Pairwise biosynthesis of ion channels stabilizes excitability and mitigates arrhythmias.

Coordinated expression of ion channels is crucial for cardiac rhythms, neural signaling, and cell cycle progression. Perturbation of this balance results in many disorders including cardiac arrhythmias. Prior work revealed association of mRNAs encoding cardiac NaV1.5 (SCN5A) and hERG1 (KCNH2), but the functional significance of this association was not established. Here, we provide a more comprehensive picture of KCNH2, SCN5A, CACNA1C, and KCNQ1 transcripts collectively copurifying with nascent hERG1, NaV1.5, CaV1.2, or KCNQ1 channel proteins. Single-molecule fluorescence in situ hybridization (smFISH) combined with immunofluorescence reveals that the channel proteins are synthesized predominantly as heterotypic pairs from discrete molecules of mRNA, not as larger cotranslational complexes. Puromycin disrupted colocalization of mRNA with its encoded protein, as expected, but remarkably also pairwise mRNA association, suggesting that transcript association relies on intact translational machinery or the presence of the nascent protein. Targeted depletion of KCHN2 by specific shRNA resulted in concomitant reduction of all associated mRNAs, with a corresponding reduction in the encoded channel currents. This co-knockdown effect, originally described for KCNH2 and SCN5A, thus appears to be a general phenomenon among transcripts encoding functionally related proteins. In multielectrode array recordings, proarrhythmic behavior arose when IKr was reduced by the selective blocker dofetilide at IC50 concentrations, but not when equivalent reductions were mediated by shRNA, suggesting that co-knockdown mitigates proarrhythmic behavior expected from the selective reduction of a single channel species. We propose that coordinated, cotranslational association of functionally related ion channel mRNAs confers electrical stability by co-regulating complementary ion channels in macromolecular complexes.

Temporal Dynamics, Discovery, and Emergence of Human-Transmissible RNA Viruses.

Transmissibility, the ability to spread within host populations, is a prerequisite for a pathogen to have epidemic or pandemic potential. Here, we estimate the phylogenies of human infectivity and transmissibility using 1,408 genome sequences from 743 distinct RNA virus species/types in 59 genera. By repeating this analysis using data sets censored by virus discovery date, we explore how temporal changes in the known diversity of RNA viruses-especially recent increases in recognized nonhuman viruses-have altered these phylogenies. Over time, we find significant increases in the proportion of RNA virus genera estimated to have a nonhuman-infective ancestral state, in the fraction of distinct human virus lineages that are purely human-transmissible or strictly zoonotic (compared to mixed lineages), and in the number of human viruses with nearest relatives known not to infect humans. Our results are consistent with viruses that are capable of spreading in human populations commonly emerging from a nonhuman reservoir. This is more likely in lineages that already contain human-transmissible viruses but is rare in lineages that contain only strictly zoonotic viruses.

An intracellular hydrophobic nexus critical for hERG1 channel slow deactivation.

Slow deactivation is a critical property of voltage-gated K+ channels encoded by the human Ether-à-go-go-Related Gene 1 (hERG). hERG1 channel deactivation is modulated by interactions between intracellular N-terminal Per-Arnt-Sim (PAS) and C-terminal cyclic nucleotide-binding homology (CNBh) domains. The PAS domain is multipartite, comprising a globular domain (gPAS; residues 26-135) and an N-terminal PAS-cap that is further subdivided into an initial unstructured "tip" (residues 1-12) and an amphipathic α-helical region (residues 13-25). Although the PAS-cap tip has long been considered the effector of slow deactivation, how its position near the gating machinery is controlled has not been elucidated. Here, we show that a triad of hydrophobic interactions among the gPAS, PAS-cap α helix, and the CNBh domains is required to support slow deactivation in hERG1. The primary sequence of this "hydrophobic nexus" is highly conserved among mammalian ERG channels but shows key differences to fast-deactivating Ether-à-go-go 1 (EAG1) channels. Combining sequence analysis, structure-directed mutagenesis, electrophysiology, and molecular dynamics simulations, we demonstrate that polar serine substitutions uncover an intermediate deactivation mode that is also mimicked by deletion of the PAS-cap α helix. Molecular dynamics simulation analyses of the serine-substituted channels show an increase in distance among the residues of the hydrophobic nexus, a rotation of the intracellular gating ring, and a retraction of the PAS-cap tip from its receptor site near the voltage sensor domain and channel gate. These findings provide compelling evidence that the hydrophobic nexus coordinates the respective components of the intracellular gating ring and positions the PAS-cap tip to control hERG1 deactivation gating.

