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N Unwin

Publications and source records attributed to N Unwin.

At least 19 recordsLinked to original sources
PubMed1995

Acetylcholine receptor channel imaged in the open state.

The structure of the open-channel form of the acetylcholine receptor has been determined from electron images of Torpedo ray postsynaptic membranes activated by brief (< 5 ms) mixing with droplets containing acetylcholine. Comparison with the closed-channel form shows that acetylcholine initiates small rotations of the subunits in the extracellular domain, which trigger a change in configuration of alpha-helices lining the membrane-spanning pore. The open pore tapers towards the intracellular membrane face, where it is shaped by a 'barrel' of alpha-helices having a pronounced right-handed twist.

Acetylcholine↗
PubMed1995

Three-dimensional location of the main immunogenic region of the acetylcholine receptor.

About two-thirds of the antibodies to the nicotinic acetylcholine (ACh) receptor in patients with the autoimmune disease myasthenia gravis bind to the main immunogenic region (MIR). This is small, well-defined region on each of the two alpha subunits, containing residues 67-76 (alpha 67-76). By determining the structure of the ACh receptor complexed with two different fragments of an MIR-directed antibody, we have determined the three-dimensional location of the MIR (and therefore residues alpha 67-76) to be at the extreme synaptic end of each alpha subunit. The antibody fragments extend from the binding site away from the receptor axis and into the synaptic cleft, minimizing any steric interference neighboring ACh receptors might have with their binding.

Animals↗
PubMed1994

Images of purified Shaker potassium channels.

BACKGROUND: Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. RESULTS: We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. CONCLUSION: These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.

Animals↗
PubMed1994

Analysis of transient structures by cryo-microscopy combined with rapid mixing of spray droplets.

A simple method to determine transient conformations of biological molecules is described. The two reactants (e.g. protein complex and ligand) are mixed rapidly by the coalescence of spray droplets containing one component, with a thin, grid-supported aqueous film containing the other. The transient state is then trapped by rapid freezing, and investigated later by cryo-microscopy. Images of conformations associated with reaction times of 1-100 ms can be achieved by adjusting the delay between the droplet impact and freezing. The droplets (typically 1 micron in diameter) are propelled onto the grid by an atomizer spray. It is shown that the droplets impinging on the liquid film spread rapidly over its surface under the influence of surface tension, and only weakly disturb the underlying film, partially displacing its contents away from the point of impact. Experiments with sprayed salt solutions, using vesicles derived from erythrocytes as micro-osmometers, indicate that rapid mixing occurs both through the film and laterally, by diffusion. The spraying process does not produce any detectable concentration changes due to drying in either the droplets or the film, and the method is applicable to high-resolution imaging.

Acetylcholine↗
PubMed1992

On your bikes.

Explore the source record for details and available documents.

Bicycling↗
PubMed1992

Structure of gap junction channels.

Gap junctions are regions of contact between adjacent cells, consisting of arrays of channels linking the cell interiors. The channels are formed by polypeptides called connexins; the amino acid sequences of many different connexins are known, and they are thought to resemble each other closely in tertiary and quarternary structure. Single channels have recently been isolated and purified, and earlier evidence has been confirmed showing that they consist of six identical subunits arranged around the central pore. Gap junction channels are known to open and close in response to changes in ligand concentrations and electrical potential; in this respect they are very similar to ligand-gated ion channels which act as receptors in the membranes of excitable cells. The similarity is shown to extend to structural features such as the amino acid residues lining the pore, and perhaps the location of the actual gate.

Amino Acid Sequence↗
PubMed1992

Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.

Animals↗
PubMed1992

New acuity test for toddlers.

Preferential looking is the technique of choice for measuring visual acuity in infants and young children. Most workers agree that the toddler age group, 1 to 3 years, is the most difficult to test. This is because of their short attention span and restlessness, but mostly because they find the grating target used in the test, frankly boring. The concept of the vanishing optotype chart offers alternative test targets, while utilizing the technique of preferential looking. We have designed a test which comprises a familiar shape (house, car etc.) on the upper or lower part of a neutral grey card. The shape is computer generated, and designed to fade completely when beyond the resolution limit. Acuity is determined by the width of the white lines making up the shape. As with conventional preferential looking, the observer notes the child's eye movements to determine the position of the target shape. Although picture naming is not required, the shapes help to maintain the child's interest in the test. The test is quick, and is successful with the toddler age group and older patients with intellectual impairment.

Child, Preschool↗
PubMed1991

Isolation and purification of gap junction channels.

This paper reports methods we have developed to solubilize gap junction channels, or connexons, from isolated gap junctions and to purify them in milligram quantities. Two sources of material are used: rat liver gap junctions and gap junctions produced by infecting insect cells with a baculovirus containing the cDNA for human liver beta 1 protein (connexin 32). Complete solubilization is obtained with long chain detergents (lauryl dimethyl amineoxide, dodecyl maltoside) and requires high ionic strength and high pH as well as reducing conditions. The purification involves chromatography on hydroxylapatite and gel filtration on Superose 6. A homogeneous product is indicated by a single band on a silver-stained gel and a homogeneous population of doughnut-shaped particles under the electron microscope. These particles have hexameric symmetry. The purified connexons have a tendency to form aggregates: filaments and sheets. The filaments grow by end-to-end association of connexons and are nonpolar, suggesting that the connexons are paired as in the cell-to-cell channel. The sheets grow by lateral association of the filaments.

Animals↗
PubMed1990

Three-dimensional structure of the acetylcholine receptor by cryoelectron microscopy and helical image reconstruction.

Long tubular vesicles have been grown from isolated Torpedo postsynaptic membranes, in which the receptors are arranged helically on the vesicle surface. The structures of these tubes have been analyzed by cryoelectron microscopy of specimens embedded in thin films of ice, combined with helical image reconstruction. Complete data sets from tubes belonging to several helical families have been obtained to a resolution of 17 A in all directions. Confirming a preliminary study (Toyoshima, C., and N. Unwin. 1988. Nature (Lond.). 336:247-250), the central ion channel has an almost constant diameter throughout the molecule except for the portion extending through the hydrophobic part of the lipid bilayer, where the pore is too small to be resolved. However, the density on the pseudo fivefold axis running through the pore is consistently highest in the cytoplasmic half of the bilayer, suggesting the gate is located in that region. The path followed by each subunit has been identified throughout the length of the receptor. The two alpha subunits follow equivalent paths. All subunits have similar features which change in character at the same level relative to the membrane.

Animals↗
PubMed1988

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.

Animals↗