Loss of sodium current caused by a Brugada syndrome-associated variant is determined by patient-specific genetic background.

BACKGROUND: Brugada syndrome (BrS) is an inherited cardiac arrhythmogenic disease that predisposes patients to sudden cardiac death. It is associated with mutations in SCN5A, which encodes the cardiac sodium channel alpha subunit (NaV1.5). BrS-related mutations have incomplete penetrance and variable expressivity within families. OBJECTIVE: The purpose of this study was to determine the role of patient-specific genetic background on the cellular and clinical phenotype among carriers of NaV1.5_p.V1525M. METHODS: We studied sodium currents from patient-specific human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and heterologously transfected human embryonic kidney (HEK) tsA201 cells using the whole-cell patch-clamp technique. We determined gene and protein expression by quantitative polymerase chain reaction, RNA sequencing, and western blot and performed a genetic panel for arrhythmogenic diseases. RESULTS: Our results showed a large reduction in INa density in hiPSC-CM derived from 2 V1525M single nucleotide variant (SNV) carriers compared with hiPSC-CM derived from a noncarrier, suggesting a dominant-negative effect of the NaV1.5_p.V1525M channel. INa was not affected in hiPSC-CMs derived from a V1525M SNV carrier who also carries the NaV1.5_p.H558R polymorphism. Heterozygous expression of V1525M in HEK-293T cells produced a loss of INa function, not observed when this variant was expressed together with H558R. In addition, the antiarrhythmic drug mexiletine rescued INa function in hiPSC-CM. SCN5A expression was increased in the V1525M carrier who also expresses NaV1.5_p.H558R. CONCLUSION: Our results in patient-specific hiPSC-CM point to a dominant-negative effect of NaV1.5_p.V1525M, which can be reverted by the presence of NaV1.5_p.H558R. Overall, our data points to a role of patient-specific genetic background as a determinant for incomplete penetrance in BrS.

Generation of the induced pluripotent stem cell line ESi108-A from a familial atrial fibrillation patient.

Tissue-specific cells differentiated from patient-derived human induced pluripotent stem cells (hiPSC) are a relevant cellular model to study several diseases. We obtained a hiPSC line from skin fibroblasts of a patient affected by familial atrial fibrillation by nucleofection of non-integrating episomal vectors. The resulting hiPSC line displays a normal karyotype, expresses pluripotency surface markers and pluripotency genes, and differentiates into cells of the 3 germ layers. Therefore, it represents a reliable model to study the disease in a physiologically relevant cellular environment.

Alterations in Calcium Handling Are a Common Feature in an Arrhythmogenic Cardiomyopathy Cell Model Triggered by Desmosome Genes Loss.

Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium. Deleterious variants in desmosomal genes are the main cause of ACM and lead to common and gene-specific molecular alterations, which are not yet fully understood. This article presents the first systematic in vitro study describing gene and protein expression alterations in desmosomes, electrical conduction-related genes, and genes involved in fibrosis and adipogenesis. Moreover, molecular and functional alterations in calcium handling were also characterized. This study was performed d with HL1 cells with homozygous knockouts of three of the most frequently mutated desmosomal genes in ACM: PKP2, DSG2, and DSC2 (generated by CRISPR/Cas9). Moreover, knockout and N-truncated clones of DSP were also included. Our results showed functional alterations in calcium handling, a slower calcium re-uptake was observed in the absence of PKP2, DSG2, and DSC2, and the DSP knockout clone showed a more rapid re-uptake. We propose that the described functional alterations of the calcium handling genes may be explained by mRNA expression levels of ANK2, CASQ2, ATP2A2, RYR2, and PLN. In conclusion, the loss of desmosomal genes provokes alterations in calcium handling, potentially contributing to the development of arrhythmogenic events in ACM.

Generation of four induced pluripotent stem cell lines from a family harboring a single nucleotide variant in SCN5A.