Conformation-sensitive antibody reveals an altered cytosolic PAS/CNBh assembly during hERG channel gating.

The human ERG (hERG) K+ channel has a crucial function in cardiac repolarization, and mutations or channel block can give rise to long QT syndrome and catastrophic ventricular arrhythmias. The cytosolic assembly formed by the Per-Arnt-Sim (PAS) and cyclic nucleotide binding homology (CNBh) domains is the defining structural feature of hERG and related KCNH channels. However, the molecular role of these two domains in channel gating remains unclear. We have previously shown that single-chain variable fragment (scFv) antibodies can modulate hERG function by binding to the PAS domain. Here, we mapped the scFv2.12 epitope to a site overlapping with the PAS/CNBh domain interface using NMR spectroscopy and mutagenesis and show that scFv binding in vitro and in the cell is incompatible with the PAS interaction with CNBh. By generating a fluorescently labeled scFv2.12, we demonstrate that association with the full-length hERG channel is state dependent. We detect Förster resonance energy transfer (FRET) with scFv2.12 when the channel gate is open but not when it is closed. In addition, state dependence of scFv2.12 FRET signal disappears when the R56Q mutation, known to destabilize the PAS-CNBh interaction, is introduced in the channel. Altogether, these data are consistent with an extensive structural alteration of the PAS/CNBh assembly when the cytosolic gate opens, likely favoring PAS domain dissociation from the CNBh domain.

Length and associated characteristics of short-term detentions: an analysis of detentions under the Mental Health Act in Scotland, 2006-2018.

PURPOSE: The Mental Health Act in Scotland is under review. Previous iterations increased patients' rights but the maximum time for short-term detentions remains unchanged, despite evolving psychiatric treatment models. We explored length, mode of ending and factors of influence on the application of short-term detention certificates (STDCs), which can last up to 28 days, across Scotland between 2006 and 2018. METHODS: Data on age, gender, ethnicity, date of commencement and ending of the STDC and detention site from all 42,493 STDCs issued to 30,464 patients over 12 years were extracted from the national repository for detentions under the Mental Health (Care and Treatment) (Scotland) Act 2003 and analysed using mixed models. RESULTS: One in five STDCs lapsed on day 28. Two in five were revoked and the remainder extended to a treatment order. STDCs that were not extended averaged 19 days, and revoked STDCs 14 days. The probability of a detention lapsing varied across hospitals and increased with patient age. The odds of a detention lapsing on day 28 were 62% lower and revoked detentions 10% shorter in 2018 relative to 2006. The odds of a detention extending decreased significantly from 2012 to 2018. Extended STDCs were associated with increased patient age, male gender, and ethnicity other than White Scottish. There was little initiation of or active revocation of STDCs on weekend days. CONCLUSION: The length of STDCs reduced over time, fewer detentions lapsed, and weekday patterning was evident in each year. These data can inform legislative and service reviews.

Environmental heterogeneity promotes individual specialisation in habitat selection in a widely distributed seabird.

