Assessment of the operational characteristics of the National Comprehensive Cancer Network distress management tool, v. 2.2018, in patients seen at the Instituto Nacional de Cancerología, Bogotá / Evaluación de las características operativas de la versión 2.2018 del instrumento de evaluación del malestar emocional de la National Comprehensive Cancer Network en pacientes atendidos en el Instituto Nacional de Cancerología, Bogotá

Introduction: Cancer patients have significant levels of emotional distress. The National Comprehensive Cancer Network (NCCN) developed the distress management tool to quickly assess significant distress in oncological patients who require intervention. For its use in Colombia, we made its cross-cultural adaptation and validation. Objective: To determine the operative characteristics of the distress management tool, version 2.2018, in patients seen at the Instituto Nacional de Cancerología (INC) in Colombia. Materials and methods: Counting with the authorization from the NCCN, we translated, made the cross-cultural adaptation, and evaluated the operational characteristics of the tool. We included 343 cancer patients seen at the INC, who filled out the cross-culturally adapted instrument. A diagnostic test study was carried out with a semi-structured interview as a reference. Results: The patients had an average age of 49.7 years (SD=15) and the majority were women (67%). The instrument had an area under the ROC curve of 0.81 (95% CI: 0.77 -0.86); its optimal cut-off point was 3.5 approached to 4 when using integers on the scale; its sensitivity was 0.81 (95% CI: 0.76 - 0.85), and its specificity, 0.69 (95% CI: 0.64 - 0.74). The agreement percentage between the result of the interview and the instrument was 73% (kappa = 0.64; p< 0.001). Conclusions: The distress management tool allowed for the detection of moderate to severe distress requiring intervention and management. This instrument was adapted and validated in cancer patients in Colombia keeping the cutoff point at ≥ 4 as in the original version.

Introducción. Los pacientes con cáncer presentan niveles significativos de malestar emocional. La National Comprehensive Cancer Network (NCCN) desarrolló un instrumento (Distress Management) para evaluarlo de forma rápida en pacientes oncológicos. Para su utilización en Colombia, se hizo la adaptación transcultural y se validó. Objetivo. Determinar las características operativas del instrumento de malestar emocional, versión 2.2018, en pacientes atendidos en el Instituto Nacional de Cancerología. Materiales y métodos. Previa autorización de la NCCN, se procedió a la traducción, adaptación transcultural y evaluación de las características operativas del instrumento. Se incluyeron 343 pacientes con diagnóstico de cáncer atendidos en el Instituto Nacional de Cancerología, quienes diligenciaron el instrumento adaptado transculturalmente. Se efectuó un estudio de prueba diagnóstica como patrón de referencia mediante una entrevista semiestructurada. Resultados. Los pacientes tenían una edad promedio de 49,7 años (DE=15) y la mayoría (67 %) eran mujeres. El instrumento tuvo un área bajo la curva ROC de 0,81 (IC95% 0,77-0,86); el punto de corte óptimo fue de 3,5, el cual se aproximó a 4; la sensibilidad fue de 0,81 (IC95% 0,76-0,85) y la especificidad de 0,69 (IC95% 0,64-0,74). El porcentaje de acuerdo entre el resultado de la entrevista y el instrumento fue de 73 % (kappa=0,64; p<0,001). Conclusiones. El instrumento de malestar emocional permitió detectar el malestar emocional moderado a grave que requiere intervención y manejo. Este instrumento fue adaptado y validado en pacientes con cáncer en Colombia, conservándose el punto de corte en ≥4 como en la versión original.