Patient-derived induced pluripotent stem cells (iPSC) are a valuable approach to model cardiovascular diseases. We nucleofected non-integrating episomal vectors in skin fibroblasts of three family members carrying a single nucleotide variant (SNV) in SCN5A, which encodes the cardiac-type sodium channel, and of a related healthy control. The SNV SCN5A_c.4573G > A had been previously identified in a Brugada Syndrome patient. The resulting iPS cell lines differentiate into cells of the 3 germ layers, display normal karyotypes and express pluripotency surface markers and genes. Thus, they are a reliable source to study the effect of the identified mutation in a physiologically relevant environment.

Generation of an induced pluripotent stem cell line from a healthy Caucasian male.

The effects of genetic mutations on protein function can be studied in a physiologically relevant environment using tissue-specific cells differentiated from patient-derived induced pluripotent stem cells (iPSC). However, it is crucial to use iPSC derived from healthy individuals as control. We generated an iPS cell line from skin fibroblasts of a healthy Caucasian male by nucleofection of non-integrating episomal vectors. This cell line has normal karyotype, expresses pluripotency surface markers and pluripotency genes, and successfully differentiates into cells of the 3 germ layers. Therefore, it can be used as control for any disease of interest that is modelled using iPSC.

Epigenetic Changes Governing Scn5a Expression in Denervated Skeletal Muscle.

The SCN5A gene encodes the α-subunit of the voltage-gated cardiac sodium channel (NaV1.5), a key player in cardiac action potential depolarization. Genetic variants in protein-coding regions of the human SCN5A have been largely associated with inherited cardiac arrhythmias. Increasing evidence also suggests that aberrant expression of the SCN5A gene could increase susceptibility to arrhythmogenic diseases, but the mechanisms governing SCN5A expression are not yet well understood. To gain insights into the molecular basis of SCN5A gene regulation, we used rat gastrocnemius muscle four days following denervation, a process well known to stimulate Scn5a expression. Our results show that denervation of rat skeletal muscle induces the expression of the adult cardiac Scn5a isoform. RNA-seq experiments reveal that denervation leads to significant changes in the transcriptome, with Scn5a amongst the fifty top upregulated genes. Consistent with this increase in expression, ChIP-qPCR assays show enrichment of H3K27ac and H3K4me3 and binding of the transcription factor Gata4 near the Scn5a promoter region. Also, Gata4 mRNA levels are significantly induced upon denervation. Genome-wide analysis of H3K27ac by ChIP-seq suggest that a super enhancer recently described to regulate Scn5a in cardiac tissue is activated in response to denervation. Altogether, our experiments reveal that similar mechanisms regulate the expression of Scn5a in denervated muscle and cardiac tissue, suggesting a conserved pathway for SCN5A expression among striated muscles.

An SCN1B Variant Affects Both Cardiac-Type (NaV1.5) and Brain-Type (NaV1.1) Sodium Currents and Contributes to Complex Concomitant Brain and Cardiac Disorders.

Voltage-gated sodium (NaV) channels are transmembrane proteins that initiate and propagate neuronal and cardiac action potentials. NaV channel ß subunits have been widely studied due to their modulatory role. Mice null for Scn1b, which encodes NaV ß1 and ß1b subunits, have defects in neuronal development and excitability, spontaneous generalized seizures, cardiac arrhythmias, and early mortality. A mutation in exon 3 of SCN1B, c.308A>T leading to ß1_p.D103V and ß1b_p.D103V, was previously found in a patient with a history of proarrhythmic conditions with progressive atrial standstill as well as cognitive and motor deficits accompanying structural brain abnormalities. We investigated whether ß1 or ß1b subunits carrying this mutation affect NaV1.5 and/or NaV1.1 currents using a whole cell patch-clamp technique in tsA201 cells. We observed a decrease in sodium current density in cells co-expressing NaV1.5 or NaV1.1 and ß1D103V compared to ß1WT. Interestingly, ß1bD103V did not affect NaV1.1 sodium current density but induced a positive shift in the voltage dependence of inactivation and a faster recovery from inactivation compared to ß1bWT. The ß1bD103V isoform did not affect NaV1.5 current properties. Although the SCN1B_c.308A>T mutation may not be the sole cause of the patient's symptoms, we observed a clear loss of function in both cardiac and brain sodium channels. Our results suggest that the mutant ß1 and ß1b subunits play a fundamental role in the observed electrical dysfunction.

