Sugar binding of sodium-glucose cotransporters analyzed by voltage-clamp fluorometry.

Sugar absorption is crucial for life and relies on glucose transporters, including sodium-glucose cotransporters (SGLTs). Although the structure of SGLTs has been resolved, the substrate selectivity of SGLTs across diverse isoforms has not been determined owing to the complex substrate-recognition processes and limited analysis methods. Therefore, this study used voltage-clamp fluorometry (VCF) to explore the substrate-binding affinities of human SGLT1 in Xenopus oocytes.

ATP modulates the activity of the voltage-gated proton channel through direct binding interaction.

ATP is an important molecule implicated in diverse biochemical processes, including the modulation of ion channel and transporter activity. The voltage-gated proton channel (Hv1) controls proton flow through the transmembrane pathway in response to membrane potential, and various molecules regulate its activity. Although it is believed that ATP is not essential for Hv1 activity, a report has indicated that cytosolic ATP may modulate Hv1.

Intermolecular functional coupling between phosphoinositides and the potassium channel KcsA.

Biological membranes are composed of a wide variety of lipids. Phosphoinositides (PIPns) in the membrane inner leaflet only account for a small percentage of the total membrane lipids but modulate the functions of various membrane proteins, including ion channels, which play important roles in cell signaling. KcsA, a prototypical K channel that is small, simple, and easy to handle, has been broadly examined regarding its crystallography, in silico molecular analysis, and electrophysiology.

Interaction between S4 and the phosphatase domain mediates electrochemical coupling in voltage-sensing phosphatase (VSP).

Voltage-sensing phosphatase (VSP) consists of a voltage sensor domain (VSD) and a cytoplasmic catalytic region (CCR), which is similar to phosphatase and tensin homolog (PTEN). How the VSD regulates the innate enzyme component of VSP remains unclear. Here, we took a combined approach that entailed the use of electrophysiology, fluorometry, and structural modeling to study the electrochemical coupling in VSP.

Rhodopsin-bestrophin fusion proteins from unicellular algae form gigantic pentameric ion channels.

Many organisms sense light using rhodopsins, photoreceptive proteins containing a retinal chromophore. Here we report the discovery, structure and biophysical characterization of bestrhodopsins, a microbial rhodopsin subfamily from marine unicellular algae, in which one rhodopsin domain of eight transmembrane helices or, more often, two such domains in tandem, are C-terminally fused to a bestrophin channel. Cryo-EM analysis of a rhodopsin-rhodopsin-bestrophin fusion revealed that it forms a pentameric megacomplex (~700 kDa) with five rhodopsin pseudodimers surrounding the channel in the center.

Heterologous functional expression of ascidian Nav1 channels and close relationship with the evolutionary ancestor of vertebrate Nav channels.

Voltage-gated sodium channels (Nav1s) are responsible for the initiation and propagation of action potentials in neurons, muscle, and endocrine cells. Many clinically used drugs such as local anesthetics and antiarrhythmics inhibit Nav1s, and a variety of inherited human disorders are caused by mutations in Nav1 genes. Nav1s consist of the main α subunit and several auxiliary β subunits.

Engineering an enhanced voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) consist of a membrane-spanning voltage sensor domain and a cytoplasmic region that has enzymatic activity toward phosphoinositides (PIs). VSP enzyme activity is regulated by membrane potential, and its activation leads to rapid and reversible alteration of cellular PIP levels. These properties enable VSPs to be used as a tool for studying the effects of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) binding to ion channels and transporters.

The mechanoreceptor DEG-1 regulates cold tolerance in Caenorhabditis elegans.

Caenorhabditis elegans mechanoreceptors located in ASG sensory neurons have been found to sense ambient temperature, which is a key trait for animal survival. Here, we show that experimental loss of xanthine dehydrogenase (XDH-1) function in AIN and AVJ interneurons results in reduced cold tolerance and atypical neuronal response to changes in temperature. These interneurons connect with upstream neurons such as the mechanoreceptor-expressing ASG.

