ABSTRACT
Voltage-dependent potassium (Kv) channels allow for the selective permeability of potassium ions in a membrane potential dependent manner, playing crucial roles in neurotransmission and muscle contraction. Kv channel is a tetramer, in which each subunit possesses a voltage-sensing domain (VSD) and a pore domain (PD). Although several lines of evidence indicated that membrane depolarization is sensed as the movement of helix S4 of the VSD, the detailed voltage-sensing mechanism remained elusive, due to the difficulty of structural analyses at resting potential. In this study, we conducted a comprehensive disulfide locking analysis of the VSD using 36 double Cys mutants, in order to identify the proximal residue pairs of the VSD in the presence or absence of a membrane potential. An intramolecular SS-bond was formed between 6 Cys pairs under both polarized and depolarized environment, and one pair only under depolarized environment. The multiple conformations captured by the SS-bond can be divided by two states, up and down, where S4 lies on the extracellular and intracellular sides of the membrane, respectively, with axial rotation of 180°. The transition between these two states is caused by the S4 translocation of 12 Å, enabling allosteric regulation of the gating at the PD.
Subject(s)
Archaeal Proteins/chemistry , Ion Channel Gating , Potassium Channels, Voltage-Gated/chemistry , Aeropyrum , Amino Acid Substitution , Archaeal Proteins/genetics , Archaeal Proteins/physiology , Bridged Bicyclo Compounds/chemistry , Cystine/chemistry , Cystine/genetics , Fluorescent Dyes/chemistry , Liposomes/chemistry , Membrane Potentials , Potassium Channels, Voltage-Gated/genetics , Potassium Channels, Voltage-Gated/physiology , Protein Conformation, alpha-Helical , Spectrometry, FluorescenceABSTRACT
Voltage-dependent K(+) (Kv) channels play crucial roles in nerve and muscle action potentials. Voltage-sensing domains (VSDs) of Kv channels sense changes in the transmembrane potential, regulating the K(+)-permeability across the membrane. Gating modifier toxins, which have been used for the functional analyses of Kv channels, inhibit Kv channels by binding to VSD. However, the structural basis for the inhibition remains elusive. Here, fluorescence and NMR analyses of the interaction between VSD derived from KvAP channel and its gating modifier toxin, VSTx1, indicate that VSTx1 recognizes VSD under depolarized condition. We identified the VSD-binding residues of VSTx1 and their proximal residues of VSD by the cross-saturation (CS) and amino acid selective CS experiments, which enabled to build a docking model of the complex. These results provide structural basis for the specific binding and inhibition of Kv channels by gating modifier toxins.
Subject(s)
Potassium Channel Blockers/chemistry , Potassium Channel Blockers/pharmacology , Potassium Channels, Voltage-Gated/antagonists & inhibitors , Potassium Channels, Voltage-Gated/chemistry , Toxins, Biological/chemistry , Toxins, Biological/pharmacology , Binding Sites , Ion Channel Gating/drug effects , Models, Molecular , Molecular Conformation , Molecular Docking Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Interaction Domains and Motifs , Structure-Activity RelationshipABSTRACT
Alginate (ALA), which is an intercellular polysaccharide associated with brown algae, is used as a food additive, a health food and a medicine. Here, we first examined the adsorption of strontium (Sr) and cesium (Cs) by ALA in vitro, and then evaluated the effects of ALA on absorption and excretion of Sr and Cs in rats, in order to evaluate its potential usefulness for minimizing radiation damage from materials released after a nuclear accident. Both Sr and Cs were concentration-dependently adsorbed by sodium alginate (ALA-Na) in vitro. In rats given diet containing either ALA-Na or calcium alginate (ALA-Ca) for two weeks, the plasma concentration of Sr gradually decreased compared with the controls (normal diet); however, in the case of Cs, the plasma concentration was decreased only in the ALA-Ca group, but not the ALA-Na group. Moreover, we examined the effect of preadministration of diet containing either ALA-Na or ALA-Ca on absorption of Sr and Cs administered orally as the chloride salts to rats. Absorption of both Sr and Cs was reduced in the ALA-Ca group, while absorption of only Sr was reduced in the ALA-Na group. Safety assessments indicated that ALA-Ca is safer than ALA-Na. These results indicate that ALA-Ca reduces absorption and promotes excretion of both Sr and Cs, while ALA-Na does so only for Sr.
Subject(s)
Alginates/pharmacology , Cesium/pharmacokinetics , Strontium/pharmacokinetics , Absorption , Alginates/toxicity , Animals , Dose-Response Relationship, Drug , Glucuronic Acid/pharmacology , Glucuronic Acid/toxicity , Hexuronic Acids/pharmacology , Hexuronic Acids/toxicity , Male , Rats , Rats, WistarABSTRACT
MD-1 is a glycoprotein that associates with a B-cell-specific RP105 protein and has a low sequence identity of 16% to MD-2 that associates with Toll-like receptor 4 and recognizes endotoxic lipopolysaccharide. MD-1 and RP105 are supposed to mediate lipopolysaccharide recognition; however, little is known about their structures and functions. Here, the crystal structure of mouse MD-1 is determined at 1.65 A resolution. MD-1 has a hydrophobic cavity sandwiched by two beta-sheets as is MD-2. The cavity is 25 A long, 5 A wide, and 10 A deep: longer, narrower, and shallower than that of MD-2. No charged residues are located on the cavity entrance. MD-1 is primarily monomeric in solution but shows a dimeric assembly in the crystal lattices, with their cavity entrances facing each other. In the cavity, electron densities attributable to phosphatidylcholine are located. Together with the binding assay with tetra-acylated lipid IVa, MD-1 is shown to be a lipid-binding coreceptor.