Sudden infant death as the most severe phenotype caused by genetic modulation in a family with atrial fibrillation.

AIMS: To assess the functional impact of two combined KCNH2 variants involved in atrial fibrillation, syncope and sudden infant death syndrome. METHODS AND RESULTS: Genetic testing of a 4-month old SIDS victim identified a rare missense heterozygous in KCNH2 variant (V483I) and a missense homozygous polymorphism (K897T) which is often described as a genetic modifier. Electrophysiological characterisation of heterologous HERG channels representing two different KCNH2 genotypes within the family, showed significant differences in both voltage and time dependence of activation and inactivation with a global gain-of-function effect of mutant versus wild type channels and, also, differences between both types of recombinant channels. CONCLUSIONS: The rare variant V483I in combination with K897T produces a gain-of-function effect that represents a pathological substrate for atrial fibrillation, syncope and sudden infant death syndrome events in this family. Ascertaining the genotype-phenotype correlation of genetic variants is imperative for the correct assessment of genetic testing and counselling. TRANSLATIONAL PERSPECTIVE: According to the current guidelines for clinical interpretation of sequence variants, functional studies are an essential tool for the ascertainment of variant pathogenicity. They are especially relevant in the context of sudden infant death syndrome and sudden cardiac death, where individuals cannot be clinically evaluated. The patch-clamp technique is a gold-standard for analysis of the biophysical mechanisms of ion channels.

Extra Virgin Olive Oil Contains a Phenolic Inhibitor of the Histone Demethylase LSD1/KDM1A.

The lysine-specific histone demethylase 1A (LSD1) also known as lysine (K)-specific demethylase 1A (KDM1A) is a central epigenetic regulator of metabolic reprogramming in obesity-associated diseases, neurological disorders, and cancer. Here, we evaluated the ability of oleacein, a biophenol secoiridoid naturally present in extra virgin olive oil (EVOO), to target LSD1. Molecular docking and dynamic simulation approaches revealed that oleacein could target the binding site of the LSD1 cofactor flavin adenosine dinucleotide with high affinity and at low concentrations. At higher concentrations, oleacein was predicted to target the interaction of LSD1 with histone H3 and the LSD1 co-repressor (RCOR1/CoREST), likely disturbing the anchorage of LSD1 to chromatin. AlphaScreen-based in vitro assays confirmed the ability of oleacein to act as a direct inhibitor of recombinant LSD1, with an IC50 as low as 2.5 µmol/L. Further, oleacein fully suppressed the expression of the transcription factor SOX2 (SEX determining Region Y-box 2) in cancer stem-like and induced pluripotent stem (iPS) cells, which specifically occurs under the control of an LSD1-targeted distal enhancer. Conversely, oleacein failed to modify ectopic SOX2 overexpression driven by a constitutive promoter. Overall, our findings provide the first evidence that EVOO contains a naturally occurring phenolic inhibitor of LSD1, and support the use of oleacein as a template to design new secoiridoid-based LSD1 inhibitors.

Experimental Models of Brugada syndrome.

