Phosphorylation and protonation of neighboring MiRP2 sites: function and pathophysiology of MiRP2-Kv3.4 potassium channels in periodic paralysis.

MinK-related peptide 2 (MiRP2) and Kv3.4 subunits assemble in skeletal muscle to create subthreshold, voltage-gated potassium channels. MiRP2 acts on Kv3.4 to shift the voltage dependence of activation, speed recovery from inactivation, suppress cumulative inactivation and increase unitary conductance. We previously found an R83H missense mutation in MiRP2 that segregated with periodic paralysis in two families and diminished the effects of MiRP2 on Kv3.4. Here we show that MiRP2 has a single, functional PKC phosphorylation site at serine 82 and that normal MiRP2-Kv3.4 function requires phosphorylation of the site. The R83H variant does not prevent PKC phosphorylation of neighboring S82; rather, the change shifts the voltage dependence of activation and endows MiRP2-Kv3.4 channels with sensitivity to changes in intracellular pH across the physiological range. Thus, current passed by single R83H channels decreases as internal pH is lowered (pK(a) approximately 7.3, consistent with histidine protonation) whereas wild-type channels are largely insensitive. These findings identify a key regulatory domain in MiRP2 and suggest a mechanistic link between acidosis and episodes of periodic paralysis

Sumoylation silences the plasma membrane leak K+ channel K2P1.

Reversible, covalent modification with small ubiquitin-related modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the SUMO pathway is shown to operate at the plasma membrane to control ion channel function. SUMO-conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 had not previously shown function despite mRNA expression in heart, brain, and kidney and sequence features like other two-P loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly rectifying channel regulated by reversible peptide linkage

Ito channels are octomeric complexes with four subunits of each Kv4.2 and K+ channel-interacting protein 2.

Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits

Three-dimensional structure of I(to); Kv4.2-KChIP2 ion channels by electron microscopy at 21 Angstrom resolution.

Regulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes to produce native voltage-gated potassium (Kv) channels like cardiac I(to) and neuronal I(A) subtypes. Here, negative stain electron microscopy (EM) and single particle averaging reveal KChIP2 to create a novel approximately 35 x 115 x 115 Angstrom, intracellular fenestrated rotunda: four peripheral columns that extend down from the membrane-embedded portion of the channel to enclose the Kv4.2 "hanging gondola" (a platform held beneath the transmembrane conduction pore by four internal columns). To reach the pore from the cytosol, ions traverse one of four external fenestrae to enter the rotundal vestibule and then cross one of four internal windows in the gondola

‘It’s like equality now; it’s not as if it’s the old days’: an investigation into gender identity development and football participation of adolescent girls

This article explores the influence participating in football has on the development of adolescent girls’ gender identity, an area which currently lacks academic attention. Data were taken from an ethnographic study with a group of adolescent girls and boys and compared to Jeanes’ research. A social constructionist framework was deployed with links to both critical theory and feminist literature. Qualitative and participatory methods were used to fully engage with the complex issue of gender identity. The girls within this study were aware of the normative gender expectations linked to ‘being a female’ but did not find this restrictive. The girls moved between many changing identities and organised their ‘web of selves’ accordingly. The apparent need to measure success by the parameters of male standards created a barrier to girls’ identity development

Three-Dimensional Structure of Ito Kv4.2-KChIP2 Ion Channels by Electron Microscopy at 21 Å Resolution

AbstractRegulatory KChIP2 subunits assemble with pore-forming Kv4.2 subunits in 4:4 complexes to produce native voltage-gated potassium (Kv) channels like cardiac Ito and neuronal IA subtypes. Here, negative stain electron microscopy (EM) and single particle averaging reveal KChIP2 to create a novel ∼35 × 115 × 115 Å, intracellular fenestrated rotunda: four peripheral columns that extend down from the membrane-embedded portion of the channel to enclose the Kv4.2 “hanging gondola” (a platform held beneath the transmembrane conduction pore by four internal columns). To reach the pore from the cytosol, ions traverse one of four external fenestrae to enter the rotundal vestibule and then cross one of four internal windows in the gondola

Attributable costs of surgical site infection and endometritis after low transverse cesarean delivery

