Mapping the conformational wave of acetylcholine receptor channel gating

Allosteric transitions allow fast regulation of protein function in living systems. Even though the end points of such conformational changes are known for many proteins, the characteristics of the paths connecting these states remain largely unexplored. Rate-equilibrium linear free-energy relationships (LFERs) provide information about such pathways by relating changes in the free energy of the transition state to those of the ground states upon systematic perturbation of the system. Here we present an LFER analysis of the gating reaction pathway of the muscle acetylcholine receptor. We studied the closed <==> open conformational change at the single-molecule level following perturbation by series of single-site mutations, agonists and membrane voltages. This method provided a snapshot of several regions of the receptor at the transition state in terms of their approximate positions along the reaction coordinate, on a scale from 0 (closed-like) to 1 (open-like). The resulting map reveals a spatial gradient of positional values, which suggests that the conformational change proceeds in a wave-like manner, with the low-to-high affinity change at the transmitter-binding sites preceding the complete opening of the pore.     

Role of acetylcholine receptor subunits in gating of the channel

The Torpedo and calf acetylcholine receptors and hybrids composed of subunits from the two species have been produced in Xenopus oocytes by the use of the cloned complementary DNAs. Single-channel current measurements indicate that these receptors form channels of similar conductance but with different gating behaviour.     

A stepwise mechanism for acetylcholine receptor channel gating

Muscle contraction is triggered by the opening of acetylcholine receptors at the vertebrate nerve-muscle synapse. The M2 helix of this allosteric membrane protein lines the channel, and contains a 'gate' that regulates the flow of ions through the pore. We used single-molecule kinetic analysis to probe the transition state of the gating conformational change and estimate the relative timing of M2 motions in the alpha-subunit of the murine acetylcholine receptor. This analysis produces a 'Phi-value' for a given residue that reflects its open-like versus closed-like character at the transition state. Here we show that most of the residues throughout the length of M2 have a Phi-value of approximately 0.64 but that some near the middle have lower Phi-values of 0.52 or 0.31, suggesting that alphaM2 moves in three discrete steps. The core of the channel serves both as a gate that regulates ion flow and as a hub that directs the propagation of the gating isomerization through the membrane domain of the acetylcholine receptor.     

A prokaryotic proton-gated ion channel from the nicotinic acetylcholine receptor family

Ligand-gated ion channels (LGICs) mediate excitatory and inhibitory transmission in the nervous system. Among them, the pentameric or 'Cys-loop' receptors (pLGICs) compose a family that until recently was found in only eukaryotes. Yet a recent genome search identified putative homologues of these proteins in several bacterial species. Here we report the cloning, expression and functional identification of one of these putative homologues from the cyanobacterium Gloeobacter violaceus. It was expressed as a homo-oligomer in HEK 293 cells and Xenopus oocytes, generating a transmembrane cationic channel that is opened by extracellular protons and shows slow kinetics of activation, no desensitization and a single channel conductance of 8 pS. Electron microscopy and cross-linking experiments of the protein fused to the maltose-binding protein and expressed in Escherichia coli are consistent with a homo-pentameric organization. Sequence comparison shows that it possesses a compact structure, with the absence of the amino-terminal helix, the canonical disulphide bridge and the large cytoplasmic domain found in eukaryotic pLGICs. Therefore it embodies a minimal structure required for signal transduction. These data establish the prokaryotic origin of the family. Because Gloeobacter violaceus carries out photosynthesis and proton transport at the cytoplasmic membrane, this new proton-gated ion channel might contribute to adaptation to pH change.     

Ion channel of acetylcholine receptor reconstructed from images of postsynaptic membranes

The nicotinic acetylcholine receptor belongs to a class of molecules that respond transiently to chemical stimuli by opening a water-filled channel through the cell membrane for cations to diffuse. This channel lies along the central axis delineated by a ring of five homologous, membrane-spanning subunits and thus has properties, such as conductance and ion selectivity, which depend on the profile created by the encircling subunits. Insight has been gained recently about the amino-acid residues implicated directly in the ion transport, and some information about the subunit configuration around the channel has come from electron microscopy studies of postsynaptic membranes crystallized in the form of flattened tubular vesicles. The resolution along the axis of the channel has, however, been limited by the restricted range of views obtainable. Here we report the structure of the channel at 17 A resolution, determined by three-dimensional image reconstruction from tubular vesicles having receptors organized in helical arrays across their surfaces. The helical symmetry is preserved by suspending the tubes in thin films of ice, and the receptors in such tubes can be seen from all angles, allowing the channel to be revealed clearly in relation to the lipid bilayer and the peripheral protein for the first time.     