Evaluación de las características operativas de la versión 2. 2018 del instrumento de evaluación del malestar emocional de la National Comprehensive Cancer Network en pacientes atendidos en el Instituto Nacional de Cancerología, Bogotá / Assessment of the operational characteristics of the National Comprehensive Cancer Network distress management tool, v. 2.2018, in patients seen at the Instituto Nacional de Cancerología, Bogotá

Resumen | Introducción. Los pacientes con cáncer presentan niveles significativos de malestar emocional. La National Comprehensive Cancer Network (NCCN) desarrolló un instrumento (Distress Management) para evaluarlo de forma rápida en pacientes oncológicos. Para su utilización en Colombia, se hizo la adaptación transcultural y se validó. Objetivo. Determinar las características operativas del instrumento de malestar emocional, versión 2.2018, en pacientes atendidos en el Instituto Nacional de Cancerología. Materiales y métodos. Previa autorización de la NCCN, se procedió a la traducción, adaptación transcultural y evaluación de las características operativas del instrumento. Se incluyeron 343 pacientes con diagnóstico de cáncer atendidos en el Instituto Nacional de Cancerología, quienes diligenciaron el instrumento adaptado transculturalmente. Se efectuó un estudio de prueba diagnóstica como patrón de referencia mediante una entrevista semiestructurada. Resultados. Los pacientes tenían una edad promedio de 49,7 años (DE=15) y la mayoría (67 %) eran mujeres. El instrumento tuvo un área bajo la curva ROC de 0,81 (IC95% 0,77-0,86); el punto de corte óptimo fue de 3,5, el cual se aproximó a 4; la sensibilidad fue de 0,81 (IC95% 0,76-0,85) y la especificidad de 0,69 (IC95% 0,64-0,74). El porcentaje de acuerdo entre el resultado de la entrevista y el instrumento fue de 73 % (kappa=0,64; p<0,001). Conclusiones. El instrumento de malestar emocional permitió detectar el malestar emocional moderado a grave que requiere intervención y manejo. Este instrumento fue adaptado y validado en pacientes con cáncer en Colombia, conservándose el punto de corte en ≥4 como en la versión original.

Abstract | Introduction: Cancer patients have significant levels of emotional distress. The National Comprehensive Cancer Network (NCCN) developed the distress management tool to quickly assess significant distress in oncological patients who require intervention. For its use in Colombia, we made its cross-cultural adaptation and validation. Objective: To determine the operative characteristics of the distress management tool, version 2.2018, in patients seen at the Instituto Nacional de Cancerología (INC) in Colombia. Materials and methods: Counting with the authorization from the NCCN, we translated, made the cross-cultural adaptation, and evaluated the operational characteristics of the tool. We included 343 cancer patients seen at the INC, who filled out the cross-culturally adapted instrument. A diagnostic test study was carried out with a semi-structured interview as a reference. Results: The patients had an average age of 49.7 years (SD=15) and the majority were women (67%). The instrument had an area under the ROC curve of 0.81 (95% CI: 0.77 - 0.86); its optimal cut-off point was 3.5 approached to 4 when using integers on the scale; its sensitivity was 0.81 (95% CI: 0.76 - 0.85), and its specificity, 0.69 (95% CI: 0.64 - 0.74). The agreement percentage between the result of the interview and the instrument was 73% (kappa = 0.64; p< 0.001). Conclusions: The distress management tool allowed for the detection of moderate to severe distress requiring intervention and management. This instrument was adapted and validated in cancer patients in Colombia keeping the cutoff point at ≥ 4 as in the original version.

Adaptación transcultural al español de la escala Distress Management del NCCN versión 2. 2018 para su utilización en pacientes oncológicos en Colombia. / Transcultural adaptation to spanish of the Distress Management NCCN scale version 2.2018 for use in cancer patients in Colombia

