Ca_V-channel dependent activation of BK channels is critical for feedback control of both calcium influx and cell excitability. Here we addressed the functional and spatial interaction between BK and Ca_V1.3 channels, unique Ca_V1 channels that activate at low voltages. We found that when BK and Ca_V1.3 channels were co-expressed in the same cell, BK channels started activating near -50 mV, ~30 mV more negative than for activation of co-expressed BK and high-voltage activated Ca_V2.2 channels. In addition, single-molecule localization microscopy revealed striking clusters of Ca_V1.3 channels surrounding clusters of BK channels and forming a multi-channel complex both in a heterologous system and in rat hippocampal and sympathetic neurons. We propose that this spatial arrangement allows tight tracking between local BK channel activation and the gating of Ca_V1.3 channels at quite negative membrane potentials, facilitating the regulation of neuronal excitability at voltages close to the threshold to fire action potentials.

Children seeking care in the emergency department (ED) for mental health conditions are at risk for prolonged length of stay (LOS). A more contemporary description of trends and visit characteristics associated with prolonged ED LOS at the national level is lacking in the literature. Our objectives were to (1) compare LOS trends for pediatric mental health versus non-mental health ED visits and (2) explore patient-level characteristics associated with prolonged LOS for mental health ED visits. We conducted an observational analysis of ED visits among children 6 to 17 years of age using the National Hospital Ambulatory Medical Care Survey (2005-2015). We assessed trends in rates of prolonged LOS and the association between prolonged LOS and demographic and clinical characteristics (race and ethnicity, payer type, and presence of a concurrent physical health diagnosis) using descriptive statistics and survey-weighted logistic regression. From 2005 to 2015, rates of prolonged LOS for pediatric mental health ED visits increased over time from 16.3% to 24.6% (LOS >6 hours) and 5.3% to 12.7% (LOS >12 hours), in contrast to non-mental health visits for which LOS remained stable. For mental health visits, Hispanic ethnicity was associated with an almost threefold odds of LOS >12 hours (odds ratio 2.74; 95% confidence interval 1.69-4.44); there was no difference in LOS by payer type. The substantial rise in prolonged LOS for mental health ED visits and disparity for Hispanic children suggest worsening and inequitable access to definitive pediatric mental health care. Policy makers and health systems should work to provide equitable and timely access to pediatric mental health care.

Introduction

Inadequately spaced pregnancies, defined as pregnancies fewer than 18 months apart, are linked to maternal, infant, and child morbidity and mortality, and adverse social, educational and economic outcomes in later life for women and children. Quantifying the relation between intimate partner violence (IPV) and women's ability to space and time their pregnancies is an important part of understanding the burden of disease related to IPV.

Methods

We applied Cox proportional hazards models to monthly data from the Demographic and Health Surveys' Reproductive Health Calendar to compare interpregnancy intervals for women who experienced physical, sexual and/or emotional IPV in 29 countries. We conducted a one-stage meta-analysis to identify the periods when women who experienced IPV were at the highest risk of unintended and incident pregnancy, and a two-stage meta-analysis to explore cross-country variations in the magnitude of the relation between women's experience of IPV and pregnancy spacing.

Results

For the one-stage analysis, considering 52 959 incident pregnancies from 90 446 women, which represented 232 394 person-years at risk, women's experience of IPV was associated with a 51% increase in the risk of pregnancy (95% CI 1.38 to 1.66), although this association decreased over time. When limiting our inference to unintended pregnancies that resulted in live births, women's experience of IPV was associated with a 30% increase in the risk of unintended pregnancy (95% CI 1.25 to 1.34; n=13 541 pregnancies, 92 848 women, 310 319 person-years at risk). In the two-stage meta-analyses, women's experience of IPV was associated with a 13% increase in the probability of incident pregnancy (95% CI 1.07 to 1.20) and a 28% increase in the likelihood of unintended pregnancy (95% CI 1.19 to 1.38).

Conclusions

Across countries, women's experience of IPV is associated with a reduction in time between pregnancies and an increase in the risk of unintended pregnancy; the magnitude of this effect varied by country and over time.

CaV1.3 channels regulate excitability in many neurons. As is the case for all voltage-gated channels, it is widely assumed that individual CaV1.3 channels behave independently with respect to voltage-activation, open probability, and facilitation. Here, we report the results of super-resolution imaging, optogenetic, and electrophysiological measurements that refute this long-held view. We found that the short channel isoform (CaV1.3S), but not the long (CaV1.3L), associates in functional clusters of two or more channels that open cooperatively, facilitating Ca(2+) influx. CaV1.3S channels are coupled via a C-terminus-to-C-terminus interaction that requires binding of the incoming Ca(2+) to calmodulin (CaM) and subsequent binding of CaM to the pre-IQ domain of the channels. Physically-coupled channels facilitate Ca(2+) currents as a consequence of their higher open probabilities, leading to increased firing rates in rat hippocampal neurons. We propose that cooperative gating of CaV1.3S channels represents a mechanism for the regulation of Ca(2+) signaling and electrical activity.

In the heart, reliable activation of Ca(2+) release from the sarcoplasmic reticulum during the plateau of the ventricular action potential requires synchronous opening of multiple CaV1.2 channels. Yet the mechanisms that coordinate this simultaneous opening during every heartbeat are unclear. Here, we demonstrate that CaV1.2 channels form clusters that undergo dynamic, reciprocal, allosteric interactions. This 'functional coupling' facilitates Ca(2+) influx by increasing activation of adjoined channels and occurs through C-terminal-to-C-terminal interactions. These interactions are initiated by binding of incoming Ca(2+) to calmodulin (CaM) and proceed through Ca(2+)/CaM binding to the CaV1.2 pre-IQ domain. Coupling fades as [Ca(2+)]i decreases, but persists longer than the current that evoked it, providing evidence for 'molecular memory'. Our findings suggest a model for CaV1.2 channel gating and Ca(2+)-influx amplification that unifies diverse observations about Ca(2+) signaling in the heart, and challenges the long-held view that voltage-gated channels open and close independently.