Brugada syndrome is an inherited, rare cardiac arrhythmogenic disease, associated with sudden cardiac death. It accounts for up to 20% of sudden deaths in patients without structural cardiac abnormalities. The majority of mutations involve the cardiac sodium channel gene SCN5A and give rise to classical abnormal electrocardiogram with ST segment elevation in the right precordial leads V1 to V3 and a predisposition to ventricular fibrillation. The pathophysiological mechanisms of Brugada syndrome have been investigated using model systems including transgenic mice, canine heart preparations, and expression systems to study different SCN5A mutations. These models have a number of limitations. The recent development of pluripotent stem cell technology creates an opportunity to study cardiomyocytes derived from patients and healthy individuals. To date, only a few studies have been done using Brugada syndrome patient-specific iPS-CM, which have provided novel insights into the mechanisms and pathophysiology of Brugada syndrome. This review provides an evaluation of the strengths and limitations of each of these model systems and summarizes the key mechanisms that have been identified to date.

Sodium channel current loss of function in induced pluripotent stem cell-derived cardiomyocytes from a Brugada syndrome patient.

Brugada syndrome predisposes to sudden death due to disruption of normal cardiac ion channel function, yet our understanding of the underlying cellular mechanisms is incomplete. Commonly used heterologous expression models lack many characteristics of native cardiomyocytes and, in particular, the individual genetic background of a patient. Patient-specific induced pluripotent stem (iPS) cell-derived cardiomyocytes (iPS-CM) may uncover cellular phenotypical characteristics not observed in heterologous models. Our objective was to determine the properties of the sodium current in iPS-CM with a mutation in SCN5A associated with Brugada syndrome. Dermal fibroblasts from a Brugada syndrome patient with a mutation in SCN5A (c.1100G>A, leading to Nav1.5_p.R367H) were reprogrammed to iPS cells. Clones were characterized and differentiated to form beating clusters and sheets. Patient and control iPS-CM were structurally indistinguishable. Sodium current properties of patient and control iPS-CM were compared. These results were contrasted with those obtained in tsA201 cells heterologously expressing sodium channels with the same mutation. Patient-derived iPS-CM showed a 33.1-45.5% reduction in INa density, a shift in both activation and inactivation voltage-dependence curves, and faster recovery from inactivation. Co-expression of wild-type and mutant channels in tsA201 cells did not compromise channel trafficking to the membrane, but resulted in a reduction of 49.8% in sodium current density without affecting any other parameters. Cardiomyocytes derived from iPS cells from a Brugada syndrome patient with a mutation in SCN5A recapitulate the loss of function of sodium channel current associated with this syndrome; including pro-arrhythmic changes in channel function not detected using conventional heterologous expression systems.

Competencia clínica de médicos de seguridad social guatemaltecos para manejar hepatitis virales en atención primaria / Clinical competences for human viral hepatitis treatment of primary healthcare physicians from the Guatemalan social security system

RESUMEN Objetivo Medir la competencia clínica para el diagnóstico y manejo de hepatitis virales en médicos de primer nivel de atención a la salud. Metodología Se efectuó un estudio transversal en el que usando un instrumento previamente validado se midió la competencia y posteriormente se comparó entre médicos adscritos a diversas unidades médicas de atención primaria a la salud (UMAPS) del Instituto Guatemalteco de Seguridad Social (IGSS). La información fue analizada mediante estadística descriptiva e inferencial no paramétrica. Se evaluaron 104 médicos de 5 UMAPS del IGSS. Resultados Se encontró un nivel muy bajo de competencia clínica para el diagnóstico y tratamiento de las hepatitis virales, dentro de un intervalo de 9 a 62 puntos obtenidos en el instrumento que tiene un valor máximo teórico de 88, sin encontrar diferencias estadísticamente significativas entre UMAPS. Conclusiones: Se requiere educación continua en los médicos de las UMAPS del IGSS para mejorar sus competencias en hepatitis virales.(AU)