Resumen Antecedentes: Los pacientes con diagnóstico de cáncer presentan niveles significativos de malestar emocional relacionados con la enfermedad oncológica. El termómetro de malestar emocional (Distress Management) es una herramienta diseñada por la National Comprehensive Cáncer Network (NCCN) para la medición del malestar emocional que requiere atención psicosocial en pacientes con cáncer como parte de los procesos usuales en la atención. Objetivo: Traducir y adaptar transculturalmente al español colombiano la versión en inglés de la escala Distress Management del NCCN versión 2.2018. Métodos: El proceso de traducción y adaptación transcultural se desarrolló siguiendo las recomendaciones metodológicas del grupo de calidad de vida EORTC (European Organization for Research and Treatment of Cáncer), realizando una traducción inicial, traducción inversa y una prueba piloto donde participaron 10 pacientes atendidos en el Instituto Nacional de Cancerología. Resultados: Se hizo la traducción de la escala Distress Management del NCCN versión 2.2018 encontrándose algunas discrepancias en la traducción directa e inversa, llegando a un consenso en cada etapa del proceso. En la prueba piloto, la escala fue respondida entre 5 a 9 minutos y se presentó dificultad en la comprensión de 6 ítems de la lista de problemas. Se decidió adicionar "cansancio" al ítem de fatiga, "psicoactivas" al ítem de uso de sustancias y realizar una explicación de las instrucciones previa a su diligenciamiento. Conclusión: Se realizó la adaptación transcultural de la escala Distress a una versión en español colombiano. Esta versión puede utilizarse para determinar sus propiedades psicométricas al aplicarse a pacientes con cáncer en Colombia.

Abstract Background: Patients diagnosed with cancer have significant levels of emotional distress related with oncological disease. The Distress Management is a tool designed by the NCCN (The National Comprehensive Cancer Network) for the measurement of emotional distress that requires psychosocial care in patients with cancer as part of the usual processes in care. Objective: Translate and cross-cultural comparison the English version of the Distress Management scale of NCCN version 2.2018 to Colombian Spanish. Methods: The transcultural translation and adaptation process was developed following the methodological recommendations of the EORTC quality of life group (European Organization for Research and Treatment of Cancer), performing an initial translation, inverse translation and a pilot test involving 10 patients treated at the National Institute of Cancerology Bogotá. Results: The translation of the thermometer was carried out, finding some discrepancies in the direct and inverse translation, reaching a consensus in each stage of the process. In the pilot test, the scale was answered between 5 to 9 minutes, there was difficulty in understanding 6 items in the list of problems. It was decided to add "tiredness" to the item of fatigue, "psychoactive" to the item of substance use and to make an explanation of the instructions prior to the patients reading and filling the instrument. Conclusion: The transcultural adaptation of the Distress Management of the NCCN version 2.2018 was made to a Colombian Spanish version. This version can be used to determine the psychometric properties when applied to cancer patients in Colombia.

MitoBKCa channel is functionally associated with its regulatory ß1 subunit in cardiac mitochondria.

KEY POINTS: Association of plasma membrane BKCa channels with BK-ß subunits shapes their biophysical properties and physiological roles; however, functional modulation of the mitochondrial BKCa channel (mitoBKCa ) by BK-ß subunits is not established. MitoBKCa -α and the regulatory BK-ß1 subunit associate in mouse cardiac mitochondria. A large fraction of mitoBKCa display properties similar to that of plasma membrane BKCa when associated with BK-ß1 (left-shifted voltage dependence of activation, V1/2  = -55 mV, 12 µm matrix Ca2+ ). In BK-ß1 knockout mice, cardiac mitoBKCa displayed a low Po and a depolarized V1/2 of activation (+47 mV at 12 µm matrix Ca2+ ) Co-expression of BKCa with the BK-ß1 subunit in HeLa cells doubled the density of BKCa in mitochondria. The present study supports the view that the cardiac mitoBKCa channel is functionally modulated by the BK-ß1 subunit; proper targeting and activation of mitoBKCa shapes mitochondrial Ca2+ handling. ABSTRACT: Association of the plasma membrane BKCa channel with auxiliary BK-ß1-4 subunits profoundly affects the regulatory mechanisms and physiological processes in which this channel participates. However, functional association of mitochondrial BK (mitoBKCa ) with regulatory subunits is unknown. We report that mitoBKCa functionally associates with its regulatory subunit BK-ß1 in adult rodent cardiomyocytes. Cardiac mitoBKCa is a calcium- and voltage-activated channel that is sensitive to paxilline with a large conductance for K+ of 300 pS. Additionally, mitoBKCa displays a high open probability (Po ) and voltage half-activation (V1/2  = -55 mV, n = 7) resembling that of plasma membrane BKCa when associated with its regulatory BK-ß1 subunit. Immunochemistry assays demonstrated an interaction between mitochondrial BKCa -α and its BK-ß1 subunit. Mitochondria from the BK-ß1 knockout (KO) mice showed sparse mitoBKCa currents (five patches with mitoBKCa activity out of 28 total patches from n = 5 different hearts), displaying a depolarized V1/2 of activation (+47 mV in 12 µm matrix Ca2+ ). The reduced activity of mitoBKCa was accompanied by a high expression of BKCa transcript in the BK-ß1 KO, suggesting a lower abundance of mitoBKCa channels in this genotype. Accordingly, BK-ß1subunit increased the localization of BKDEC (i.e. the splice variant of BKCa that specifically targets mitochondria) into mitochondria by two-fold. Importantly, both paxilline-treated and BK-ß1 KO mitochondria displayed a more rapid Ca2+ overload, featuring an early opening of the mitochondrial transition pore. We provide strong evidence that mitoBKCa associates with its regulatory BK-ß1 subunit in cardiac mitochondria, ensuring proper targeting and activation of the mitoBKCa channel that helps to maintain mitochondrial Ca2+ homeostasis.

