Novel FGF9 variant contributes to multiple synostoses syndrome 3.

Multiple synostoses syndromes (SYNS) are autosomal dominant syndromes characterized by multiple joint fusions commonly involving the carpal-tarsal, interphalangeal, humeroradial, and cervical spine joints. They display genetic heterogeneity with pathogenic variants reported in four separate genes (NOG, GDF5, FGF9, and GDF6) defining four different SYNS forms. FGF9 variants have been reported in SYNS3, a SYNS with multiple synostoses, normal cognition, normal hearing, and craniosynostosis. Here, we report a novel FGF9 c.569G > C p.(Arg190Thr) variant identified by whole-exome sequencing in a patient with multiple bony abnormalities. The patient initially presented with elbow instability and decreased range of motion. Imaging revealed bilateral radial head deformities, carpal-tarsal fusions, brachydactyly, and osteoarthritis of the sacroiliac joints. In silico protein modeling of the identified FGF9 variant predicts decreased stability of ligand-receptor binding supporting the pathogenicity of this finding. This finding expands the repertoire of FGF9 variants and phenotypic information reported for SYNS3 and suggest that genotype phenotype correlations due to localization seem less likely and more so due to the consequence of the pathogenic variant on the receptor. This is useful in the counseling in families as more de novo variants emerge.

Structures of KcsA in complex with symmetrical quaternary ammonium compounds reveal a hydrophobic binding site.

Potassium channels allow for the passive movement of potassium ions across the cell membrane and are instrumental in controlling the membrane potential in all cell types. Quaternary ammonium (QA) compounds block potassium channels and have long been used to study the functional and structural properties of these channels. Here we describe the interaction between three symmetrical hydrophobic QAs and the prokaryotic potassium channel KcsA. The structures demonstrate the presence of a hydrophobic pocket between the inner helices of KcsA and provide insight into the binding site and blocking mechanism of hydrophobic QAs. The structures also reveal a structurally hidden pathway between the central cavity and the outside membrane environment reminiscent of the lateral fenestration observed in sodium channels that can be accessed through small conformational changes in the pore wall. We propose that the hydrophobic binding pocket stabilizes the alkyl chains of long-chain QA molecules and may play a key role in hydrophobic drug binding in general.

Electron spin-echo envelope modulation (ESEEM) reveals water and phosphate interactions with the KcsA potassium channel.

Electron spin-echo envelope modulation (ESEEM) spectroscopy is a well-established technique for the study of naturally occurring paramagnetic metal centers. The technique has been used to study copper complexes, hemes, enzyme mechanisms, micellar water content, and water permeation profiles in membranes, among other applications. In the present study, we combine ESEEM spectroscopy with site-directed spin labeling (SDSL) and X-ray crystallography in order to evaluate the technique's potential as a structural tool to describe the native environment of membrane proteins. Using the KcsA potassium channel as a model system, we demonstrate that deuterium ESEEM can detect water permeation along the lipid-exposed surface of the KcsA outer helix. We further demonstrate that (31)P ESEEM is able to identify channel residues that interact with the phosphate headgroup of the lipid bilayer. In combination with X-ray crystallography, the (31)P data may be used to define the phosphate interaction surface of the protein. The results presented here establish ESEEM as a highly informative technique for SDSL studies of membrane proteins.

Structural basis of TEA blockade in a model potassium channel.

Potassium channels catalyze the selective transfer of potassium across the cell membrane and are essential for setting the resting potential in cells, controlling heart rate and modulating the firing pattern in neurons. Tetraethylammonium (TEA) blocks ion conduction through potassium channels in a voltage-dependent manner from both sides of the membrane. Here we show the structural basis of TEA blockade by cocrystallizing the prokaryotic potassium channel KcsA with two selective TEA analogs. TEA binding at both sites alters ion occupancy in the selectivity filter; these findings underlie the mutual destabilization and voltage-dependence of TEA blockade. We propose that TEA blocks potassium channels by acting as a potassium analog at the dehydration transition step during permeation.