Individual specialisations in behaviour are predicted to arise where divergence benefits fitness. Such specialisations are more likely in heterogeneous environments where there is both greater ecological opportunity and competition-driven frequency dependent selection. Such an effect could explain observed differences in rates of individual specialisation in habitat selection, as it offers individuals an opportunity to select for habitat types that maximise resource gain while minimising competition; however, this mechanism has not been tested before. Here, we use habitat selection functions to quantify individual specialisations while foraging by black-legged kittiwakes Rissa tridactyla, a marine top predator, at 15 colonies around the United Kingdom and Ireland, along a gradient of environmental heterogeneity. We find support for the hypothesis that individual specialisations in habitat selection while foraging are more prevalent in heterogeneous environments. This trend was significant across multiple dynamic habitat variables that change over short time-scales and did not arise through site fidelity, which highlights the importance of environmental processes in facilitating behavioural adaptation by predators. Individual differences may drive evolutionary processes, and therefore these results suggest that there is broad scope for the degree of environmental heterogeneity to determine current and future population, species and community dynamics.

hERG Function in Light of Structure.

The human ether-a-go-go-related gene1 (hERG) ion channel has been the subject of fascination since it was identified as a target of long QT syndrome more than 20 years ago. In this Biophysical Perspective, we look at what makes hERG intriguing and vexingly unique. By probing recent high-resolution structures in the context of functional and biochemical data, we attempt to summarize new insights into hERG-specific function and articulate important unanswered questions. X-ray crystallography and cryo-electron microscopy have revealed features not previously on the radar-the "nonswapped" transmembrane architecture, an "intrinsic ligand," and hydrophobic pockets off a pore cavity that is surprisingly small. Advances in our understanding of drug block and inactivation mechanisms are noted, but a full picture will require more investigation.

Localization and functional consequences of a direct interaction between TRIOBP-1 and hERG proteins in the heart.

Reduced levels of the cardiac human (h)ERG ion channel protein and the corresponding repolarizing current IKr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native IKr and disrupted action potential repolarization. Ca2+ currents were also somewhat reduced and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, IKr magnitude and cardiac membrane excitability.

Environmental heterogeneity decreases reproductive success via effects on foraging behaviour.

Environmental heterogeneity shapes the uneven distribution of resources available to foragers, and is ubiquitous in nature. Optimal foraging theory predicts that an animal's ability to exploit resource patches is key to foraging success. However, the potential fitness costs and benefits of foraging in a heterogeneous environment are difficult to measure empirically. Heterogeneity may provide higher-quality foraging opportunities, or alternatively could increase the cost of resource acquisition because of reduced patch density or increased competition. Here, we study the influence of physical environmental heterogeneity on behaviour and reproductive success of black-legged kittiwakes, Rissa tridactyla. From GPS tracking data at 15 colonies throughout their British and Irish range, we found that environments that were physically more heterogeneous were associated with longer trip duration, more time spent foraging while away from the colony, increased overlap of foraging areas between individuals and lower breeding success. These results suggest that there is greater competition between individuals for finite resources in more heterogeneous environments, which comes at a cost to reproduction. Resource hotspots are often considered beneficial, as individuals can learn to exploit them if sufficiently predictable. However, we demonstrate here that such fitness gains can be countered by greater competition in more heterogeneous environments.

Cotranslational association of mRNA encoding subunits of heteromeric ion channels.

Oligomers of homomeric voltage-gated potassium channels associate early in biogenesis as the nascent proteins emerge from the polysome. Less is known about how proteins emerging from different polysomes associate to form hetero-oligomeric channels. Here, we report that alternate mRNA transcripts encoding human ether-à-go-go-related gene (hERG) 1a and 1b subunits, which assemble to produce ion channels mediating cardiac repolarization, are physically associated during translation. We show that shRNA specifically targeting either hERG 1a or 1b transcripts reduced levels of both transcripts, but only when they were coexpressed heterologously. Both transcripts could be copurified with an Ab against the nascent hERG 1a N terminus. This interaction occurred even when translation of 1b was prevented, indicating the transcripts associate independent of their encoded proteins. The association was also demonstrated in cardiomyocytes, where levels of both hERG transcripts were reduced by either 1a or 1b shRNA, but native KCNE1 and ryanodine receptor 2 (RYR2) transcripts were unaffected. Changes in protein levels and membrane currents mirrored changes in transcript levels, indicating the targeted transcripts were undergoing translation. The physical association of transcripts encoding different subunits provides the spatial proximity required for nascent proteins to interact during biogenesis, and may represent a general mechanism facilitating assembly of heteromeric protein complexes involved in a range of biological processes.