The mitochondrial BKCa channel cardiac interactome reveals BKCa association with the mitochondrial import receptor subunit Tom22, and the adenine nucleotide translocator.

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.

Skeletal muscle action of estrogen receptor α is critical for the maintenance of mitochondrial function and metabolic homeostasis in females.

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.

Rescue of Pressure Overload-Induced Heart Failure by Estrogen Therapy.

BACKGROUND: Estrogen 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 RESULTS: HF 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<0.05) were significantly reduced in HF. Estrogen therapy increased cardiac estrogen 3-fold and restored aromatase transcripts. Estrogen also rescued HF by restoring ejection fraction to 53.1±1.3% (P<0.001) and improving cardiac hemodynamics both in male and female mice. Estrogen therapy stimulated angiogenesis as capillary density increased from 0.66±0.07 in HF to 2.83±0.14 (P<0.001, normalized to control) and reversed the fibrotic scarring observed in HF (45.5±2.8% in HF versus 5.3±1.0%, P<0.001). Stimulation of angiogenesis by estrogen seems to be one of the key mechanisms, since in the presence of an angiogenesis inhibitor estrogen failed to rescue HF (ejection fraction=29.3±2.1%, P<0.001 versus E2). CONCLUSIONS: Estrogen rescues pre-existing HF by restoring cardiac estrogen and aromatase, stimulating angiogenesis, and suppressing fibrosis.

Resonant Scanning with Large Field of View Reduces Photobleaching and Enhances Fluorescence Yield in STED Microscopy.

Photobleaching is a major limitation of superresolution Stimulated Depletion Emission (STED) microscopy. Fast scanning has long been considered an effective means to reduce photobleaching in fluorescence microscopy, but a careful quantitative study of this issue is missing. In this paper, we show that the photobleaching rate in STED microscopy can be slowed down and the fluorescence yield be enhanced by scanning with high speed, enabled by using large field of view in a custom-built resonant-scanning STED microscope. The effect of scanning speed on photobleaching and fluorescence yield is more remarkable at higher levels of depletion laser irradiance, and virtually disappears in conventional confocal microscopy. With ≥6 GW∙cm(-2) depletion irradiance, we were able to extend the fluorophore survival time of Atto 647N and Abberior STAR 635P by ~80% with 8-fold wider field of view. We confirm that STED Photobleaching is primarily caused by the depletion light acting upon the excited fluorophores. Experimental data agree with a theoretical model. Our results encourage further increasing the linear scanning speed for photobleaching reduction in STED microscopy.