TRPV4 (transient receptor potential vanilloid 4) channels are Ca²⁺-permeable channels that play a key role in regulating vascular tone. In arterial myocytes, opening of TRPV4 channels creates local increases in Ca²⁺ influx, detectable optically as "TRPV4 sparklets." TRPV4 sparklet activity can be enhanced by the action of the vasoconstrictor angiotensin II (AngII). This modulation depends on the activation of subcellular signaling domains that comprise protein kinase C α (PKCα) bound to the anchoring protein AKAP150. Here, we used super-resolution nanoscopy, patch-clamp electrophysiology, Ca²⁺ imaging, and mathematical modeling approaches to test the hypothesis that AKAP150-dependent modulation of TRPV4 channels is critically dependent on the distance between these two proteins in the sarcolemma of arterial myocytes. Our data show that the distance between AKAP150 and TRPV4 channel clusters varies with sex and arterial bed. Consistent with our hypothesis, we further find that basal and AngII-induced TRPV4 channel activity decays exponentially as the distance between TRPV4 and AKAP150 increases. Our data suggest a maximum radius of action of ∼200 nm for local modulation of TRPV4 channels by AKAP150-associated PKCα.

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) are at the center of new cell-based therapies for cardiac disease, but may also serve as a useful in vitro model for cardiac cell development. An intriguing feature of hESC-CMs is that although they express contractile proteins and have sarcomeres, they do not develop transverse-tubules (T-tubules) with adult-like Ca²⁺ release units (CRUs). We tested the hypothesis that expression of the protein BIN1 in hESC-CMs promotes T-tubules formation, facilitates Ca_V 1.2 channel clustering along the tubules, and results in the development of stable CRUs. Using electrophysiology, [Ca²⁺ ]_i imaging, and super resolution microscopy, we found that BIN1 expression induced T-tubule development in hESC-CMs, while increasing differentiation toward a more ventricular-like phenotype. Voltage-gated Ca_V 1.2 channels clustered along the surface sarcolemma and T-tubules of hESC-CM. The length and width of the T-tubules as well as the expression and size of Ca_V 1.2 clusters grew, as BIN1 expression increased and cells matured. BIN1 expression increased Ca_V 1.2 channel activity and the probability of coupled gating within channel clusters. Interestingly, BIN1 clusters also served as sites for sarcoplasmic reticulum (SR) anchoring and stabilization. Accordingly, BIN1-expressing cells had more Ca_V 1.2-ryanodine receptor junctions than control cells. This was associated with larger [Ca²⁺ ]_i transients during excitation-contraction coupling. Our data support the view that BIN1 is a key regulator of T-tubule formation and Ca_V 1.2 channel delivery. By studying the role of BIN1 during the differentiation of hESC-CMs, we show that BIN1 is also important for Ca_V 1.2 channel clustering, junctional SR organization, and the establishment of excitation-contraction coupling. Stem Cells 2019;37:54-64.

Ion channels are often found arranged into dense clusters in the plasma membranes of excitable cells, but the mechanisms underlying the formation and maintenance of these functional aggregates are unknown. Here, we tested the hypothesis that channel clustering is the consequence of a stochastic self-assembly process and propose a model by which channel clusters are formed and regulated in size. Our hypothesis is based on statistical analyses of the size distributions of the channel clusters we measured in neurons, ventricular myocytes, arterial smooth muscle, and heterologous cells, which in all cases were described by exponential functions, indicative of a Poisson process (i.e., clusters form in a continuous, independent, and memory-less fashion). We were able to reproduce the observed cluster distributions of five different types of channels in the membrane of excitable and tsA-201 cells in simulations using a computer model in which channels are "delivered" to the membrane at randomly assigned locations. The model's three parameters represent channel cluster nucleation, growth, and removal probabilities, the values of which were estimated based on our experimental measurements. We also determined the time course of cluster formation and membrane dwell time for Ca_V1.2 and TRPV4 channels expressed in tsA-201 cells to constrain our model. In addition, we elaborated a more complex version of our model that incorporated a self-regulating feedback mechanism to shape channel cluster formation. The strong inference we make from our results is that Ca_V1.2, Ca_V1.3, BK, and TRPV4 proteins are all randomly inserted into the plasma membranes of excitable cells and that they form homogeneous clusters that increase in size until they reach a steady state. Further, it appears likely that cluster size for a diverse set of membrane-bound proteins and a wide range of cell types is regulated by a common feedback mechanism.

Nanobodies (nAbs) are small, minimal antibodies that have distinct attributes that make them uniquely suited for certain biomedical research, diagnostic and therapeutic applications. Prominent uses include as intracellular antibodies or intrabodies to bind and deliver cargo to specific proteins and/or subcellular sites within cells, and as nanoscale immunolabels for enhanced tissue penetration and improved spatial imaging resolution. Here, we report the generation and validation of nAbs against a set of proteins prominently expressed at specific subcellular sites in mammalian brain neurons. We describe a novel hierarchical validation pipeline to systematically evaluate nAbs isolated by phage display for effective and specific use as intrabodies and immunolabels in mammalian cells including brain neurons. These nAbs form part of a robust toolbox for targeting proteins with distinct and highly spatially-restricted subcellular localization in mammalian brain neurons, allowing for visualization and/or modulation of structure and function at those sites.