Location of a delta-subunit region determining ion transport through the acetylcholine receptor channel

The combination of complementary DNA expression and single-channel current analysis provides a powerful tool for studying the structure-function relationship of the nicotinic acetylcholine receptor (AChR) (refs 1-5). We have previously shown that AChR channels consisting of subunits from different species, expressed in the surface membrane of Xenopus oocytes, can be used to relate functional properties to individual subunits. Here we report that, in extracellular solution of low divalent cation concentration, the bovine AChR channel has a smaller conductance than the Torpedo AChR channel. Replacement of the delta-subunit of the Torpedo AChR by the bovine delta-subunit makes the channel conductance similar to that of the bovine AChR channel. To locate the region in the delta-subunit responsible for this difference, we have constructed chimaeric delta-subunit cDNAs with different combinations of the Torpedo and bovine counterparts. The conductances of AChR channels containing chimaeric delta-subunits suggest that a region comprising the putative transmembrane segment M2 and the adjacent bend portion between segments M2 and M3 is involved in determining the rate of ion transport through the open channel.     

Multiple conductance channels in type-2 cerebellar astrocytes activated by excitatory amino acids

L-GLUTAMATE and L-aspartate are thought to have a widespread function as synaptic transmitters in the mammalian central nervous system and there are at least three types of neuronal glutamate receptors, which can be activated by the selective agonists N-methyl-D-aspartate (NMDA), quisqualate and kainate. Recent experiments indicate that glutamate receptors also occur in astrocytes. We have used patch-clamp methods to determine whether one type of macroglial cell, the type-2 astrocyte, possesses glutamate receptors, as previously proposed from neurochemical studies. We find that glutamate and related amino acids can evoke whole-cell and single-channel currents in type-2 astrocytes from rat cerebellum. Although these cells are found mainly in white matter, where neurotransmission does not occur, their processes are closely associated with axons at nodes of Ranvier, suggesting that such receptors are involved in neuronal-glial signalling at the node. Our experiments show that glial cells possess quisqualate- and kainate-receptor channels but lack receptors for NMDA. Interestingly, these glutamate channels exhibit multiple conductance levels that are similar in amplitude to the neuronal glutamate channels.     

Transport of cationic amino acids by the mouse ecotropic retrovirus receptor.

Susceptibility of rodent cells to infection by ecotropic murine leukaemia viruses (MuLV) is determined by binding of the virus envelope to a membrane receptor that has multiple membrane-spanning domains. Cells infected by ecotropic MuLV synthesize envelope protein, gp70, which binds to this receptor, thereby preventing additional infections. The consequences of envelope-MuLV receptor binding for the infected host cell have not been directly determined, partly because the cellular function of the MuLV receptor protein is unknown. Here we report a coincidence in the positions of the first eight putative membrane-spanning domains found in the virus receptor and in two related proteins, the arginine and histidine permeases of Saccharomyces cerevisiae (Fig. 1), but not in any other proteins identified by computer-based sequence comparison of the GenBank data base. Xenopus oocytes injected with receptor-encoding messenger RNA show increased uptake of L-arginine, L-lysine and L-ornithine. The transport properties and the expression pattern of the virus receptor behave in ways previously attributed to y+, the principal transporter of cationic L-amino acids in mammalian cells.     

Quaternary structure of the acetylcholine receptor

The five membrane-spanning subunits of the acetylcholine receptor have been resolved in electron microscope images and are shown to lie at pentagonally symmetrical positions around the channel over a large fraction of their length. The channel consists of a wide synaptic portion and a narrow portion extending through the membrane into the interior of the cell.     