Resonant-scanning dual-color STED microscopy with ultrafast photon counting: A concise guide.

STED (stimulated emission depletion) is a popular super-resolution fluorescence microscopy technique. In this paper, we present a concise guide to building a resonant-scanning STED microscope with ultrafast photon-counting acquisition. The STED microscope has two channels, using a pulsed laser and a continuous-wave (CW) laser as the depletion laser source, respectively. The CW STED channel preforms time-gated detection to enhance optical resolution in this channel. We use a resonant mirror to attain high scanning speed and ultrafast photon counting acquisition to scan a large field of view, which help reduce photobleaching. We discuss some practical issues in building a STED microscope, including creating a hollow depletion beam profile, manipulating polarization, and monitoring optical aberration. We also demonstrate a STED image enhancement method using stationary wavelet expansion and image analysis methods to register objects and to quantify colocalization in STED microscopy.

BK Channels Alleviate Lysosomal Storage Diseases by Providing Positive Feedback Regulation of Lysosomal Ca2+ Release.

Promoting lysosomal trafficking represents a promising therapeutic approach for lysosome storage diseases. Efficient Ca(2+) mobilization from lysosomes is important for lysosomal trafficking. Ca(2+) release from lysosomes could generate a negative potential in the lumen to disturb subsequent Ca(2+) release in the absence of counter ion flux. Here we report that lysosomes express big-conductance Ca(2+)-activated potassium (BK) channels that form physical and functional coupling with the lysosomal Ca(2+) release channel, TRPML1. Ca(2+) release via TRPML1 causes BK activation, which in turn facilitates further lysosomal Ca(2+) release and membrane trafficking. Importantly, BK overexpression rescues the impaired TRPML1-mediated Ca(2+) release and abnormal lysosomal storage in cells from Niemann-Pick C1 patients. Therefore, we have identified a lysosomal K(+) channel that provides a positive feedback mechanism to facilitate TRPML1-mediated Ca(2+) release and membrane trafficking. Our findings suggest that upregulating BK may be a potential therapeutic strategy for certain lysosomal storage diseases and common neurodegenerative disorders.

Mitochondrial BKCa channel.

Since its discovery in a glioma cell line 15 years ago, mitochondrial BKCa channel (mitoBKCa) has been studied in brain cells and cardiomyocytes sharing general biophysical properties such as high K(+) conductance (~300 pS), voltage-dependency and Ca(2+)-sensitivity. Main advances in deciphering the molecular composition of mitoBKCa have included establishing that it is encoded by the Kcnma1 gene, that a C-terminal splice insert confers mitoBKCa ability to be targeted to cardiac mitochondria, and evidence for its potential coassembly with ß subunits. Notoriously, ß1 subunit directly interacts with cytochrome c oxidase and mitoBKCa can be modulated by substrates of the respiratory chain. mitoBKCa channel has a central role in protecting the heart from ischemia, where pharmacological activation of the channel impacts the generation of reactive oxygen species and mitochondrial Ca(2+) preventing cell death likely by impeding uncontrolled opening of the mitochondrial transition pore. Supporting this view, inhibition of mitoBKCa with Iberiotoxin, enhances cytochrome c release from glioma mitochondria. Many tantalizing questions remain open. Some of them are: how is mitoBKCa coupled to the respiratory chain? Does mitoBKCa play non-conduction roles in mitochondria physiology? Which are the functional partners of mitoBKCa? What are the roles of mitoBKCa in other cell types? Answers to these questions are essential to define the impact of mitoBKCa channel in mitochondria biology and disease.

A strategy to improve treatment-related mortality and abandonment of therapy for childhood ALL in a developing country reveals the impact of treatment delays.