Functional analysis of a double-point mutation in the KCNJ2 gene identified in a family with Andersen-Tawil syndrome.

Andersen-Tawil syndrome (ATS) is a skeletal muscle channelopathy with autosomal dominant inheritance resulting in periodic paralysis, arrhythmia characterized by QT prolongation, and dysmorphic features. The KCNJ2 gene has been identified as the causative gene of ATS. Herein, we reported 2 cases of a 21-year-old man and his mother, with episodic paralytic attacks and/or arrhythmia, which are characteristic of ATS.

The hydrophobic nature of a novel membrane interface regulates the enzyme activity of a voltage-sensing phosphatase.

Voltage-sensing phosphatases (VSP) contain a voltage sensor domain (VSD) similar to that of voltage-gated ion channels but lack a pore-gate domain. A VSD in a VSP regulates the cytoplasmic catalytic region (CCR). However, the mechanisms by which the VSD couples to the CCR remain elusive.

Voltage-Sensing Phosphatases: Biophysics, Physiology, and Molecular Engineering.

Voltage-sensing phosphatase (VSP) contains a voltage sensor domain (VSD) similar to that in voltage-gated ion channels, and a phosphoinositide phosphatase region similar to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). The VSP gene is conserved from unicellular organisms to higher vertebrates. Membrane depolarization induces electrical driven conformational rearrangement in the VSD, which is translated into catalytic enzyme activity.

Domain-to-domain coupling in voltage-sensing phosphatase.

Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization.

Comparison between mouse and sea urchin orthologs of voltage-gated proton channel suggests role of S3 segment in activation gating.

The voltage-gated proton channel, Hv1, is expressed in blood cells, airway epithelium, sperm and microglia, playing important roles in diverse biological contexts including phagocytosis or sperm maturation through its regulation of membrane potential and pH. The gene encoding Hv1, HVCN1, is widely found across many species and is also conserved in unicellular organisms such as algae or dinoflagellates where Hv1 plays role in calcification or bioluminescence. Voltage-gated proton channels exhibit a large variation of activation rate among different species.

Voltage-dependent motion of the catalytic region of voltage-sensing phosphatase monitored by a fluorescent amino acid.

The cytoplasmic region of voltage-sensing phosphatase (VSP) derives the voltage dependence of its catalytic activity from coupling to a voltage sensor homologous to that of voltage-gated ion channels. To assess the conformational changes in the cytoplasmic region upon activation of the voltage sensor, we genetically incorporated a fluorescent unnatural amino acid, 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap), into the catalytic region of Ciona intestinalis VSP (Ci-VSP). Measurements of Anap fluorescence under voltage clamp in Xenopus oocytes revealed that the catalytic region assumes distinct conformations dependent on the degree of voltage-sensor activation.

Effects of unsaturated fatty acids on the kinetics of voltage-gated proton channels heterologously expressed in cultured cells.

Arachidonic acid (AA) greatly enhances the activity of the voltage-gated proton (Hv) channel, although its mechanism of action and physiological function remain unclear. In the present study, we analysed the effects of AA on proton currents through Hv channels heterologously expressed in HEK293T cells. The dramatic increase in proton current amplitude elicited by AA was accompanied by accelerated activation kinetics and a leftward shift in the voltage-dependence of activation.

Evaluation of bifidobacterial adhesion to acidic sugar chains of porcine colonic mucins.

The aim of this study was to assess the adhesion of Bifidobacterium strains to acidic carbohydrate moieties of porcine colonic mucin. Mucins were extracted and purified via gel filtration chromatography followed by density-gradient ultracentrifugation. The presence of sulfated and sialylated carbohydrates in mucins was shown by enzyme-linked immunosorbent assays using PGM34 and HMC31 monoclonal antibodies (mAbs), respectively.

X-ray crystal structure of voltage-gated proton channel.