Enhancement of hERG channel activity by scFv antibody fragments targeted to the PAS domain.

The human human ether-à-go-go-related gene (hERG) potassium channel plays a critical role in the repolarization of the cardiac action potential. Changes in hERG channel function underlie long QT syndrome (LQTS) and are associated with cardiac arrhythmias and sudden death. A striking feature of this channel and KCNH channels in general is the presence of an N-terminal Per-Arnt-Sim (PAS) domain. In other proteins, PAS domains bind ligands and modulate effector domains. However, the PAS domains of KCNH channels are orphan receptors. We have uncovered a family of positive modulators of hERG that specifically bind to the PAS domain. We generated two single-chain variable fragments (scFvs) that recognize different epitopes on the PAS domain. Both antibodies increase the rate of deactivation but have different effects on channel activation and inactivation. Importantly, we show that both antibodies, on binding to the PAS domain, increase the total amount of current that permeates the channel during a ventricular action potential and significantly reduce the action potential duration recorded in human cardiomyocytes. Overall, these molecules constitute a previously unidentified class of positive modulators and establish that allosteric modulation of hERG channel function through ligand binding to the PAS domain can be attained.

Breeding density, fine-scale tracking, and large-scale modeling reveal the regional distribution of four seabird species.

Population-level estimates of species' distributions can reveal fundamental ecological processes and facilitate conservation. However, these may be difficult to obtain for mobile species, especially colonial central-place foragers (CCPFs; e.g., bats, corvids, social insects), because it is often impractical to determine the provenance of individuals observed beyond breeding sites. Moreover, some CCPFs, especially in the marine realm (e.g., pinnipeds, turtles, and seabirds) are difficult to observe because they range tens to ten thousands of kilometers from their colonies. It is hypothesized that the distribution of CCPFs depends largely on habitat availability and intraspecific competition. Modeling these effects may therefore allow distributions to be estimated from samples of individual spatial usage. Such data can be obtained for an increasing number of species using tracking technology. However, techniques for estimating population-level distributions using the telemetry data are poorly developed. This is of concern because many marine CCPFs, such as seabirds, are threatened by anthropogenic activities. Here, we aim to estimate the distribution at sea of four seabird species, foraging from approximately 5,500 breeding sites in Britain and Ireland. To do so, we GPS-tracked a sample of 230 European Shags Phalacrocorax aristotelis, 464 Black-legged Kittiwakes Rissa tridactyla, 178 Common Murres Uria aalge, and 281 Razorbills Alca torda from 13, 20, 12, and 14 colonies, respectively. Using Poisson point process habitat use models, we show that distribution at sea is dependent on (1) density-dependent competition among sympatric conspecifics (all species) and parapatric conspecifics (Kittiwakes and Murres); (2) habitat accessibility and coastal geometry, such that birds travel further from colonies with limited access to the sea; and (3) regional habitat availability. Using these models, we predict space use by birds from unobserved colonies and thereby map the distribution at sea of each species at both the colony and regional level. Space use by all four species' British breeding populations is concentrated in the coastal waters of Scotland, highlighting the need for robust conservation measures in this area. The techniques we present are applicable to any CCPF.

hERG 1b is critical for human cardiac repolarization.

