An OxPLORE Initiative Evaluating Children’s Surgery Resources Worldwide: A Cross-sectional Implementation of the OReCS Document

World Journal of Surgery - The Global Initiative for Children's Surgery (GICS) group produced the Optimal Resources for Children’s Surgery (OReCS) document in 2019, listing standards of...     

Tricyclic antidepressant overdose in a toddler treated with intravenous lipid emulsion

We report a case that involves the use of intravenous lipid emulsion as an antidote for a drug overdose involving a 20-month-old girl who had ingested a potentially lethal amount of the tricyclic antidepressant (TCA) dothiepin. The patient's condition continued to deteriorate despite implementation of standard pediatric treatment recommendations for TCA toxicity. Administration of intravenous lipid emulsion in addition to standard therapy (including sodium bicarbonate) and direct-current cardioversion for ventricular arrhythmia led to a successful outcome. The case report is followed by a review of the current evidence underlying this novel therapy and the background on its use. TCA toxicity is addressed specifically.     

STIM protein coupling in the activation of Orai channels

STIM proteins are sensors of endoplasmic reticulum (ER) luminal Ca²⁺ changes and rapidly translocate into near plasma membrane (PM) junctions to activate Ca²⁺ entry through the Orai family of highly Ca²⁺-selective “store-operated” channels (SOCs). Dissecting the STIM–Orai coupling process is restricted by the abstruse nature of the ER–PM junctional domain. To overcome this problem, we studied coupling by using STIM chimera and cytoplasmic C-terminal domains of STIM1 and STIM2 (S1ct and S2ct) and identifying a fundamental action of the powerful SOC modifier, 2-aminoethoxydiphenyl borate (2-APB), the mechanism of which has eluded recent scrutiny. We reveal that 2-APB induces profound, rapid, and direct interactions between S1ct or S2ct and Orai1, effecting full Ca²⁺ release-activated Ca²⁺ (CRAC) current activation. The short 235-505 S1ct coiled-coil region was sufficient for functional Orai1 coupling. YFP-tagged S1ct or S2ct fragments cleared from the cytosol seconds after 2-APB addition, binding avidly to Orai1-CFP with a rapid increase in FRET and transiently increasing CRAC current 200-fold above basal levels. Functional S1ct–Orai1 coupling occurred in STIM1/STIM2^−/− DT40 chicken B cells, indicating ct fragments operate independently of native STIM proteins. The 2-APB-induced S1ct–Orai1 and S2-ct–Orai1 complexes undergo rapid reorganization into discrete colocalized PM clusters, which remain stable for >100 s, well beyond CRAC activation and subsequent deactivation. In addition to defining 2-APB''s action, the locked STIMct–Orai complex provides a potentially useful probe to structurally examine coupling.     

Acquired C1 esterase inhibitor deficiency: An atypical first presentation intra-operatively

We report a case of airway difficulties encountered as a result of blistering, oedema and bleeding from the oropharyngeal mucosa of an 85-year-old female undergoing an elective excision of a submandibular neck lump. This led to a delay in extubation by several hours. The patient was subsequently found to have an acquired form of C1 esterase inhibitor deficiency considered to be consequent of haematological malignancy.     

