ACETYLCHOLINE BINDING PROTEIN

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    Annu. Rev. Biophys. Biomol. Struct. 2003. 32:31134doi: 10.1146/annurev.biophys.32.110601.142536

    Copyright c 2003 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on February 21, 2003

    ACETYLCHOLINE BINDING PROTEIN (AChBP):A Secreted Glial Protein That Providesa

    High-Resolution Model for theExtracellular

    Domain of Pentameric Ligand-Gated Ion Channels

    Titia K. Sixma1 and August B. Smit21Division of Molecular Carcinogenesis, Netherlands Cancer Institute, Plesmanlaan 121,

    1066 CX Amsterdam, The Netherlands; email: t.sixma@nki.nl2Department of Molecular and Cellular Neurobiology, Research Institute Neurosciences

    Vrije Universiteit, Faculty of Biology, De Boelelaan 1087, 1081 HV Amsterdam,

    The Netherlands; email: absmit@bio.vu.nl

    Key Words nicotinic acetylcholine receptor, GABAA, 5HT3, glycine receptor,Cys-loop

    s Abstract Acetylcholine binding protein (AChBP) has recently been identifiedfrom molluskan glial cells. Glial cells secrete it into cholinergic synapses, where itplays a role in modulating synaptic transmission. This novel mechanism resembles glia-dependent modulation of glutamate synapses, with several key differences. AChBP is ahomolog of theligandbinding domain of thepentameric ligand-gatedion-channels. Thecrystal structure of AChBP provides the first high-resolution structure for this familyof Cys-loop receptors. Nicotinic acetylcholine receptors and related ion-channels suchas GABAA, serotonin 5HT3, and glycine can be interpreted in the light of the 2.7 AAChBP structure. The structural template provides critical details of the binding siteand helps create models for toxin binding, mutational effects, and molecular gating.

    CONTENTS

    INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

    AChBP IN LYMNAEA STAGNALIS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

    The Role of Glial Cells in Synaptic Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . 312

    Production and Release of AChBP from Molluskan Glia . . . . . . . . . . . . . . . . . . . . . 313

    AChBP-Mediated Modulation of Synaptic Transmission . . . . . . . . . . . . . . . . . . . . . 313

    THE ACETYLCHOLINE BINDING PROTEIN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

    AChBP in Other Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315AChBP Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

    THE LIGAND-GATED ION CHANNEL SUPERFAMILY . . . . . . . . . . . . . . . . . . . . 316

    AChBP Sequence Compared with LGIC N-Terminal Domains . . . . . . . . . . . . . . . . 316

    Nicotinic Acetylcholine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

    5HT3 Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

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    312 SIXMA SMIT

    GABA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325

    Glycine Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

    CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

    INTRODUCTION

    In this review we discuss the remarkable physiology of acetylcholine binding pro-

    tein (AChBP) in the great pond snail, Lymnaea stagnalis, its secretion by glial

    cells, and its possible role in molluskan cholinergic synapses. Current views of

    the presence of related proteins in other organisms are discussed. We use the

    crystal structure of AChBP to shed light on structure/function aspects of pen-

    tameric ligand-gated ion-channels (LGICs), which mediate and modulate chem-

    ical synaptic transmission. This family of transmembrane receptors includes thenicotinic acetylcholine (nAChR), serotonin 5HT3, -aminobutyric acid (GABAAand GABAC), and glycine receptors (86) as well as invertebrate glutamate (27)

    and histamine (130) channels. These receptors are, apart from their endogenous

    neuronal ligands, receptive to diverse compounds such as nicotine, alcohol, and

    various snake and snail venoms. In addition, they are prime targets for pharmaceu-

    ticals such as barbiturates, benzodiazepines, and anti-emetics. Mutations in these

    receptors are involved in diseases such as congenital myasthenia gravis, epilepsy,

    startle syndrome, and in sensitivity to alcohol (120). Also, nAChRs mediate nico-

    tine addiction in chronic tobacco users. Because LGICs are involved in importantaspects of brain functioning and brain diseases, they are considered prime targets

    for novel drug discovery programs.