ABSTRACT Objective To measure the clinical competence for diagnosis and treatment of human viral hepatitis in primary health care physicians. Methodology Cross-sectional study in which a previously validated instrument to measure competences was used, and subsequent comparison between physicians at various primary health care units (PHCT) from the Guatemalan Institute of Social Security (GISS). This information was analyzed using descriptive and non-parametrical statistics. 104 physicians, from 5 PHCT ascribed to GISS were analyzed. Results A low level of clinical competence for diagnosis and treatment of human viral hepatitis in this physicians group was found, within a range of 9 to 62 points obtained through an instrument with a maximum theoretical value of 88; no significant statistical difference between PHCT was found. Conclusions PHCT physicians from require continuing education to improve their clinical competence on human viral hepatitis.(AU)

[Clinical competences for human viral hepatitis treatment of primary healthcare physicians from the Guatemalan social security system]. / Competencia clínica de médicos de seguridad social guatemaltecos para manejar hepatitis virales en atención primaria.

OBJECTIVE: To measure the clinical competence for diagnosis and treatment of human viral hepatitis in primary health care physicians. METHODOLOGY: Cross-sectional study in which a previously validated instrument to measure competences was used, and subsequent comparison between physicians at various primary health care units (PHCT) from the Guatemalan Institute of Social Security (GISS). This information was analyzed using descriptive and non-parametrical statistics. 104 physicians, from 5 PHCT ascribed to GISS were analyzed. RESULTS: A low level of clinical competence for diagnosis and treatment of human viral hepatitis in this physicians group was found, within a range of 9 to 62 points obtained through an instrument with a maximum theoretical value of 88; no significant statistical difference between PHCT was found. CONCLUSIONS: PHCT physicians from require continuing education to improve their clinical competence on human viral hepatitis.

OBJETIVO: Medir la competencia clínica para el diagnóstico y manejo de hepatitis virales en médicos de primer nivel de atención a la salud. METODOLOGÍA: Se efectuó un estudio transversal en el que usando un instrumento previamente validado se midió la competencia y posteriormente se comparó entre médicos adscritos a diversas unidades médicas de atención primaria a la salud (UMAPS) del Instituto Guatemalteco de Seguridad Social (IGSS). La información fue analizada mediante estadística descriptiva e inferencial no paramétrica. Se evaluaron 104 médicos de 5 UMAPS del IGSS. RESULTADOS: Se encontró un nivel muy bajo de competencia clínica para el diagnóstico y tratamiento de las hepatitis virales, dentro de un intervalo de 9 a 62 puntos obtenidos en el instrumento que tiene un valor máximo teórico de 88, sin encontrar diferencias estadísticamente significativas entre UMAPS. Conclusiones: Se requiere educación continua en los médicos de las UMAPS del IGSS para mejorar sus competencias en hepatitis virales.

Clinical and molecular characterization of a cardiac ryanodine receptor founder mutation causing catecholaminergic polymorphic ventricular tachycardia.

BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a difficult-to-diagnose cause of sudden cardiac death (SCD). We identified a family of 1400 individuals with multiple cases of CPVT, including 36 SCDs during youth. OBJECTIVES: We sought to identify the genetic cause of CPVT in this family, to preventively treat and clinically characterize the mutation-positive individuals, and to functionally characterize the pathogenic mechanisms of the mutation. METHODS: Genetic testing was performed for 1404 relatives. Mutation-positive individuals were preventively treated with ß-blockers and clinically characterized with a serial exercise treadmill test (ETT) and Holter monitoring. In vitro functional studies included caffeine sensitivity and store overload-induced calcium release activity of the mutant channel in HEK293 cells. RESULTS: We identified the p.G357S_RyR2 mutation, in the cardiac ryanodine receptor, in 179 family members and in 6 SCD cases. No SCD was observed among treated mutation-positive individuals over a median follow-up of 37 months; however, 3 relatives who had refused genetic testing (confirmed mutation-positive individuals) experienced SCD. Holter monitoring did not provide relevant information for CPVT diagnosis. One single ETT was unable to detect complex cardiac arrhythmias in 72% of mutation-positive individuals, though the serial ETT improved the accuracy. Functional studies showed that the G357S mutation increased caffeine sensitivity and store overload-induced calcium release activity under conditions that mimic catecholaminergic stress. CONCLUSION: Our study supports the use of genetic testing to identify individuals at risk of SCD to undertake prophylactic interventions. We also show that the pathogenic mechanisms of p.G357S_RyR2 appear to depend on ß-adrenergic stimulation.

