copper and cadmium ions on different biosorbents was PAOP>HPOP>OP. The maximum adsorption capacity of copper and cadmium, was obtained as 0.48 and 0.10 mmol/g, respectively, for PAOP. Metal biosorption tests in presence of Na(+) in solution were also carried out in order to evaluate the effect of chemical treatment on biomass selectivity. These data showed that PAOP is more selective for cadmium than the other sorbents, while similar selectivity was observed for copper.

Lymphoma transformation of tumor infiltrating lymphocytes observed in testicular patient­derived xenograft models.

Testicular germ cell tumors (TGCTs) are highly sensitive to cisplatin­based chemotherapy. Nevertheless, there are metastatic tumors that do not completely respond to front­line chemotherapy. For these tumors, surgical resection of residual masses is necessary to achieve long­term disease control. Resected tissues represent valuable clinical material, which may be used for the engraftment into immunocompromised mice to produce patient­derived xenografts (PDXs). They typically maintain similarities to the parental tumors and therefore serve as more realistic preclinical models. Moreover, a correlation between PDX treatment outcomes and clinical response to chemotherapy has been previously described. The aim of the present study was to establish and characterize TGCT patient­derived xenografts. These originated from retroperitoneal lymph node metastases infiltrated with TGCTs following previous cisplatin­based chemotherapy, in order to analyze novel treatment options for cisplatin­resistant testicular tumors. We generated two testicular patient­derived xenograft models in SCID beige male mice. Immunohistochemical analyses demonstrated that histological characteristics of the primary tumor were not retained, and transformation into lymphoma, and eventually plasmocytoma, was observed. A potential explanation for the lymphoma transformation observed in PDXs may include tumor­infiltrating lymphocytes (TILs) in xenografted samples of patients, which are transformed following engraftment into immunodeficient recipient mice. Based on these data, we indicated that lymphomagenesis prevention and terminal differentiation represent new challenges in the establishment of PDX models derived from patients with germ cell tumors.

Endocytic trafficking signals in KCNMB2 regulate surface expression of a large conductance voltage and Ca(2+)-activated K+ channel.

Large conductance voltage and calcium-activated K(+) channels play critical roles in neuronal excitability and vascular tone. Previously, we showed that coexpression of the transmembrane beta2 subunit, KCNMB2, with the human pore-forming alpha subunit of the large conductance voltage and Ca(2+)-activated K(+) channel (hSlo) yields inactivating currents similar to those observed in hippocampal neurons [Hicks GA, Marrion NV (1998) Ca(2+)-dependent inactivation of large conductance Ca(2+)-activated K(+) (BK) channels in rat hippocampal neurones produced by pore block from an associated particle. J Physiol (Lond) 508 (Pt 3):721-734; Wallner M, Meera P, Toro L (1999b) Molecular basis of fast inactivation in voltage and Ca(2+)-activated K(+) channels: A transmembrane beta-subunit homolog. Proc Natl Acad Sci U S A 96:4137-4142]. Herein, we report that coexpression of beta2 subunit with hSlo can also modulate hSlo surface expression levels in HEK293T cells. We found that, when expressed alone, beta2 subunit appears to reach the plasma membrane but also displays a distinct intracellular punctuated pattern that resembles endosomal compartments. beta2 Subunit coexpression with hSlo causes two biological effects: i) a shift of hSlo's intracellular expression pattern from a relatively diffuse to a distinct punctated cytoplasmic distribution overlapping beta2 expression; and ii) a decrease of hSlo surface expression that surpassed an observed small decrease in total hSlo expression levels. beta2 Site-directed mutagenesis studies revealed two putative endocytic signals at the C-terminus of beta2 that can control expression levels of hSlo. In contrast, a beta2 N-terminal consensus endocytic signal had no effect on hSlo expression levels. Thus, beta2 subunit not only can influence hSlo currents but also has the ability to limit hSlo surface expression levels via an endocytic mechanism. This new mode of beta2 modulation of hSlo may depend on particular coregulatory mechanisms in different cell types.

Toxic elements at a disused mine district: Particle size distribution and total concentration in stream sediments and mine tailings.

