The primary functional unit of the nervous system is the synapse, a highly specialized and tightly regulated connection between one nerve cell and another excitable cell. Neurotransmitter-gated channels are the pivotal molecular components that mediate communication between the pair of cells; however, little has been learned about their structures from x-ray crystallography or solution NMR spectroscopy because they are membrane proteins. We have begun to study the structural details of the functional 25 residue peptides corresponding to the pore forming elements of the ion-channels of the acetylcholine receptor and the NMDA receptor. The sequences of the second transmembrane segment, M2, are highly conserved among all members of the superfamily of neurotransmitter-gated channels. Significantly, the model of the Torpedo acetylcholine receptor has a pore formed by a bundle of five ?-helices, one from each protein subunit formed from M2. It has been shown that a pentameric bundle of M2 helices is sufficient to display several functional properties of the cholinergic receptor as analyzed by reconstitution of purified peptides in planar lipid bilayers, measured by single-channel recordings under voltage-clamp conditions. We have expressed these peptides as fusion proteins in bacteria and obtained uniformly labeled samples for solution NMR studies in micelles and solid-state multidimensional solution NMR spectroscopy can be used to determine three-dimensional structures based on many short-range distance measurements. In contrast, in solid state NMR spectroscopy of oriented samples it is the determinations of the orientations of individual peptide planes that are used to characterize the structures. The two approaches provide independent paths to the structure of biopolymers, which is important in order to verify the results of the new solid-state NMR method on these systems. A direct comparison of the results from solution NMR studies of micelle samples and solid-state NMR studies of bilayer samples of the acetylcholine M2 peptide yield virtually identical structures. All of the NMR data can be summarized with the model of the peptide where the monomeric M2 peptides self-assemble in bilayers to function much as they do in the intact proteins. The next step is to determine the structures of substantially larger segments of the membrane domains of the neurotransmitter-gated channels to find the role of the larger polypeptide environment on the structure of the peptides. We have recently obtained preliminary spectra from a sample of a designed potassium channel protein with 120 residues.
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NIH ExPorter