BACKGROUND: Treatment-related mortality and abandonment of therapy are major barriers to successful treatment of childhood acute lymphoblastic leukemia (ALL) in the developing world. PROCEDURE: A collaboration was undertaken between Instituto Nacional de Cancerologia (Bogota, Colombia), which serves a poor patient population in an upper-middle income country, and Dana-Farber/Boston Children's Cancer and Blood Disorders Center (Boston, USA). Several interventions aimed at reducing toxic deaths and abandonment were implemented, including a reduced-intensity treatment regimen and a psychosocial effort targeting abandonment. We performed a cohort study to assess impact. RESULTS: The Study Population comprised 99 children with ALL diagnosed between 2007 and 2010, and the Historic Cohort comprised 181 children treated prior to the study interventions (1995-2004). Significant improvements were achieved in the rate of deaths in complete remission (13% to 3%; P = 0.005), abandonment (32% to 9%; P < 0.001), and event-free survival with abandonment considered an event (47% to 65% at 2 years; P = 0.016). However, relapse rate did not improve. Medically unnecessary treatment delays were common, and landmark analysis revealed that initiating the PIII phase of therapy ≥4 weeks delayed predicted markedly inferior disease-free survival (P = 0.016). Conversely, patients who received therapy without excessive delays had outcomes approaching those achieved in high-income countries. CONCLUSIONS: Implementation of a twinning program was followed by reductions in abandonment and toxic deaths, but relapse rate did not improve. Inappropriate treatment delays were common and strongly predicted treatment failure. These findings highlight the importance of adherence to treatment schedule for effective therapy of ALL.

Regulation of transcriptional activation function of rat estrogen receptor α (ERα) by novel C-terminal splice inserts.

Estrogen receptor α (ERα) mediates estrogen diverse actions on tissues. ERα gene has eight constitutively expressing exons and is known to have multiple isoforms generated by alternative initiation of transcription and splicing events including exon skipping. We have discovered two novel exons inserted between exon 5 and 6 of rat ERα that can add independently or in tandem 18 and 14 amino acids to the estrogen binding/activator function 2 domain of the receptor. Their transcript expression is three to six fold higher in heart compared to brain, aorta, liver, ovary and uterus. In heart, the new variants increased ~2 fold with animal growth from prenatal to adulthood, and had a minor increment in aged animals (28 months). Inclusion of these exons yields a receptor with practically no binding capacity for estrogen and reduced dimerization. The new variants show nuclear localization but are less efficient in binding to estrogen responsive elements (EREs) and failed to transcriptionally activate promoters containing EREs (mSlo, KCNE2). Thus, the new variants can regulate the wild-type receptor function and may contribute to the regulatory action of estrogen, especially in the maturing heart where they are more abundant.

Ultrafast photon counting applied to resonant scanning STED microscopy.

To take full advantage of fast resonant scanning in super-resolution stimulated emission depletion (STED) microscopy, we have developed an ultrafast photon counting system based on a multigiga sample per second analogue-to-digital conversion chip that delivers an unprecedented 450 MHz pixel clock (2.2 ns pixel dwell time in each scan). The system achieves a large field of view (∼50 × 50 µm) with fast scanning that reduces photobleaching, and advances the time-gated continuous wave STED technology to the usage of resonant scanning with hardware-based time-gating. The assembled system provides superb signal-to-noise ratio and highly linear quantification of light that result in superior image quality. Also, the system design allows great flexibility in processing photon signals to further improve the dynamic range. In conclusion, we have constructed a frontier photon counting image acquisition system with ultrafast readout rate, excellent counting linearity, and with the capacity of realizing resonant-scanning continuous wave STED microscopy with online time-gated detection.

Both transmembrane domains of BK ß1 subunits are essential to confer the normal phenotype of ß1-containing BK channels.

Voltage/Ca²âº(i)-gated, large conductance K+ (BK) channels result from tetrameric association of α (slo1) subunits. In most tissues, BK protein complexes include regulatory ß subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK ß types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK ß modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK ß1 drastically increases the channel's apparent Ca²âº(i) sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK ß1 regions responsible for the characteristic phenotype of ß1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant ß1, neuron-abundant ß4, or chimeras constructed by swapping ß1 and ß4 regions, and determined the contribution of specific ß1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from ß1 and the remaining regions ("background") from ß4 showed a phenotype (V(half), τ(act), τ(deact)) identical to that of complexes containing wt ß1. This phenotype could not be evoked by complexes that included chimeras combining either ß1 TM1 or ß1 TM2 with a ß4 background. Likewise, ß "halves" (each including ß1 TM1 or ß1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between ß1 TMs via the EC loop is also necessary for proper channel function.