The human ether-à-go-go-related gene (hERG; or KCNH2) encodes the voltage-gated potassium channel underlying IKr, a repolarizing current in the heart. Mutations in KCNH2 or pharmacological agents that reduce IKr slow action potential (AP) repolarization and can trigger cardiac arrhythmias associated with long QT syndrome. Two channel-forming subunits encoded by KCNH2 (hERG 1a and 1b) are expressed in cardiac tissue. In heterologous expression systems, these subunits avidly coassemble and exhibit biophysical and pharmacological properties distinct from those of homomeric hERG 1a channels. Despite these findings, adoption of hERG 1a/1b heteromeric channels as a model for cardiac IKr has been hampered by the lack of evidence for a direct functional role for the 1b subunit in native tissue. In this study, we measured IKr and APs at physiological temperature in cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs). We found that specific knockdown of the 1b subunit using shRNA caused reductions in 1b mRNA, 1b protein levels, and IKr magnitude by roughly one-half. AP duration was increased and AP variability was enhanced relative to controls. Early afterdepolarizations, considered cellular substrates for arrhythmia, were also observed in cells with reduced 1b expression. Similar behavior was elicited when channels were effectively converted from heteromers to 1a homomers by expressing a fragment corresponding to the 1a-specific N-terminal Per-Arnt-Sim domain, which is omitted from hERG 1b by alternate transcription. These findings establish that hERG 1b is critical for normal repolarization and that loss of 1b is proarrhythmic in human cardiac cells.

Prospective relationship between poor sleep and substance-related problems in a national sample of adolescents.

BACKGROUND: Previous studies showed that poor sleep prospectively predicted alcohol-related problems and illicit drug use in adolescents and young adults (Wong and Brower, 2012; Wong et al., 2010). However, more work needs to be done to elucidate the nature of these problems. The purpose of this study was to examine whether sleep difficulties and hours of sleep prospectively predicted several serious substance-related problems, for example, binge drinking, driving under the influence of alcohol, and risky sexual behavior. METHODS: Study participants were 6,504 adolescents from the National Longitudinal Study of Adolescent Health. Data were collected from interviews and questionnaires. This study analyzed data from the first 3 waves of data (T1: 1994 to 1995; T2: 1996; T3: 2001 to 2002). In all analyses, we used sleep difficulties at a previous wave to predict substance-related problems at a subsequent wave, while controlling for substance-related problems at a previous wave. RESULTS: Holding T1 alcohol-related problems constant, sleep difficulties at T1 significantly predicted alcohol-related interpersonal problems, binge drinking, gotten drunk or very high on alcohol, driving under the influence of alcohol, getting into a sexual situation one later regretted due to drinking, ever using any illicit drugs, and drug-related problems at T2. T1 hours of sleep negatively predicted T2 alcohol-related interpersonal problems and binge drinking. The relationship between T2 sleep variables and T3 substance-related problems was consistent with previous waves, although the effect was weaker. CONCLUSIONS: Sleep difficulties and hours of sleep are a significant predictor of a number of substance-related problems. It may be useful to educate adolescents about the importance of sleep, sleep hygiene, and the potential consequences of poor sleep on drinking and related behaviors.

Brief report: Peer group influences and adolescent internalizing problems as mediated by effortful control.

Internalizing problems in adolescence encompass behaviors directed inward at the self (Colman, Wadsworth, Croudace, & Jones, 2007). Several predictors have been linked to internalizing problems including antisocial and prosocial peers (Cartwright, 2007; Chung, 2010). Effortful control, a component of self-regulation, is one factor that could mediate the relationship between peer behaviors and individual outcomes. This study assessed the relationship between peer behaviors, effortful control, and adolescent internalizing problems. Participants were 151 middle school adolescents (M = 12.16 years old) who completed self-report questionnaires regarding behaviors of their peers, perceptions of effortful control, and experiences of internalizing problems. Structural equation modeling (SEM) yielded a significant negative relationship between antisocial peers and effortful control, and a significant positive relationship between prosocial peers and effortful control. In addition, effortful control significantly mediated the relationship between prosocial peers and internalizing problems, but not for antisocial peers. Implications for interventions related to adolescent health were discussed.

Self-Reported Sexual Behavior of Transgender Youth.

