Transport mechanism of DgoT, a bacterial homolog of SLC17 organic anion transporters.

The solute carrier 17 (SLC17) family contains anion transporters that accumulate neurotransmitters in secretory vesicles, remove carboxylated monosaccharides from lysosomes, or extrude organic anions from the kidneys and liver. We combined classical molecular dynamics simulations, Markov state modeling and hybrid first principles quantum mechanical/classical mechanical (QM/MM) simulations with experimental approaches to describe the transport mechanisms of a model bacterial protein, the D-galactonate transporter DgoT, at atomic resolution. We found that protonation of D46 and E133 precedes galactonate binding and that substrate binding induces closure of the extracellular gate, with the conserved R47 coupling substrate binding to transmembrane helix movement.

Expanding the genetic and phenotypic relevance of CLCN4 variants in neurodevelopmental condition: 13 new patients.

Objectives: CLCN4 variations have recently been identified as a genetic cause of X-linked neurodevelopmental disorders. This study aims to broaden the phenotypic spectrum of CLCN4-related condition and correlate it with functional consequences of CLCN4 variants.

Methods: We described 13 individuals with CLCN4-related neurodevelopmental disorder.

Elevator-like movements of prestin mediate outer hair cell electromotility.

The outstanding acuity of the mammalian ear relies on cochlear amplification, an active mechanism based on the electromotility (eM) of outer hair cells. eM is a piezoelectric mechanism generated by little-understood, voltage-induced conformational changes of the anion transporter homolog prestin (SLC26A5). We used a combination of molecular dynamics (MD) simulations and biophysical approaches to identify the structural dynamics of prestin that mediate eM.

Electrophysiological properties of layer 2/3 pyramidal neurons in the primary visual cortex of a retinitis pigmentosa mouse model ().

Retinal degeneration is one of the main causes of visual impairment and blindness. One group of retinal degenerative diseases, leading to the loss of photoreceptors, is collectively termed retinitis pigmentosa. In this group of diseases, the remaining retina is largely spared from initial cell death making retinal ganglion cells an interesting target for vision restoration methods.

Domain- and state-specific shape of the electric field tunes voltage sensing in voltage-gated sodium channels.

The ability to sense transmembrane voltage underlies most physiological roles of voltage-gated sodium (Nav) channels. Whereas the key role of their voltage-sensing domains (VSDs) in channel activation is well established, the molecular underpinnings of voltage coupling remain incompletely understood. Voltage-dependent energetics of the activation process can be described in terms of the gating charge that is defined by coupling of charged residues to the external electric field.

Proton channels in molluscs: A new bivalvian-specific minimal H 4 channel.

Recently, three proton channels (H ) have been identified and characterized in Aplysia californica (AcH 1-3). Focusing on AcH 1 and AcH 2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified H homologous channels in other molluscs: channels homologous to AcH 1 and to AcH 2 were found in 90 species (56 full-length sequences) and in 33 species (18 full-length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, H 4.

Nitric Oxide Sensing by a Blue Fluorescent Protein.

S-Nitrosylation of cysteine residues is an important molecular mechanism for dynamic, post-translational regulation of several proteins, providing a ubiquitous redox regulation. Cys residues are present in several fluorescent proteins (FP), including members of the family of Green Fluorescent Protein (GFP)-derived FPs, where two highly conserved cysteine residues contribute to a favorable environment for the autocatalytic chromophore formation reaction. The effect of nitric oxide on the fluorescence properties of FPs has not been investigated thus far, despite the tremendous role FPs have played for 25 years as tools in cell biology.

Development and Characterization of Flavin-Binding Fluorescent Proteins, Part II: Advanced Characterization.

Flavin-based fluorescent proteins (FbFPs), a class of small fluorescent proteins derived from light-oxygen-voltage (LOV) domains, bind ubiquitous endogenous flavins as chromophores. Due to their unique properties, they can be used as versatile in vivo reporter proteins under aerobic and anaerobic conditions. This chapter presents methodologies for in-depth characterization of the biochemical, spectroscopic, photophysical, and photochemical properties of FbFPs.

A Novel Homozygous KLHL3 Mutation as a Cause of Autosomal Recessive Pseudohypoaldosteronism Type II Diagnosed Late in Life.

Introduction: Pseudohypoaldosteronism type II (PHA II) is a Mendelian disorder, featuring hyperkalemic acidosis and low plasma renin levels, typically associated with hypertension. Mutations in WNK1, WNK4, CUL3, and KLHL3 cause PHA II, with dominant mutations in WNK1, WNK4, and CUL3 and either dominant or recessive mutations in KLHL3. Fourteen families with recessive KLHL3 mutations have been reported, with diagnosis at the age of 3 months to 56 years, typically in individuals with normal kidney function.

Voltage-gated proton channels in polyneopteran insects.

Voltage-gated proton channels (H 1) are expressed in eukaryotes, including basal hexapods and polyneopteran insects. However, currently, there is little known about H 1 channels in insects. A characteristic aspartate (Asp) that functions as the proton selectivity filter (SF) and the RxWRxxR voltage-sensor motif are conserved structural elements in H 1 channels.

Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases.

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments.

Simultaneous recording of multiple cellular signaling events by frequency- and spectrally-tuned multiplexing of fluorescent probes.

Fluorescent probes that change their spectral properties upon binding to small biomolecules, ions, or changes in the membrane potential (V) are invaluable tools to study cellular signaling pathways. Here, we introduce a novel technique for simultaneous recording of multiple probes at millisecond time resolution: (FAST). Different from present multiplexing approaches, FAST uses phase-sensitive signal detection, which renders various combinations of common probes for V and ions accessible for multiplexing.

Accessing Mitochondrial Protein Import in Living Cells by Protein Microinjection.