A missense mutation in the sodium channel ß1b subunit reveals SCN1B as a susceptibility gene underlying long QT syndrome.

BACKGROUND: Long QT syndrome (LQTS) is associated with sudden cardiac death and the prolongation of the QT interval on the electrocardiogram. A comprehensive screening of all genes previously associated with this disease leaves 30% of the patients without a genetic diagnosis. Pathogenic mutations in the sodium channel ß subunits have been associated with cardiac channelopathies, including SCN4B mutations in LQTS. OBJECTIVE: To evaluate the role of mutations in the sodium channel ß subunits in LQTS. METHODS: We screened for mutations in the genes encoding the 5 sodium ß subunits (SCN1B isoforms a and b, SCN2B, SCN3B, and SCN4B) from 30 nonrelated patients who were clinically diagnosed with LQTS without mutations in common LQTS-related genes. We used the patch-clamp technique to study the properties of sodium currents and the action potential duration in human embryonic kidney and HL-1 cells, respectively, in the presence of ß1b subunits. RESULTS: The genetic screening revealed a novel mutation in the SCN1Bb gene (ß1bP213T) in an 8-year-old boy. Our electrophysiological analysis revealed that ß1bP213T increases late sodium current. In addition, ß1bP213T subtly altered Nav1.5 function by shifting the window current, accelerating recovery from inactivation, and decreasing the slow inactivation rate. Moreover, experiments using HL-1 cells revealed that the action potential duration significantly increases when the mutant ß1b was overexpressed compared with ß1bWT. CONCLUSION: These data revealed SCN1Bb as a susceptibility gene responsible for LQTS, highlighting the importance of continuing the search for new genes and mechanisms to decrease the percentage of patients with LQTS remaining without genetic diagnosis.

The smooth muscle-type ß1 subunit potentiates activation by DiBAC4(3) in recombinant BK channels.

Large-conductance Ca(2+)-activated (BK) channels, expressed in a variety of tissues, play a fundamental role in regulating and maintaining arterial tone. We recently demonstrated that the slow voltage indicator DiBAC4(3) does not depend, as initially proposed, on the ß 1 or ß 4 subunits to activate native arterial smooth muscle BK channels. Using recombinant mslo BK channels, we now show that the ß 1 subunit is not essential to this activation but exerts a large potentiating effect. DiBAC4(3) promotes concentration-dependent activation of BK channels and slows deactivation kinetics, changes that are independent of Ca(2+). Kd values for BK channel activation by DiBAC4(3) in 0 mM Ca(2+) are approximately 20 µM (α) and 5 µM (α+ß 1), and G-V curves shift up to -40 mV and -110 mV, respectively. ß1 to ß2 mutations R11A and C18E do not interfere with the potentiating effect of the subunit. Our findings should help refine the role of the ß 1 subunit in cardiovascular pharmacology.

Complete home smoking bans and antitobacco contingencies: a natural experiment.