Heavy metal and metalloid pollution at a disused pyrite mine was investigated. Five solid samples collected in the area (three stream sediments with different soil texture, a background soil sample and a mine tailing) were characterised by mineral and element composition, particle size distribution (by wet and dry sieving and laser diffraction) and total concentration by acid digestion (Mn, Zn, Cu, Pb, Hg, Cd, Sb and As). X-ray and element analyses denoted a common mineralogical and chemical composition of mainly quartz, clinochlore, muscovite, anorthite, and hematite. Particle size distributions of the five samples showed that stream sediments were characterised by larger percentage of sand range classes (2000-60 microm) while background sample and tailing are mainly characterised by gravel particles (>2000 microm). Wet and dry sieving procedures gave different particle size distributions, which can be interpreted by laser diffraction analysis and represented by Rosin-Rammler model. Concentrations of Zn, Cu and Cd were higher in the stream sediments than the tailing and background soil, while Mn, As, Sb and Hg are mainly concentrated in the tailing sample. Metal concentrations in the three stream sediment samples are correlated with both particle size dimensions (D(63.2)) and concentration of geochemical normalizers (iron and aluminium). These correlations are observed also for the pollutants that are mainly concentrated in tailing sample (Mn and As), denoting the importance of surface interactions also for the binding of these elements onto stream sediments.

Effectiveness of environmental control measures to decrease the risk of invasive aspergillosis in acute leukaemia patients during hospital building work.

BACKGROUND: Invasive aspergillosis (IA) is a significant problem in acute leukaemia patients. Construction work near hospital wards caring for immunocompromised patients is one of the main risk factors for developing invasive pulmonary aspergillosis (IPA). AIM: To assess the impact of environmental control measures used during hospital construction for the prevention of IA in acute leukaemia patients. METHODS: A retrospective cohort study was developed to evaluate the IA incidence in acute leukaemia patients with different environmental control measures employed during hospital construction. We used European Organisation for the Research and Treatment of Cancer (EORTC) criterial diagnosis parameters for definition of IA. FINDINGS: A total of 175 episodes of inpatient care were evaluated, 62 of which did not have any environmental control measures (when an outbreak occurred), and 113 that were subject to environmental control measures directed to preventing IA. The study showed an IA incidence of 25.8% for the group without environmental control measures vs 12.4% for those who did receive environmental control measures (P=0.024). The relative risk for IA was 0.595 (95% confidence interval: 0.394-0.897) for the group with environmental control measures. CONCLUSION: The current study suggests that the implementation of environmental control measures during a hospital construction has a positive impact for prevention of IA in patients hospitalized with acute leukaemia.

KCNMB1 regulates surface expression of a voltage and Ca2+-activated K+ channel via endocytic trafficking signals.

Voltage-dependent and calcium-activated K(+) (MaxiK, BK) channels are ubiquitously expressed and have various physiological roles including regulation of neurotransmitter release and smooth muscle tone. Coexpression of the pore-forming alpha (hSlo) subunit of MaxiK channels with a regulatory beta1 subunit (KCNMB1) produces noninactivating currents that are distinguished by high voltage/Ca(2+) sensitivities and altered pharmacology [McManus OB, Helms LM, Pallanck L, Ganetzky B, Swanson R, Leonard RJ (1995) Functional role of the beta subunit of high conductance calcium-activated potassium channels. Neuron 14:645-650; Wallner M, Meera P, Ottolia M, Kaczorowski G, Latorre R, Garcia ML, Stefani E, Toro L (1995) Characterization of and modulation by a beta-subunit of a human maxi K(Ca) channel cloned from myometrium. Receptors Channels 3:185-199]. We now show that beta1 can regulate hSlo traffic as well, resulting in decreased hSlo surface expression. beta1 subunit expressed alone is able to reach the plasma membrane; in addition, it exhibits a distinct intracellular punctated pattern that colocalizes with an endosomal marker. Coexpressing beta1 subunit with hSlo, switches hSlo's rather diffuse intracellular expression to a punctate cytoplasmic localization that overlaps beta1 expression. Furthermore, coexpressed beta1 subunit reduces steady-state hSlo surface expression. Site-directed mutagenesis underscores a role of a putative endocytic signal at the beta1 C-terminus in the control of hSlo surface expression. We propose that aside from its well-established role as regulator of hSlo electrical activity, beta1 can regulate hSlo expression levels by means of an endocytic mechanism. This highlights a new beta1 subunit feature that regulates hSlo channels by a trafficking mechanism.