BACKGROUND: Accurate data on costs attributable to hospital-acquired infections are needed in order to determine their economic impact and the cost-benefit of potential preventive strategies. OBJECTIVE: Determine the attributable costs of surgical site infection (SSI) and endometritis (EMM) after cesarean section using two different methods. DESIGN: Retrospective cohort. SETTING: Barnes-Jewish Hospital, a 1250-bed academic tertiary care hospital. PATIENTS: 1,605 women who underwent low transverse cesarean section from 7/1999 – 6/2001. METHODS: Attributable costs of SSI and EMM were determined by generalized least squares (GLS) and propensity score matched-pairs using administrative claims data to define underlying comorbidities and procedures. For the matched-pairs analyses, uninfected control patients were matched to patients with SSI or with EMM based on their propensity to develop infection, and the median difference in costs calculated. RESULTS: The attributable total hospital cost of SSI calculated by GLS was $3,529 and by propensity score matched-pairs was$2,852. The attributable total hospital cost of EMM calculated by GLS was $3,956 and by propensity score matched-pairs was$3,842. The majority of excess costs were associated with room and board and pharmacy costs. CONCLUSIONS: The costs of SSI and EMM were lower than SSI costs reported after more extensive operations. The attributable costs of EMM calculated using the two methods were very similar, while the costs of SSI calculated using propensity score matched-pairs were lower than the costs calculated by GLS. The difference in costs determined by the two methods needs to be considered by investigators performing cost analyses of hospital-acquired infections

Toward high-throughput engineering techniques for improving CAR intracellular signaling domains

Chimeric antigen receptors (CAR) are generated by linking extracellular antigen recognition domains with one or more intracellular signaling domains derived from the T-cell receptor complex or various co-stimulatory receptors. The choice and relative positioning of signaling domains help to determine chimeric antigen receptors T-cell activity and fate in vivo. While prior studies have focused on optimizing signaling power through combinatorial investigation of native intracellular signaling domains in modular fashion, few have investigated the prospect of sequence engineering within domains. Here, we sought to develop a novel in situ screening method that could permit deployment of directed evolution approaches to identify intracellular domain variants that drive selective induction of transcription factors. To accomplish this goal, we evaluated a screening approach based on the activation of a human NF-κB and NFAT reporter T-cell line for the isolation of mutations that directly impact T cell activation in vitro. As a proof-of-concept, a model library of chimeric antigen receptors signaling domain variants was constructed and used to demonstrate the ability to discern amongst chimeric antigen receptors containing different co-stimulatory domains. A rare, higher-signaling variant with frequency as low as 1 in 1000 could be identified in a high throughput setting. Collectively, this work highlights both prospects and limitations of novel mammalian display methods for chimeric antigen receptors signaling domain discovery and points to potential strategies for future chimeric antigen receptors development

Effect of Oral Iron Repletion on Exercise Capacity in Patients With Heart Failure With Reduced Ejection Fraction and Iron Deficiency: The IRONOUT HF Randomized Clinical Trial.

Importance: Iron deficiency is present in approximately 50% of patients with heart failure with reduced left ventricular ejection fraction (HFrEF) and is an independent predictor of reduced functional capacity and mortality. However, the efficacy of inexpensive readily available oral iron supplementation in heart failure is unknown. Objective: To test whether therapy with oral iron improves peak exercise capacity in patients with HFrEF and iron deficiency. Design, Setting, and Participants: Phase 2, double-blind, placebo-controlled randomized clinical trial of patients with HFrEF ( Interventions: Oral iron polysaccharide (n = 111) or placebo (n = 114), 150 mg twice daily for 16 weeks. Main Outcomes and Measures: The primary end point was a change in peak oxygen uptake (V̇o2) from baseline to 16 weeks. Secondary end points were change in 6-minute walk distance, plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, and health status as assessed by Kansas City Cardiomyopathy Questionnaire (KCCQ, range 0-100, higher scores reflect better quality of life). Results: Among 225 randomized participants (median age, 63 years; 36% women) 203 completed the study. The median baseline peak V̇o2 was 1196 mL/min (interquartile range [IQR], 887-1448 mL/min) in the oral iron group and 1167 mL/min (IQR, 887-1449 mL/min) in the placebo group. The primary end point, change in peak V̇o2 at 16 weeks, did not significantly differ between the oral iron and placebo groups (+23 mL/min vs -2 mL/min; difference, 21 mL/min [95% CI, -34 to +76 mL/min]; P = .46). Similarly, at 16 weeks, there were no significant differences between treatment groups in changes in 6-minute walk distance (-13 m; 95% CI, -32 to 6 m), NT-proBNP levels (159; 95% CI, -280 to 599 pg/mL), or KCCQ score (1; 95% CI, -2.4 to 4.4), all P \u3e .05. Conclusions and Relevance: Among participants with HFrEF with iron deficiency, high-dose oral iron did not improve exercise capacity over 16 weeks. These results do not support use of oral iron supplementation in patients with HFrEF. Trial Registration: clinicaltrials.gov Identifier: NCT02188784