The voltage-gated proton channel Hv1 (or VSOP) has a voltage-sensor domain (VSD) with dual roles of voltage sensing and proton permeation. Its gating is sensitive to pH and Zn(2+). Here we present a crystal structure of mouse Hv1 in the resting state at 3.

Comparative FTIR study of a new fungal rhodopsin.

Bacteriorhodopsin (BR) is a light-driven proton pump of halophilic Archaea , and BR-like proton-pumping rhodopsins have been discovered in Bacteria and Eucarya as well. Leptosphaeria rhodopsin (LR) and Phaeosphaeria Rhodopsin 2 (PhaeoRD2) are both fungal rhodopsins in such a functional class, even though they belong to different branches of the phylogenetic tree. In this study, we compared light-induced structural changes in the K, L, and M photointermediates for PhaeoRD2, LR, and BR using low-temperature Fourier transform infrared (FTIR) spectroscopy.

Anomalous pH Effect of Blue Proteorhodopsin.

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (B-PR; λmax ≈ 490 nm) and green-absorbing PR (G-PR; λmax ≈ 525 nm). In this report, we present conversion of B-PR into G-PR using anomalous pH effect. B-PR in LC1-200, marine γ-proteobacteria, absorbs 497 and 513 nm maximally at pH 7 and 4, respectively, whose pH titration was reversible (pKa = 4.

Diversity and functional analysis of proteorhodopsin in marine Flavobacteria.

Proteorhodopsin (PR) genes are widely distributed among marine prokaryotes and functions as light-driven proton pump when expressed heterologously in Escherichia coli, suggesting that light energy passing through PR may be substantial in marine environment. However, there are no data on PR proton pump activities in native marine bacteria. Here, we demonstrate light-driven proton pump activity (c.

Structural characteristics around the β-ionone ring of the retinal chromophore in Salinibacter sensory rhodopsin I.

Organisms sense and respond to environmental stimuli through membrane-embedded receptors and transducers. Sensory rhodopsin I (SRI) and sensory rhodopsin II (SRII) are the photoreceptors for the positive and negative phototaxis in microorganisms, respectively. They form signaling complexes in the membrane with their cognate transducer proteins, HtrI and HtrII, and these SRI-HtrI and SRII-HtrII complexes transmit a light signal through their cytoplasmic sensory signaling system, inducing opposite effects (i.

Chimeric microbial rhodopsins containing the third cytoplasmic loop of bovine rhodopsin.

G-protein-coupled receptors transmit stimuli (light, taste, hormone, neurotransmitter, etc.) to the intracellular signaling systems, and rhodopsin (Rh) is the most-studied G-protein-coupled receptor. Rh possesses an 11-cis retinal as the chromophore, and 11-cis to all-trans photoisomerization leads to the protein structural changes in the cytoplasmic loops to activate G-protein.

An inward proton transport using Anabaena sensory rhodopsin.

ATP is synthesized by an enzyme that utilizes proton motive force and thus nature creates various proton pumps. The best understood proton pump is bacteriorhodopsin (BR), an outward-directed light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity.

Importance of alanine at position 178 in proteorhodopsin for absorption of prevalent ambient light in the marine environment.

It is usually assumed that only amino acids located near the retinal chromophore are responsible for color tuning of rhodopsins. However, we recently found that replacement of Ala178 with Arg in the E-F loop of proteorhodopsin (PR), an archaeal-type rhodopsin in marine bacteria, shifts the lambda(max) from 525 to 545 nm at neutral pH [Yoshitsugu, M., Shibata, M.

Engineering an inward proton transport from a bacterial sensor rhodopsin.

ATP is synthesized by an enzyme that utilizes proton motive force, and thus, nature has created various proton pumps. The best-understood proton pump is bacteriorhodopsin (BR), an outward-directed, light-driven proton pump in Halobacterium salinarum. Many archaeal and eubacterial rhodopsins are now known to show similar proton transport activity.