Decreased expression of voltage- and Ca(2+)-activated K(+) channels in coronary smooth muscle during aging.

Aging is the main risk factor for coronary artery disease. One characteristic of aging coronary arteries is their enhanced contractile responses to endothelial vasoconstricting factors, which increase the risk of coronary vasospasm in older people. Because large-conductance voltage- and Ca(2+)-activated K(+) channels (MaxiK) are key regulators of vascular tone, we explored the possibility that this class of channels is diminished with increasing age. Using site-directed antibodies recognizing the pore-forming alpha subunit and electrophysiological methods, we demonstrate that the number of MaxiK channels is dramatically diminished in aged coronary arteries from old F344 rats. Channel density was reduced from 52+/-9 channels/pF (3 months old) to 18+/-5 channels/pF (25 to 30 months old), which represents a 65% reduction in the older population. Pixel intensity of Western blots was also diminished by approximately 50%. Moreover, the age-related decrease in the channel protein expression was also evident in humans, which showed approximately 80% reduction in 61- to 70-year-old subjects compared with 3- to 18-year-old youngsters and approximately 45% reduction compared with 19- to 56-year-old adults. In agreement with a reduction of MaxiK channel numbers in aging coronary arteries, old coronary arteries from F344 rats contract less effectively ( approximately 70% reduction) than young coronary arteries when exposed to the MaxiK channel blocker iberiotoxin. The contraction studies indicate that under physiological conditions, MaxiK channels are tonically active, serving as a hyperpolarizing force that opposes contraction. Thus, reduced expression of MaxiK channels in aged coronary arteries would lead to a decreased vasodilating capacity and increased risk of coronary spasm and myocardial ischemia in older people.

Ionic strength effect on copper biosorption by Sphaerotilus natans: equilibrium study and dynamic modelling in membrane reactor.

Biosorption of copper by Sphaerotilus natans in different conditions of ionic strength and pH was studied by performing sorption tests in batch and membrane reactors. Equilibrium batch tests evidenced the negative effect of ionic strength and the positive effect of pH on biosorption performances: the highest determined value for copper specific uptake, q, was about 60 mg/g at pH 6 and about 15 mg/g at pH 4. A competitive equilibrium model was successfully fitted to experimental data at different ionic strength levels to account for copper-sodium competition. In membrane reactor tests, experimental profiles of copper concentration in the permeate vs. time did not evidence a significant effect of ionic strength at low pH values (4 and 5). On the other hand a more remarkable effect of ionic strength on copper concentration in the permeate was observed at pH 6. Experimental profiles of continuous biosorption in the membrane reactors were successfully simulated by developing a dynamic model accounting for Cu-Na competition and for binding ability of cells fragments.

MaxiK channel interactome reveals its interaction with GABA transporter 3 and heat shock protein 60 in the mammalian brain.

Large conductance voltage and calcium-activated potassium (MaxiK) channels are activated by membrane depolarization and elevated cytosolic Ca(2+). In the brain, they localize to neurons and astrocytes, where they play roles such as resetting the membrane potential during an action potential, neurotransmitter release, and neurovascular coupling. MaxiK channels are known to associate with several modulatory proteins and accessory subunits, and each of these interactions can have distinct physiological consequences. To uncover new players in MaxiK channel brain physiology, we applied a directed proteomic approach and obtained MaxiK channel pore-forming α subunit brain interactome using specific antibodies. Controls included immunoprecipitations with rabbit immunoglobulin G (IgG) and with anti-MaxiK antibodies in wild type and MaxiK channel knockout mice (Kcnma1(-/-)), respectively. We have found known and unreported interactive partners that localize to the plasma membrane, extracellular space, cytosol and intracellular organelles including mitochondria, nucleus, endoplasmic reticulum and Golgi apparatus. Localization of MaxiK channel to mitochondria was further confirmed using purified brain mitochondria colabeled with MitoTracker. Independent proof of MaxiK channel interaction with previously unidentified partners is given for GABA transporter 3 (GAT3) and heat shock protein 60 (HSP60). In human embryonic kidney 293 cells containing SV40 T-antigen (HEK293T) cells, both GAT3 and HSP60 coimmunoprecipitated and colocalized with MaxiK channel; colabeling was observed mainly at the cell periphery with GAT3 and intracellularly with HSP60 with protein proximity indices of ∼ 0.6 and ∼ 0.4, respectively. In rat primary hippocampal neurons, colocalization index was identical for GAT3 (∼ 0.6) and slightly higher for HSP60 (∼ 0.5) association with MaxiK channel. The results of this study provide a complete interactome of MaxiK channel the mouse brain, further establish the localization of MaxiK channel in the mouse brain mitochondria and demonstrate the interaction of MaxiK channel with GAT3 and HSP60 in neurons. The interaction of MaxiK channel with GAT3 opens the possibility of a role of MaxiK channel in GABA homeostasis and signaling.

