Dept. of Molecular & Cellular Neurobiology - Room C-340
Research Institute of Neurosciences
Vrije Universiteit
De Boelelaan 1085
1081 HV Amsterdam, The Netherlands
Phone: +31 (0)20-5987116, Fax: +31 (0)20-5989281
E-mail: guus.smit@cncr.vu.nl

Guus Smit
Head of Department MCN
Teamleader Research line 5

Lingand Gated Ion Channels

 
BACKGROUND

The nicotinic acetylcholine receptor (nAChR) is the prototype of the ligand-gated ion channels, also known as Cys-loop receptors. This superfamily also includes the GABAA and GABAC, the glycine and the 5HT3 serotonin receptors. These transmembrane receptors form pentamers of related subunits, in which each subunit consists of an N-terminal ligand-binding domain, four membrane spanning regions (M1-M4) and a short intracellular region. The receptors of this superfamily play an important role in the nervous system and have been shown to be implicated in brain diseases, such as Alzheimers’ and nicotine addiction.
Extensive biochemical studies have defined the complicated ligand-binding sites that are found at the subunit interface. Structurally the proteins are well defined by the work of Nigel Unwin and colleagues, who have studied the Torpedo receptor up to a resolution of 4.6 Å but an atomic model has been lacking (see photo, right).
 
In my group, we have been using the central nervous system of the freshwater snail Lymnaea stagnalis for studying cholinergic transmission. It has large neurons that have been characterized very extensively. This precise knowledge allowed in vitro culture of cholinergic neurons and the study of their synapses. An electrophysiological analysis of an in vitro neuronal system showed that glia cells modulate cholinergic synaptic transmission. After an extensive series of studies we demonstrated that this phenomena is mediated by secretion of a novel glial protein, called acetylcholine binding protein (AChBP) (Smit et al., 2001).
Left to right: • Lymnaea stagnalis • The Lymnaea CNS • Neurons (large), glia (small & AChBP in red).
The AChBP protomer contains 210 amino acid residues and shows high homology (~24% sequence identity) with the N-terminal domain of the a-subunits of the nAChRs but it lacks the transmembrane region entirely. The conserved regions are the hallmarks of the superfamily of ligand-gated ion channels (15-20% sequence identity with e.g. GABA and 5HT3 serotonin receptors). The expressed recombinant AChBP protein assembles into stable homopentamers. Furthermore, AChBP contains all the amino acid residues needed for the ligand binding showing pharmacology that is similar to a homopentameric nAChR, such as the neuronal a7 receptor. In conclusion, AChBP is a soluble homolog of a ligand-binding domain of the nicotinic receptors and can be used for the design of novel ligands for this family of receptors.
PROJECTS

Crystall structure of AChBP

In the group of my collaborator Dr. Titia Sixma (NKI), the recombinant AChBP has been crystallized and its structure was determined (Brejc et al 2001). The electron density was improved by non-crystallographic and multi-crystal averaging across three different crystal forms. The AChBP model was refined with one pentamer per asymmetric unit at 2.7 Å resolution. The AChBP homopentamer, composed of five protomers, forms a doughnut-like structure with a radius of 80 Å and height of 62 Å, conforming to the electron microscopy data for the nAChR
The residues involved in ligand binding in the muscle and Torpedo nAChR subtypes have been extensively investigated, and are found at the interface between an a-subunit and a neighboring subunit, forming primary and complementary part of the binding pocket, respectively. The a-subunit residues important for binding are clustered in 'loop' regions, A-C. The a-subunit contributes the primary binding site, consisting of loop A, B and C. The neighboring subunit, contributes ‘loops’ D-F; in homopentamers the equivalent loops will be contributed by a-subunits.
In the AChBP structure all these ‘loop’ regions are indeed found to form a single pocket region at the subunit interface, with ‘loops’ A-C contributed from one subunit and D-F from another. The binding pocket is lined by aromatic residues of loop A-D, while the hydrophobic components of the residues in loop C form the lid of the pocket. On the left a contact of Trp 143 of the primary part of the binding pocket and nicotine is shown.
AChBP as a tool for drug discovery
Being the only available structural model for the cys-loop family of receptors at high resolution, the AChBP structure is very suitable for rational drug design. In particular novel X-ray structures with high resolution offer the possibility to work at atomic detail. In the context of the Drug Discovery Center VU (DDC-VU) and the Top-Institute Pharma we are pursuing this line of investigation. Also, AChBP might prove to be a good starting point for mutational analysis, which would mimic the binding sites of subtypes of nAChRs and other ligand-gated ion channels. Also, these studies have potential opportunities for rational drug design and screening of candidate ligands.
PUBLICATIONS (selected)

Dutertre S, Ulens C, Büttner R, Fish A, van Elk R, Kendel Y, Hopping G, Alewood PF, Schroeder C, Nicke A, Smit AB, Sixma TK, Lewis RJ. AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity. EMBO J. 2007 Aug 22;26(16):3858-67.

Ulens C, Hogg RC, Celie PH, Bertrand D, Tsetlin V, Smit AB, Sixma TK. Structural determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP. Proc Natl Acad Sci U S A. 2006 Mar 7;103(10):3615-20.

Smit, A.B., Syed, N.I., Schaap, D., Van Minnen, J., Klumperman, J., Kits, K.S., Lodder, H., Van der Schors, R.C., Van Elk, R., Sorgedrager, B., Brejc, K., Sixma, T.K. and Geraerts, W.P.M. (2001) A glia-derived modulator of cholinergic synaptic transmission. Nature 411, 261-268.

Brejc, K., Van Dijk, W.J., Klaassen, R.V., Schuurmans, M., Van der Oost, J., Smit, A.B. & Sixma, T.K. (2001) Crystal structure of AChBP reveals the ligand-binding domain of nicotinic receptors. Nature 411, 269-276.

Harel, M., Kasher, R., Nicolas, A., Guss, J.M., Balass, M., Fridkin, M., Katchalski-Katzir , E., Smit, A.B., Brejc, K., Sixma, T.K., Sussman, J., and Fuchs, S. The structure of the binding site of the acetylcholine receptor as visualized in the X-ray structure of a complex between _-bungarotoxin and a high affinity mimotope peptide. Neuron 32, 265-75.

Smit A.B., Brejc, K., Syed N.I., Sixma T.K. (2003) Structure and function of AChBP, homologue of the ligand-binding domain of the nicotinic acetylcholine receptor. Ann. N Y Acad. Sci. 998:81-92. Review

Sixma T.K, Smit A.B. (2003) Acetylcholine binding protein (AChBP): a secreted glial protein that provides a high-resolution model for the extracellular domain of pentameric ligand-gated ion channels. Annu. Rev. Biophys. Biomol. Struct. 32:311-34. Review.

Celie, P., van Rossum-Fikkert, S., van Dijk, W.J, Brejc, K., Smit, A.B. and Sixma, T.K. (2003) Nicotine and carbamoylcholine binding to nicotinic acetylcholine receptors as studied by AChBP crystal structures. Neuron 41(6):907-14