Initial steps of inactivation at the K+ channel selectivity filter

K⁺ efflux through K⁺ channels can be controlled by C-type inactivation, which is thought to arise from a conformational change near the channel’s selectivity filter. Inactivation is modulated by ion binding near the selectivity filter; however, the molecular forces that initiate inactivation remain unclear. We probe these driving forces by electrophysiology and molecular simulation of MthK, a prototypical K⁺ channel. Either Mg²⁺ or Ca²⁺ can reduce K⁺ efflux through MthK channels. However, Ca²⁺, but not Mg²⁺, can enhance entry to the inactivated state. Molecular simulations illustrate that, in the MthK pore, Ca²⁺ ions can partially dehydrate, enabling selective accessibility of Ca²⁺ to a site at the entry to the selectivity filter. Ca²⁺ binding at the site interacts with K⁺ ions in the selectivity filter, facilitating a conformational change within the filter and subsequent inactivation. These results support an ionic mechanism that precedes changes in channel conformation to initiate inactivation.Potassium (K⁺) channels are activated and opened by a variety of stimuli, including ligand binding and transmembrane voltage, to enable K⁺ efflux and thus, modulate physiological processes related to electrical excitability, such as regulation of action potential firing, smooth muscle contraction, and hormone secretion (1). In addition, many K⁺ channels are further controlled by a gating phenomenon known as C-type inactivation, in which K⁺ conduction is stopped, despite the continued presence of an activating stimulus (2). The mechanisms underlying C-type inactivation in voltage-gated K⁺ channels (Kv channels) are linked to both intracellular and extracellular permeant ion concentrations, and several lines of evidence have suggested that C-type inactivation is associated with a conformational change near the external mouth of the K⁺ channel pore (i.e., at the canonical K⁺ channel selectivity filter) (3–11).In Shaker Kv channels, C-type inactivation is known to be enhanced and recovery from inactivation is slowed by impermeant cations accessing the cytoplasmic side of the channel (5, 6, 10). Enhancement of inactivation by these cations suggests a working hypothesis, in which the impermeant ion prevents refilling of the selectivity filter with K⁺ (6). Thus, K⁺ presumably dissociates from the filter to the external solution, and this vacancy leaves the filter susceptible to a conformational change that underlies the nonconducting, inactivated state. However, the physical basis for the relation between ion movements and C-type inactivation as well as the structural underpinnings of the mechanism remain unclear.Here, we use divalent metal cations (Mg²⁺, Ca²⁺, and Sr²⁺) as probes of inactivation mechanisms in MthK, a model K⁺ channel of known structure (Fig. 1) (12–14). Specifically, we analyze conduction and gating of single MthK channels by electrophysiology combined with analysis of ion and protein movements by molecular simulation. Our electrophysiological experiments indicate that, although each of these divalent metal ions can reduce the size of single channel currents, only Ca²⁺ and Sr²⁺ can enhance inactivation, whereas Mg²⁺ does not. Using molecular simulation and potential of mean force (PMF) calculations, we find that Ca²⁺, but not Mg²⁺, can shed its hydration shell waters to access a site, termed S5, at the entry to the channel’s selectivity filter (Fig. 1C) after displacement of K⁺ ions to the extracellular side of the channel. Subsequent dissociation of a K⁺ ion from the filter, in turn, favors a conformational change within the selectivity filter, contributing to enhanced inactivation. These results support a working hypothesis that directly relates dissociation of K⁺ with a structural change in the selectivity filter to initiate inactivation of K⁺ channels.Open in a separate windowFig. 1.Structure and activation properties of MthK. (A) Presumed biological structure of MthK shown as a Cα-trace [Protein Data Bank (PDB) ID code 3RBZ]. The channel consists of a transmembrane pore domain tethered to a ring of RCK domains, which mediate channel activation by cytoplasmic Ca²⁺ (green spheres). The gray-shaded region represents the presumed plasma membrane; dashed lines represent the linker region between the pore and RCK gating ring that is unresolved in the crystal structure. (B) High-resolution structure of the MthK pore domain, with the selectivity filter shown in ball and stick representation (PDB ID code 3LDC). Subunits in the front and back have been removed for clear visualization of the conduction pathway (inside dashed rectangle), with K⁺ ions shown as purple spheres and ordered water molecules shown as red spheres. (C) –Magnified view of the MthK conduction pathway (boxed region in B) with potential ion binding sites (S0–S_cav) indicated. (D) Po vs. [Ca²⁺] (black symbols) and [Cd²⁺] (red symbols) from currents recorded at −100 mV. MthK activation requires ∼20-fold lower [Cd²⁺] compared with [Ca²⁺]. Curves represent fits with a Hill equation with the following parameters: EC₅₀ = 1.0 mM and n_H = 9.5 for Ca²⁺; EC₅₀ = 49 μM and n_H = 8.4 for Cd²⁺. (E) Representative single channel currents from reconstituted MthK at depolarized voltages with 200 mM KCl at both sides of the membrane and Ca²⁺ or Cd²⁺ at the cytoplasmic side of the channel as indicated. Cd²⁺ can fully activate MthK at concentrations that produce much less fast blockade than Ca²⁺. O and C indicate open and closed current levels, respectively. (F) Unitary current vs. voltage for MthK channels activated with 30 and 100 μM Cd²⁺ (green and red, respectively) and 2 mM Ca²⁺ (black). Smooth curves are drawn for display only; 100 μM Cd²⁺ results in nominal levels of fast blockade, yielding large outward current.