Generation of a calmodulin-based EPR calcium indicator.

Calmodulin is a ubiquitous calcium dependent protein. In the presence of calcium, calmodulin adopts an altered conformation that leads to the generation of downstream cellular calcium signals. Here we describe the introduction of nitroxide EPR probes into calmodulin by means of site-directed spin labeling. These probes sense the calcium-dependent conformational change of calmodulin and, therefore, can serve as calcium indicators. In combination with a light-sensitive calcium chelator, spin-labeled calmodulin can be used to demonstrate calcium release by flash photolysis. These results provide an important step toward describing the molecular dynamics of calcium-induced conformational changes in proteins using EPR spectroscopy.

The structure of the lipid-embedded potassium channel voltage sensor determined by double-electron-electron resonance spectroscopy.

A four-pulse electron paramagnetic resonance experiment was used to measure long-range inter-subunit distances in reconstituted KvAP, a voltage-dependent potassium (Kv) channel. The measurements have allowed us to reach the following five conclusions about the native structure of the voltage sensor of KvAP. First, the S1 helix of the voltage sensor engages in a helix packing interaction with the pore domain. Second, the crystallographically observed antiparallel helix-turn-helix motif of the voltage-sensing paddle is retained in the membrane-embedded voltage sensor. Third, the paddle is oriented in such a way as to expose one face to the pore domain and the opposite face to the membrane. Fourth, the paddle and the pore domain appear to be separated by a gap that is sufficiently wide for lipids to penetrate between the two domains. Fifth, the critical voltage-sensing arginine residues on the paddle appear to be lipid exposed. These results demonstrate the importance of the membrane for the native structure of Kv channels, suggest that lipids are an integral part of their native structure, and place the voltage-sensing machinery into a complex lipid environment near the pore domain.

Bending membranes into different shapes.

BAR domains bend membranes by imposing their curved shape. In this issue, Isas et al. show the structural differences in the interaction of the BAR domain protein amphiphysin with vesicles and tubes. They find that superficial interactions lead to vesicles, whereas more penetrating interactions of a more crowded protein lead to tubes.

Spin Labeling of Potassium Channels.

Potassium channels are the ion channels most extensively studied by structural techniques. Whereas high-resolution crystal structures have provided key insights into the molecular architecture of these channels, spin labeling studies have helped to unveil the dynamic structural aspects underlying their function. From a practical standpoint, the popularity of spin labeling studies of potassium channels lies in their small size and relative ease of overexpression. The inherent fourfold symmetry of most potassium channels has also greatly facilitated spin labeling studies. This chapter focuses on the overexpression, purification, spin labeling, and subsequent reconstitution of modified potassium channels. It will discuss the general methods used to produce a suitable spin-labeled potassium channel sample and highlight some of the common pitfalls that can occur along the way. At the end of the chapter, we provide detailed methods to produce spin-labeled samples of KcsA and KvAP, the two most commonly studied potassium channels.

Uso de la escala LRINEC en pacientes con infecciones necrosantes en el miembro superior y su correlación con la morbimortalidad ortopédica / LRINEC score diagnostic value for necrotizing infections of the upper extremity and correlation with morbidity and mortality in orthopedics patients