Mitochondrial protein biogenesis relies almost exclusively on the expression of nuclear-encoded polypeptides. The current model postulates that most of these proteins have to be delivered to their final mitochondrial destination after their synthesis in the cytoplasm. However, the knowledge of this process remains limited due to the absence of proper experimental real-time approaches to study mitochondria in their native cellular environment.

Molecular mechanisms of ion conduction and ion selectivity in TMEM16 lipid scramblases.

TMEM16 lipid scramblases transport lipids and also operate as ion channels with highly variable ion selectivities and various physiological functions. However, their molecular mechanisms of ion conduction and selectivity remain largely unknown. Using computational electrophysiology simulations at atomistic resolution, we identified the main ion-conductive state of TMEM16 lipid scramblases, in which an ion permeation pathway is lined by lipid headgroups that directly interact with permeating ions in a voltage polarity-dependent manner.

The molecular and phenotypic spectrum of CLCN4-related epilepsy.

Objective: This study was undertaken to expand the phenotypic and genetic spectrum of CLCN4-related epilepsy and to investigate genotype-phenotype correlations.

Methods: We systematically reviewed the phenotypic and genetic spectrum of newly diagnosed and previously reported patients with CLCN4-related epilepsy. Three novel variants identified in four patients reported in this study were evaluated through in silico prediction and functional analysis by Western blot, immunofluorescence, and electrophysiological measurements.

Mechanisms Underlying Proton Release in CLC-type F/H Antiporters.

The CLC family of anion channels and transporters includes Cl/H exchangers (blocked by F) and F/H exchangers (or CLCs). CLCs contain a glutamate (E318) in the central anion-binding site that is absent in CLC Cl/H exchangers. The X-ray structure of the protein from (CLC-eca) shows that E318 tightly binds to F when the gating glutamate (E118; highly conserved in the CLC family) faces the extracellular medium.

g_elpot: A Tool for Quantifying Biomolecular Electrostatics from Molecular Dynamics Trajectories.

Electrostatic forces drive a wide variety of biomolecular processes by defining the energetics of the interaction between biomolecules and charged substances. Molecular dynamics (MD) simulations provide trajectories that contain ensembles of structural configurations sampled by biomolecules and their environment. Although this information can be used for high-resolution characterization of biomolecular electrostatics, it has not yet been possible to calculate electrostatic potentials from MD trajectories in a way allowing for quantitative connection to energetics.

Ca β controls the endocytic turnover of Ca 1.2 L-type calcium channel.

Membrane depolarization activates the multisubunit Ca 1.2 L-type calcium channel initiating various excitation coupling responses. Intracellular trafficking into and out of the plasma membrane regulates the channel's surface expression and stability, and thus, the strength of Ca 1.

Na-dependent gate dynamics and electrostatic attraction ensure substrate coupling in glutamate transporters.

Excitatory amino acid transporters (EAATs) harness [Na], [K], and [H] gradients for fast and efficient glutamate removal from the synaptic cleft. Since each glutamate is cotransported with three Na ions, [Na] gradients are the predominant driving force for glutamate uptake. We combined all-atom molecular dynamics simulations, fluorescence spectroscopy, and x-ray crystallography to study Na:substrate coupling in the EAAT homolog Glt A lipidic cubic phase x-ray crystal structure of wild-type, Na-only bound Glt at 2.

Mapping the conducting channels formed along extended defects in SrTiO by means of scanning near-field optical microscopy.

Mixed ionic-electronic-conducting perovskites such as SrTiO are promising materials to be employed in efficient energy conversion or information processing. These materials exhibit a self-doping effect related to the formation of oxygen vacancies and electronic charge carriers upon reduction. It has been found that dislocations play a prominent role in this self-doping process, serving as easy reduction sites, which result in the formation of conducting filaments along the dislocations.

Uncoupling sodium channel dimers restores the phenotype of a pain-linked Na 1.7 channel mutation.

Background And Purpose: The voltage-gated sodium channel Na 1.7 is essential for adequate perception of painful stimuli. Mutations in the encoding gene, SCN9A, cause various pain syndromes in humans.

Single-nucleotide variants in human CD81 influence hepatitis C virus infection of hepatoma cells.

An estimated number of 71 million people are living with chronic hepatitis C virus (HCV) infection worldwide and 400,000 annual deaths are related to the infection. HCV entry into the hepatocytes is complex and involves several host factors. The tetraspanin human CD81 (hCD81) is one of the four essential entry factors and is composed of one large extracellular loop, one small extracellular loop, four transmembrane domains, one intracellular loop and two intracellular tails.

Molecular Basis of CLC Antiporter Inhibition by Fluoride.

CLC channels and transporters conduct or transport various kinds of anions, with the exception of fluoride, which acts as an effective inhibitor. Here, we performed sub-nanosecond DFT-based QM/MM simulations of the anion/proton exchanger ClC-ec1 and observed that fluoride binds incoming protons within the selectivity filter, with excess protons shared with the gating glutamate E148. Depending on E148 conformation, the competition for the proton can involve either a direct F/E148 interaction or the modulation of water molecules bridging the two anions.

Murine Intraepithelial Dendritic Cells Interact With Phagocytic Cells During -Induced Inflammation.

People are constantly exposed to airborne fungal spores, including conidia that can cause life-threatening conditions in immunocompromised patients or acute exacerbations in allergics. However, immunocompetent hosts do not exhibit mycoses or systemic inflammation, due to the sufficient but not excessive antifungal immune response that prevent fungal invasion. Intraepithelial dendritic cells (IE-DCs) of the conducting airway mucosa are located in the primary site of the inhalant pathogen entry; these cells can sense conidia and maintain homeostasis.