氨基酸衍生物的电化学研究及碱性氨基酸的极谱测定

本文研究了多种氨基酸与水杨醛在碱性介质中的衍生化及其产物（席夫碱）的电化学行为。结果表明：席夫碱的生成和氨基酸的测定灵敏度受氨基酸的性质影响很大，碱性氨基酸能形成很好的极谐波，检测限低于１×１０－６ｍｏｌ／Ｌ，用此法测定了食品中的赖氨酸含量和血清中蛋白质总量，得到较满意的结果．     

Topographical rearrangement of acetylcholine receptors alters channel kinetics

Plasma membranes are dynamic structures of proteins and lipids. Protein-protein or protein-lipid interactions within the membrane are believed to have important roles in many membrane functions, including ion transport, enzyme activity and signal reception. The acetylcholine (ACh) receptor-channel complex in skeletal muscle membrane is one of the best known integral membrane proteins. Its ion transport function is accessible to direct measurement at the single-channel level by the use of the 'giga-seal' patch recording technique. Here we used an in situ electrophoresis technique to rearrange the topography of pre-existing ACh receptor-channels in the muscle membrane, and measured the single-channel kinetics of ACh-activated channels in two different molecular environments within the membrane: those in the diffusely distributed region and those in the ACh receptor clusters induced by the applied field. We found that the channel kinetics are significantly prolonged in the ACh receptor cluster compared with the non-clustered region of the same cell. This result strongly supports the notion that the function of a membrane ionic channel depends on the local molecular environment.     

Preparation and characterization of some surface negatively charged residue mutants of cytochrome b5

Site-directed mutagenesis was used to obtain seven variants of tryptic fragment of bovine liver cytochrome b_m5 (cyt b₅), in which the negatively charged residues around the heme exposed edge of cyt b₅ were replaced by hydrophobic amino acid alanine. Double-site mutants, triple-site mutants and even quadruple-site mutants were obtained. DNA sequencing and molecular weight measurements of the mutant proteins both confirmed that these site-directed mutagenesises were successfully performed. Spectroelectrochemistry of these mutant proteins revealed that the apparent redox potentials of these mutant proteins caused a positive shift of 2–10 mV. The global structure of these mutant proteins did not show much difference from that of the wild type cyt b₅, providing a solid base for the further study on the roles of the proteins’ surface charges.     

Structure and gating mechanism of the acetylcholine receptor pore

The nicotinic acetylcholine receptor controls electrical signalling between nerve and muscle cells by opening and closing a gated, membrane-spanning pore. Here we present an atomic model of the closed pore, obtained by electron microscopy of crystalline postsynaptic membranes. The pore is shaped by an inner ring of 5 alpha-helices, which curve radially to create a tapering path for the ions, and an outer ring of 15 alpha-helices, which coil around each other and shield the inner ring from the lipids. The gate is a constricting hydrophobic girdle at the middle of the lipid bilayer, formed by weak interactions between neighbouring inner helices. When acetylcholine enters the ligand-binding domain, it triggers rotations of the protein chains on opposite sides of the entrance to the pore. These rotations are communicated through the inner helices, and open the pore by breaking the girdle apart.     

Immunochemical tests of acetylcholine receptor subunit models

Acetylcholine receptors of fish electric organs and mammalian skeletal muscle comprise four structurally homologous glycoprotein subunits in the mole ratio alpha 2 beta gamma delta (refs 1-4). All four subunits have leader sequences and are exposed on both sides of the membrane. From amino acid sequencing, three groups have predicted that each subunit has four hydrophobic alpha-helical transmembranous domains. Because the N-terminus of each subunit is thought to remain on the extracellular surface after cleavage of the leader sequence, this model predicts that the N- and C- termini are both on the extracellular side. An alternative model proposed by two other groups predicts that there is, in addition, a fifth amphipathic transmembranous domain which would place the C-terminus on the cytoplasmic side. Here, using anti-subunit sera and monoclonal antibodies and their reaction with synthetic subunit peptides, we demonstrate that the C-terminus is in fact on the cytoplasmic surface. We also show that, contrary to other predictions, the most hydrophilic sequence on the extracellular domain of alpha-subunits is not the main immunogenic region.