Objetivo: Utilizar la escala LRINEC en pacientes con infecciones necrosantes de miembros superiores y evaluar su correlación con la morbimortalidad ortopédica. Materiales y Métodos: Se llevó a cabo una revisión sistemática de las historias clínicas de los pacientes operados por nuestro equipo, entre el 1 de marzo de 2015 y el 1 de marzo de 2020. Se registraron los puntajes de la escala LRINEC de cada paciente operado con diagnóstico clínico y posoperatorio de infección necrosante de partes blandas, así como sus antecedentes clínicos, el microorganismo, las complicaciones y la morbimortalidad ortopédica, y otros datos clínicos importantes (tiempo de internación en terapia intensiva, necesidad de asistencia respiratoria mecánica y de diálisis, cantidad de cirugías), y se los comparó con el puntaje. Resultados: Se analizaron 4126 historias clínicas de pacientes operados por nuestro equipo. Tres tuvieron infecciones necrosantes del miembro superior. El puntaje aplicado en forma retrospectiva determinó que todos tenían una alta probabilidad de sufrir una infección necrosante. Los pacientes con puntaje más alto desarrollaron más comorbilidades ortopédicas y clínicas. Conclusiones: La escala LRINEC es un instrumento reproducible para el diagnóstico de infecciones necrosantes de partes blandas y está relacionada con el número de complicaciones y la morbilidad ortopédica, aunque no necesariamente con la cantidad de cirugías realizadas. Nivel de Evidencia: II

Objective: To use the LRINEC scoring system for necrotizing infections of the upper extremity and study its correlation with morbidity and mortality in Orthopedics patients Materials and Methods: We conducted a systematic review of the medical records of patients operated on by our team between March 1, 2015, and March 1, 2020. Data collection included the LRINEC scores of every patient who underwent surgery and had a clinical and postoperative diagnosis of necrotizing soft tissue infection, as well as their clinical history, causative organism, complications, Orthopedics-related morbidity and mortality data, and other significant clinical data (length of intensive care stay, need for mechanical respiratory assistance, need for dialysis, number of surgeries), which were then compared with their respective LRINEC score. Results: The review included 4126 medical records of patients who had undergone surgery by our team. There were three recorded cases of necrotizing infections in the upper extremity. Their LRINEC scores were retrospectively calculated and all of them showed a high risk of developing a necrotizing infection. The patients with the highest scores developed more Orthopedics and other clinical conditions. Conclusions: The LRINEC score is a reproducible method for the diagnosis of necrotizing soft tissue infections and is related to the number of complications and orthopedic conditions, although not necessarily with the number of surgeries. Level of Evidence: II

Potassium channel gating observed with site-directed mass tagging.

Potassium channels allow the selective flow of K+ ions across otherwise impermeable membranes. During a process called gating, these channels undergo a conformational change that proceeds from a closed to an open state. The closed state of KcsA, a prokaryotic potassium channel, has been structurally well characterized with equilibrium structural techniques. However, attempts to obtain a structural description of the gating transition of the channel have been hampered because the open state is only transiently occupied and, therefore, not readily accessible to such techniques. Here we describe a non-equilibrium technique that we call site-directed mass tagging and use this technique to probe the conformational change that KcsA undergoes during gating. The results indicate that KcsA is a dynamically modular molecule; the extracellular half of the membrane-spanning region is held rigid during gating, while the intracellular half undergoes a significant conformational change.

Identification of protein side chains near the membrane-aqueous interface: a site-directed spin labeling study of KcsA.

This study presents an approach to identifying surface residues on membrane proteins that are exposed toward the membrane-aqueous interface. The method employs a lipid Ni(II) chelate that localizes the metal ion to a region near the membrane-aqueous interface. Lateral diffusion of the lipid chelate results in Heisenberg exchange (HE) with nitroxide side chains in the protein only if direct contact occurs between the paramagnetic species during a collision. Thus, HE serves as a signature for residues facing the bilayer in the neighborhood of the membrane-aqueous interface. To evaluate the method, 13 surface residues on the extracellular half of KcsA, a prokaryotic potassium channel of known structure, were examined for HE with the Ni(II) chelate. The HE rate between the two species is found to depend strongly on the vertical position of the nitroxide with respect to the membrane-aqueous interface. Nitroxides introduced near the interface experience relatively high HE rates, whereas nitroxides that are immersed in the bilayer interior or sterically sheltered from collision experience low or undetectable rates. The results indicate that residues near the interface can be identified on the basis of their high rates of collision with the headgroup region of the bilayer.