GTP-dependent regulation of myometrial KCa channels incorporated into lipid bilayers.

The regulation of calcium-activated K (KCa) channels by a G protein-mediated mechanism was studied. KCa channels were reconstituted in planar lipid bilayers by fusion of membrane vesicles from rat or pig myometrium. The regulatory process was studied by exploring the actions of GTP and GTP gamma S on single channel activity. KCa channels had a conductance of 260 +/- 6 pS (n = 25, +/- SE, 250/50 mM KCl gradient) and were voltage dependent. The open probability (Po) vs. voltage relationships were well fit by a Boltzmann distribution. The slope factor (11 mV) was insensitive to internal Ca2+. The half activation potential (V1/2) was shifted -70 mV by raising internal Ca2+ from pCa 6.2 to pCa 4. Addition of GTP or GTP gamma S activated channel activity only in the presence of Mg2+, a characteristic typical of G protein-mediated mechanisms. The Po increased from 0.18 +/- 0.08 to 0.49 +/- 0.07 (n = 7, 0 mV, pCa 6 to 6.8). The channel was also activated (Po increased from 0.03 to 0.37) in the presence of AMP-PNP, a nonphosphorylating ATP analogue, suggesting a direct G protein gating of KCa channels. Upon nucleotide activation, mean open time increased by a factor of 2.7 +/- 0.7 and mean closed time decreased by 0.2 +/- 0.07 of their initial values (n = 6). Norepinephrine (NE) or isoproterenol potentiated the GTP-mediated activation of KCa channels (Po increased from 0.17 +/- 0.06 to 0.35 +/- 0.07, n = 10). These results suggest that myometrium possesses beta-adrenergic receptors coupled to a GTP-dependent protein that can directly gate KCa channels. Furthermore, KCa channels, beta-adrenergic receptors, and G proteins can be reconstituted in lipid bilayers as a stable, functionally coupled, molecular complex.

Correlation between charge movement and ionic current during slow inactivation in Shaker K+ channels.

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.

Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle.

We have studied potassium currents through a cloned Ca(2+)-dependent K+ channel (hslo) from human myometrium. Currents were recorded in inside-out macropatches from membranes of Xenopus laevis oocytes. In particular, the inactivation-like process that these channels show at high positive potentials was assessed in order to explore its molecular nature. This current inhibition conferred a bell shape to the current-voltage curves. The kinetic and voltage dependence of this process suggested the possibility of a Ba2+ block. There were the following similarities between the inactivation process observed at zero-added Ba2+ and the internal Ba2+ block of hslo channels: (a) in the steady state, the voltage dependence of the current inhibition observed at zero-added Ba2+ was the same as the voltage dependence of the Ba2+ block; (b) the time constant for recovery from current decay at zero-added Ba2+ was the same as the time constant for current recovery from Ba2+ blockade; and (c) current decay was largely suppressed in both cases by adding a Ba2+ chelator [(+)-18-crown-6-tetracarboxylic acid] to the internal solution. In our experimental conditions, we determined that the Kd for the complex chelator-Ba2+ is 1.6 x 10(-10) M. We conclude that the current decay observed at zero-added Ba2+ to the internal solution is due to contaminant Ba2+ present in our solutions (approximately 70 nM) and not to an intrinsic gating process. The Ba2+ blocking reaction in hslo channels is bimolecular. Ba2+ binds to a site (Kd = 0.36 +/- 0.05 mM at zero applied voltage) that senses 92 +/- 25% of the potential drop from the internal membrane surface.