INTRODUCTION: The California antitobacco culture may have influenced home smoking bans in Mexico. Based on the Behavioral Ecological Model, exposure to socially reinforcing contingencies or criticism may explain adoption of home smoking bans in Tijuana, Mexico, approximating rates relative to San Diego, California, and higher than those in Guadalajara, Mexico. METHODS: A representative cross-sectional population survey of Latinos (N = 1,901) was conducted in San Diego, Tijuana, and Guadalajara between June 2003 and September 2004. Cities were selected to represent high-, medium-, and low-level exposure to antitobacco social contingencies of reinforcement in a quasiexperimental analysis of possible cultural influences across borders. RESULTS: Complete home smoking ban prevalence was 91% in San Diego, 66% in Tijuana, and 38% in Guadalajara (p < .001). Sample cluster-adjusted logistic regression showed significantly lower odds of complete home smoking bans in Guadalajara (odds ratio [OR] = .048) and in Tijuana (OR = .138) compared to San Diego after control for demographics. Odds of complete home smoking bans in both Guadalajara and Tijuana in comparison with San Diego were weakened when mediators for bans were controlled in predictive models. Direction of association was consistent with theory. When theoretical mediators were explored as possible moderators, weak and nonsignificant associations were obtained for all interaction terms. Bootstrap analyses demonstrated that our multivariable logistic regression results were reliable. CONCLUSIONS: Results suggest that California antismoking social contingencies mediate complete home smoking bans in all 3 cities and may account for the greater effects in Tijuana contrasted with Guadalajara.

Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel.

The α-subunit of the cardiac voltage-gated sodium channel (NaV1.5) plays a central role in cardiomyocyte excitability. We have recently reported that NaV1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate NaV1.5, and to describe the role of arginine methylation on NaV1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate NaV1.5 in vitro, interact with NaV1.5 in human embryonic kidney (HEK) cells, and increase NaV1.5 current density by enhancing NaV1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.

Molecular heterogeneity of large-conductance calcium-activated potassium channels in canine intracardiac ganglia.

Large conductance calcium-activated potassium (BK) channels are widely expressed in the nervous system. We have recently shown that principal neurons from canine intracardiac ganglia (ICG) express a paxilline- and TEA-sensitive BK current, which increases neuronal excitability. In the present work, we further explore the molecular constituents of the BK current in canine ICG. We found that the ß1 and ß4 regulatory subunits are expressed in ICG. Single channel voltage-dependence at different calcium concentrations suggested that association of the BKα with a particular ß subunit was not enough to explain the channel activity in this tissue. Indeed, we detected the presence of several splice variants of the BKα subunit. In conclusion, BK channels in canine ICG may result from the arrangement of different BKα splice variants, plus accessory ß subunits. The particular combinations expressed in canine IC neurons likely rule the excitatory role of BK current in this tissue.

DiBAC4(3) hits a "sweet spot" for the activation of arterial large-conductance Ca²âº-activated potassium channels independently of the ß1-subunit.

The voltage-sensitive dye bis-(1,3-dibutylbarbituric acid)trimethine oxonol [DiBAC4(3)] has been reported as a novel large-conductance Ca²âº-activated K⁺ (BK) channel activator with selectivity for its ß1- or ß4-subunits. In arterial smooth muscle, BK channels are formed by a pore-forming α-subunit and a smooth muscle-abundant regulatory ß1-subunit. This tissue specificity has driven extensive pharmacological research aimed at regulating arterial tone. Using animals with a disruption of the gene for the ß1-subunit, we explored the effects of DiBAC4(3) in native channels from arterial smooth muscle. We tested the hypothesis that, in native BK channels, activation by DiBAC4(3) relies mostly on its α-subunit. We studied BK channels from wild-type and transgenic ß1-knockout mice in excised patches. BK channels from brain arteries, with or without the ß1-subunit, were similarly activated by DiBAC4(3). In addition, we found that saturating concentrations of DiBAC4(3) (~30 µM) promote an unprecedented persistent activation of the channel that negatively shifts its voltage dependence by as much as -300 mV. This "sweet spot" for persistent activation is independent of Ca²âº and/or the ß1₋4-subunits and is fully achieved when DiBAC4(3) is applied to the intracellular side of the channel. Arterial BK channel response to DiBAC4(3) varies across species and/or vascular beds. DiBAC4(3) unique effects can reveal details of BK channel gating mechanisms and help in the rational design of BK channel activators.