    AChBP IN LYM NAEA STAGNALI S

    Lymnaea stagnalis was analyzed because it has large easily identifiable neurons

    that have been characterized extensively. This precise knowledge allowed in vitro

    culture of specific synapses (128). The AChBP protein was found in a study thatconcentrated on the role of glial cells in cholinergic synapses in L. stagnalis (111).

    The Role of Glial Cells in Synaptic Transmission

    Glia are the most numerous cells in the central nervous system (CNS). Their main

    role was seen as providing metabolic and trophic support to neurons. In recent

    years this classical view on the role of glia has been challenged, and recent find-

    ings indicate active glial involvement in the modulation of synaptic transmission.

    Various studies have supported the view that the neuron-glia communication is

    bidirectional: Glial cells receive neuronal input and may also release transmitter

    onto neurons thereby affecting neuronal excitation and synaptic transmission (13,

    123). Compelling examples of this come from glutamatergic signaling between

    neurons and glia in the hippocampus, from GABAergic synapses, where GABA

    released by astrocytes potentiates inhibitory synaptic transmission (54), and from

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    ACETYLCHOLINE BINDING PROTEIN 315

    in Figure 1c), only the low AChBP titer would yield a free ACh concentration of

    16 M, which is probably adequate to activate postsynaptic receptors. As such,

    the actual synaptic concentration of AChBP might critically determine whether

    transmission is either fully active or suppressed.Various transmitter or hormone systems modulate glial cells in the mammalian

    brain. Many receptor types on glia, e.g., amino-methyl proprionic acid (AMPA)-

    (31), GABAB- (54), 1-adrenoreceptors (63), and also different nAChR subunits,

    have been found in oligodendrocytes (96) and astrocytes (51). Thus molluskan

    synaptic glial cells might integrate various signals. These might adjust the synaptic

    concentration of AChBP at which modulation of synaptic transmission will occur

    (Figure 1c).

    THE ACETYLCHOLINE BINDING PROTEIN

    The AChBP sequence encodes a 210-residue mature protein, including two disul-

    fide bonds. An N-terminal signal sequence is cleaved off in the mature protein.

    There is one glycosylation site at residue Asn66. The recombinant AChBP protein,

    expressed in the yeast Pichia pastoris, assembles into stable homopentamers as

    shown by gel filtration (111) and analytical ultracentrifugation (K. Brejc, M.H.

    Lamers & and T.K. Sixma, unpublished data).

    AChBP in Other Species

    Related AChBP genes have been found in the mollusk Aplysia californica (Acc.

    nr: AF364899) and in the leech Haementeria ghilianii (98). Based on the absence

    of typical -subunit sequence features, the latter protein is named -subunit-

    like. Lymnaea and Aplysia AChBP have 33% sequence identity, Lymnaea and

    Haementeria only 18.2%. Thus, sequence conservation is low, even within the

    molluskan phylum, in which leeches are nowadays often included. Although lig-

    and binding characteristics for the Aplysia protein are not yet available, it is prob-

    ably a functional homolog of Lymnaea AChBP, but with different ligand bindingcharacteristics. The Lymnaea and Aplysia AChBP have lower sequence identity to

    the two -subunits of nAChRs identified in Aplysia (25% and 26%, respectively)

    than to each other (33%). An important question remains whether orthologs of

    AChBP will be found in other animal phyla. The cloning of AChBP orthologs

    in more distantly related species is obviously hindered by the low degree of se-

    quence conservation. Database searches in the genomes ofCaenorhabditis elegans,

    Drosophila, and human have not yielded AChBP orthologs so far.

    AChBP StructureThe crystal structure of AChBP was determined to 2.7 A resolution (18). The

    AChBP homopentamer forms a doughnut-like structure with a radius of 80 A

    and a height of 62 A (Figure 3). Each AChBP monomer folds into an N-

    terminal helix and a curled extended -sandwich with modified immuno-

    globulin topology Where a conserved tyrosine corner is found in different

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