Soluble, Oligomeric, and Ligand-binding Extracellular Domain of the Human α7 Acetylcholine Receptor Expressed in Yeast

Abstract

The N-terminal extracellular domain (ECD; amino ac-ids 1–208) of the neuronal nicotinic acetylcholine recep-tor (AChR) ␣7 subunit, the only human AChR subunit known to assemble as a homopentamer, was expressed as a glycosylated form in the yeast Pichia pastoris in order to obtain a native-like model of the extracellular part of an intact pentameric nicotinic AChR. This mol-ecule, ␣7-ECD, although able to bind the specific ligand ␣-bungarotoxin, existed mainly in the form of microag-gregates. Substitution of Cys-116 in the ␣7-ECD with serine led to a decrease in microaggregate size. A second mutant form, ␣7-ECD(C116S,Cys-loop), was generated in which, in addition to the C116S mutation, the hydropho-bic Cys-loop (Cys 128 –Cys 142) was replaced by the corre-sponding hydrophilic Cys-loop from the snail glial cell acetylcholine-binding protein. This second mutant pro-tein was water-soluble, expressed at a moderate level (0.5 ؎ 0.1 mg/liter), and had a size corresponding approx-imately to a pentamer as judged by gel filtration and electron microscopy studies. It also bound 125 I-␣-bunga-rotoxin with relatively high affinity (K d ‫؍‬ 57 nM), the binding being inhibited by unlabeled ␣-bungarotoxin, d-tubocurarine, or nicotine (K i ‫؍‬ 0.8 ؋ 10 ؊7 M, K i ‫؍‬ 1 ؋ 10 ؊5 M, and K i ‫؍‬ 0.9 ؋ 10 ؊2 M, respectively). All three constructs were expressed as glycosylated forms, but in vitro deglycosylation reduced the heterogeneity with-out affecting their ligand binding properties. These re-sults show that ␣7-ECD(C116S,Cys-loop) was expressed in P. pastoris as an oligomer (probably a pentamer) with a near native conformation and that its deglycosylated form seems to be suitable starting material for struc-tural studies on the ligand-binding domain of a neuro-transmitter receptor. Nicotinic acetylcholine receptors (AChRs) 1 are the prototypic members of the Cys-loop LGIC superfamily, which also in-cludes serotonin (5-hydroxytryptamine 3), glycine, ␥-aminobu-tyric acid type A, and ␥-aminobutyric acid type C receptors (1). These membrane glycoproteins, which mediate rapid chemical synaptic transmission, form either homo-or heteropentamers made up of homologous subunits that show significant similar-ities in amino acid sequence, transmembrane topology, and overall secondary, tertiary, and quaternary structure, implying a common evolutionary origin (2, 3). AChRs, which are classified into muscle and neuronal types, are the best characterized members of the LGIC superfamily. Muscle-type AChRs, found at the vertebrate neuromuscular junction and in fish electric organ, have a fixed stoichiometry ((␣1) 2 ␤1␥␦ in Torpedo and embryonic mammalian AChR or (␣1) 2 ␤1⑀␦ in adult mammalian AChR), mediate neuromuscular transmission, and are implicated in the autoimmune disease myasthenia gravis. Neuronal AChRs, widely distributed in the pre-, post-, and peri-synaptic nerve terminals of the central and peripheral nervous system (4, 5), exist either as heteropentam-ers containing two or three ␣-subunits (␣2– 6) plus 2 or 3 ␤-subunits (␤2– 4) or as homopentamers (␣7–9), with ␣7 being the only human subunit known to form a homopentamer (5). Neuronal AChRs play key roles in various neuron-neuron in-teractions and are therefore involved in many functions (re-viewed in Ref. 5) such as ganglionic transmission, modulation of the release of various neurotransmitters (␥-aminobutyric acid, acetylcholine, serotonin, glutamate, dopamine, and nor-adrenaline) (6), attention, learning, memory consolidation, arousal, sensory perception, the control of locomotor activity, pain perception, and body temperature regulation (7). They are, thus, implicated in a number of serious neurological dis-orders including Alzheimer's disease, Parkinson's disease, schizophrenia, depression, autism, and forms of epilepsy as well as nicotine addiction (8). Because of this, neuronal AChRs have attracted much recent attention. The thorough understanding of AChR function and its ma-nipulation for therapeutic approaches requires the elucidation of its structure at high resolution. Currently available struc-tural information on AChRs has been mainly derived from elegant cryo-electron microscopy studies on two-dimensional crystals of Torpedo AChR, which can be purified in large amounts. Such studies on membrane-bound Torpedo AChRs have recently provided the 4.6-and 4-Å resolution structures, respectively, of the extracellular and transmembrane domains of the AChR (9 –11). However, no x-ray structure of any AChR or any other LGIC has yet been obtained, although very small, non-diffracting three-dimensional crystals of Torpedo AChR have been reported (12, 13). Attempts by several investigators to obtain diffraction quality three-dimensional crystals of this large membrane protein have met with little success. The use of hydrophilic AChR polypeptide fragments, rather than full-length subunits, seems to be a more realistic