The angiotensin II type 1 receptor (AT1R) closely interacts with large conductance voltage- and Ca2+-activated K+ (BK) channels and inhibits their activity independent of G-protein activation.

Angiotensin II (ANG-II) and BK channels play important roles in the regulation of blood pressure. In arterial smooth muscle, ANG-II inhibits BK channels, but the underlying molecular mechanisms are unknown. Here, we first investigated whether ANG-II utilizes its type 1 receptor (AT1R) to modulate BK activity. Pharmacological, biochemical, and molecular evidence supports a role for AT1R. In renal arterial myocytes, the AT1R antagonist losartan (10 µM) abolished the ANG-II (1 µM)-induced reduction of whole cell BK currents, and BK channels and ANG-II receptors were found to co-localize at the cell periphery. We also found that BK inhibition via ANG-II-activated AT1R was independent of G-protein activation (assessed with 500 µM GDPßS). In BK-expressing HEK293T cells, ANG-II (1 µM) also induced a reduction of BK currents, which was contingent on AT1R expression. The molecular mechanisms of AT1R and BK channel coupling were investigated in co-transfected cells. Co-immunoprecipitation showed formation of a macromolecular complex, and live immunolabeling demonstrated that both proteins co-localized at the plasma membrane with high proximity indexes as in arterial myocytes. Consistent with a close association, we discovered that the sole AT1R expression could decrease BK channel voltage sensitivity. Truncated BK proteins revealed that the voltage-sensing conduction cassette is sufficient for BK-AT1R association. Finally, C-terminal yellow and cyan fluorescent fusion proteins, AT1R-YFP and BK-CFP, displayed robust co-localized Förster resonance energy transfer, demonstrating intermolecular interactions at their C termini. Overall, our results strongly suggest that AT1R regulates BK channels through a close protein-protein interaction involving multiple BK regions and independent of G-protein activation.

MaxiK channel and cell signalling.

The large-conductance Ca2+- and voltage-activated K+ (MaxiK, BK, BKCa, Slo1, KCa1.1) channel role in cell signalling is becoming apparent as we learn how the channel interacts with a multiplicity of proteins not only at the plasma membrane but also in intracellular organelles including the endoplasmic reticulum, nucleus, and mitochondria. In this review, we focus on the interactions of MaxiK channels with seven-transmembrane G protein-coupled receptors and discuss information suggesting that, the channel big C-terminus may act as the nucleus of signalling molecules including kinases relevant for cell death and survival. Increasing evidence indicates that the channel is able to associate with a variety of receptors including ß-adrenergic receptors, G protein-coupled estrogen receptors, acetylcholine receptors, thromboxane A2 receptors, and angiotensin II receptors, which highlights the varied functions that the channel has (or may have) not only in regulating contraction/relaxation of muscle cells or neurotransmission in the brain but also in cell metabolism, proliferation, migration, and gene expression. In line with this view, MaxiK channels have been implicated in obesity and in brain, prostate, and mammary cancers. A better understanding on the molecular mechanisms underlying or triggered by MaxiK channel abnormalities like overexpression in certain cancers may lead to new therapeutics to prevent devastating diseases.

MitoBK(Ca) is encoded by the Kcnma1 gene, and a splicing sequence defines its mitochondrial location.