INTRODUCTION: Research indicates that transgender/gender diverse (TGD) youth are more likely to engage in sexual behavior, have more sexual partners, and initiate sexual behavior earlier than their cisgender peers. However, no gender-inclusive self-report survey questionnaires (ie, those that do not assume the gender of sexual partners or body parts used for sex) exist to assess the sexual behavior of TGD youth. The current study illustrates a questionnaire with nuanced wording to more accurately portray the sexual behavior of TGD youth presenting for gender-affirming medical care compared with national adolescent norms. METHODS: A retrospective chart review was conducted of 323 youth, ages 13-18, presenting to a pediatric gender clinic between 2015 and 2021. The youth self-reported their gender identity (ie, masculine, feminine, gender queer, questioning/unsure), sexual behaviors, and partners via a REDCAP survey. RESULTS: Rates of dating among TGD youth were significantly lower than national norms (33.7% vs 68.3%; χ2= 172.644, P < .0001), as was sexual behavior (14.9% vs. 39.5% χ2= 80.419, P < .0001). Rates of self-reported involuntary sexual activity among TGD youth did not differ significantly from national norms (7.1% vs. 6.9%, ns). The body parts used for sex, the number of sexual partners, and the gender identity of sexual partners are reported. DISCUSSION: The results suggest that rates of dating and sexual behavior among TGD youth are significantly lower than national norms, supporting a need for screening of sexual health among TGD youth utilizing gender-inclusive measures. A standardized gender-inclusive questionnaire of sexual behavior is needed to improve data accuracy and help develop inclusive programs to address the sexual health needs of TGD youth.

Validating hidden Markov models for seabird behavioural inference.

Understanding animal movement and behaviour can aid spatial planning and inform conservation management. However, it is difficult to directly observe behaviours in remote and hostile terrain such as the marine environment. Different underlying states can be identified from telemetry data using hidden Markov models (HMMs). The inferred states are subsequently associated with different behaviours, using ecological knowledge of the species. However, the inferred behaviours are not typically validated due to difficulty obtaining 'ground truth' behavioural information. We investigate the accuracy of inferred behaviours by considering a unique data set provided by Joint Nature Conservation Committee. The data consist of simultaneous proxy movement tracks of the boat (defined as visual tracks as birds are followed by eye) and seabird behaviour obtained by observers on the boat. We demonstrate that visual tracking data is suitable for our study. Accuracy of HMMs ranging from 71% to 87% during chick-rearing and 54% to 70% during incubation was generally insensitive to model choice, even when AIC values varied substantially across different models. Finally, we show that for foraging, a state of primary interest for conservation purposes, identified missed foraging bouts lasted for only a few seconds. We conclude that HMMs fitted to tracking data have the potential to accurately identify important conservation-relevant behaviours, demonstrated by a comparison in which visual tracking data provide a 'gold standard' of manually classified behaviours to validate against. Confidence in using HMMs for behavioural inference should increase as a result of these findings, but future work is needed to assess the generalisability of the results, and we recommend that, wherever feasible, validation data be collected alongside GPS tracking data to validate model performance. This work has important implications for animal conservation, where the size and location of protected areas are often informed by behaviours identified using HMMs fitted to movement data.

Caveolar Compartmentalization of Pacemaker Signaling is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure.

Background: Heart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). Methods: Biochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. Results: In both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across ~15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (I f) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with ~2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. Conclusions: SAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

A Drosophila behavioral mutant, down and out (dao), is defective in an essential regulator of Erg potassium channels.

To signal properly, excitable cells must establish and maintain the correct balance of various types of ion channels that increase or decrease membrane excitability. The mechanisms by which this balance is regulated remain largely unknown. Here, we describe a regulatory mechanism uncovered by a Drosophila behavioral mutant, down and out (dao). At elevated temperatures, dao loss-of-function mutants exhibit seizures associated with spontaneous bursts of neural activity. This phenotype closely resembles that of seizure mutations, which impair activity of ether-a-go-go-related gene (Erg)-type potassium channels. Conversely, neural over-expression of wild-type Dao confers dominant temperature-sensitive paralysis with kinetics reminiscent of paralytic sodium-channel mutants. The over-expression phenotype of dao is suppressed in a seizure mutant background, suggesting that Dao acts by an effect on Erg channels. In support of this hypothesis, functional expression of Erg channels in a heterologous system is dependent on the presence of Dao. These results indicate that Dao has an important role in establishing the proper level of neuronal membrane excitability by regulating functional expression of Erg channels.