The large-conductance Ca(2+)- and voltage-activated K(+) channel (BK(Ca), MaxiK), which is encoded by the Kcnma1 gene, is generally expressed at the plasma membrane of excitable and nonexcitable cells. However, in adult cardiomyocytes, a BK(Ca)-like channel activity has been reported in the mitochondria but not at the plasma membrane. The putative opening of this channel with the BK(Ca) agonist, NS1619, protects the heart from ischemic insult. However, the molecular origin of mitochondrial BK(Ca) (mitoBK(Ca)) is unknown because its linkage to Kcnma1 has been questioned on biochemical and molecular grounds. Here, we unequivocally demonstrate that the molecular correlate of mitoBK(Ca) is the Kcnma1 gene, which produces a protein that migrates at ∼140 kDa and arranges in clusters of ∼50 nm in purified mitochondria. Physiological experiments further support the origin of mitoBK(Ca) as a Kcnma1 product because NS1619-mediated cardioprotection was absent in Kcnma1 knockout mice. Finally, BKCa transcript analysis and expression in adult cardiomyocytes led to the discovery of a 50-aa C-terminal splice insert as essential for the mitochondrial targeting of mitoBK(Ca).

The ß1-subunit of the MaxiK channel associates with the thromboxane A2 receptor and reduces thromboxane A2 functional effects.

The large conductance voltage- and Ca(2+)-activated K(+) channel (MaxiK, BK(Ca), BK) is composed of four pore-forming α-subunits and can be associated with regulatory ß-subunits. One of the functional roles of MaxiK is to regulate vascular tone. We recently found that the MaxiK channel from coronary smooth muscle is trans-inhibited by activation of the vasoconstricting thromboxane A(2) prostanoid receptor (TP), a mechanism supported by MaxiK α-subunit (MaxiKα)-TP physical interaction. Here, we examined the role of the MaxiK ß1-subunit in TP-MaxiK association. We found that the ß1-subunit can by itself interact with TP and that this association can occur independently of MaxiKα. Subcellular localization analysis revealed that ß1 and TP are closely associated at the cell periphery. The molecular mechanism of ß1-TP interaction involves predominantly the ß1 extracellular loop. As reported previously, TP activation by the thromboxane A(2) analog U46619 caused inhibition of MaxiKα macroscopic conductance or fractional open probability (FP(o)) as a function of voltage. However, the positive shift of the FP(o) versus voltage curve by U46619 relative to the control was less prominent when ß1 was coexpressed with TP and MaxiKα proteins (20 ± 6 mV, n = 7) than in cells expressing TP and MaxiKα alone (51 ± 7 mV, n = 7). Finally, ß1 gene ablation reduced the EC(50) of the U46619 agonist in mediating aortic contraction from 18 ± 1 nm (n = 12) to 9 ± 1 nm (n = 12). The results indicate that the ß1-subunit can form a tripartite complex with TP and MaxiKα, has the ability to associate with each protein independently, and diminishes U46619-induced MaxiK channel trans-inhibition as well as vasoconstriction.

Intracellular BK(Ca) (iBK(Ca)) channels.

The large conductance calcium- and voltage-activated potassium channel (BK(Ca)) is widely expressed at the plasma membrane. This channel is involved in a variety of fundamental cellular functions including excitability, smooth muscle contractility, and Ca(2+) homeostasis, as well as in pathological situations like proinflammatory responses in rheumatoid arthritis, and cancer cell proliferation. Immunochemical, biochemical and pharmacological studies from over a decade have intermittently shown the presence of BK(Ca) in intracellular organelles. To date, intracellular BK(Ca) (iBK(Ca)) has been localized in the mitochondria, endoplasmic reticulum, nucleus and Golgi apparatus but its functional role remains largely unknown except for the mitochondrial BK(Ca) whose opening is thought to play a role in protecting the heart from ischaemic injury. In the nucleus, pharmacology suggests a role in regulating nuclear Ca(2+), membrane potential and eNOS expression. Establishing the molecular correlates of iBK(Ca), the mechanisms defining iBK(Ca) organelle-specific targeting, and their modulation are challenging questions. This review summarizes iBK(Ca) channels, their possible functions, and efforts to identify their molecular correlates.