The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most-characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nmX8 nm) were deduced from electronmicroscopy studies. Its subunits are arranged around a five-fold axis of pseudosymmetry in the order (clockwise) aI,yaLGP. Its two agonistkompetitive-antagonist-binding sites have been localized by photolabelling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane-spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand-gated ion channels. The binding site for regulatory non-competitive antagonists has been localized by photolabelling and site-directed-mutagenesis studies within this ion channel.Therefore a three-dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/competitive-antagonist-binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.

The nuclear envelope (NE) is one of the least characterized structures of eukaryotic cells. The study of its functional roles is hampered by the small number of proteins known to be specifically located to it. Here, we present a comprehensive characterization of the NE proteome. We applied different fractionation procedures and isolated protein subsets derived from distinct NE compartments. We identified 148 different proteins by 16-benzyl dimethyl hexadecyl ammonium chloride (16-BAC) gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry; among them were 19 previously unknown or noncharacterized. The identification of known proteins in particular NE fractions enabled us to assign novel proteins to NE substructures. Thus, our subcellular proteomics approach retains the screening character of classical proteomic studies, but also allows a number of predictions about subcellular localization and interactions of previously noncharacterized proteins. We demonstrate this result by showing that two novel transmembrane proteins, a 100-kDa protein with similarity to Caenorhabditis elegans Unc-84A and an unrelated 45-kDa protein we named LUMA, reside in the inner nuclear membrane and likely interact with the nuclear lamina. The utility of our approach is not restricted to the investigation of the NE. Our approach should be applicable to the analysis of other complex membrane structures of the cell as well.T he identification of predicted gene products at the protein level bridges the gap between genome sequencing data and protein function, and is referred to as ''functional genomics'' (1, 2). In this respect, the combination of subcellular fractionation and mass spectrometrical techniques (''subcellular proteomics'') is a powerful strategy for the initial identification of previously unknown protein components and for their assignment to particular subcellular structures (3, 4).The nuclei of eukaryotic cells contain several compartments defined by their morphological appearance in electron microscopy and by the distribution of a limited number of marker proteins (5). Because the structural and functional organization of nuclei seems to be intimately linked to the epigenetic control of gene expression, the characterization of such nuclear compartments at the molecular level is of great importance (6). The nuclear envelope (NE) is one of the least characterized compartments of the nucleus. It comprises an outer and inner nuclear membrane (ONM and INM, respectively), the pore membrane, the nuclear pore complexes, and the nuclear lamina (7). These subcompartments differ with respect to their protein components, but a thorough molecular characterization has not yet been achieved. Furthermore, a two-dimensional separation of NE membrane proteins by isoelectric focusing and SDS͞PAGE fails because the separation system discriminates against integral membrane proteins (8). Therefore, the characterization of the NE at the protein level has to overcome general analytical challenges; the NE con...

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP') was localized in the cc-, /I-and b-chains of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata electric tissue. The photolabel was found in homologous positions of the highly conserved sequence helix II, tl 248,j 254, and 6 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position 6 262 independent of whether photolabeling was performed with the receptor in its resting, desensitized or antagonist state. A model of the AChR ion channel is proposed, according to which the channel is formed by the five helices II contributed by the five receptor subunits.

Abstract. Nsplp interacts with nuclear pore proteins Nup49p, Nup57p and Nic96p in a stable complex which participates in nucleocytoplasmic transport. An additional p80 component is associated with Nsplp, but does not co-purify with tagged Nup57p, Nup49p and Nic96p. The p80 gene was cloned and encodes a novel essential nuclear pore protein named Nup82p. Immunoprecipitation of tagged Nup82p reveals that it is physically associated with a fraction of Nsplp which is distinct from Nsplp found in a complex with Nup57p, Nic96p and Nup49p. The Nup82 protein can be divided into at least two different domains both required for the essential function, but it is only the carboxy-terminal domain, exhibiting heptad repeats, which binds to Nsplp. Yeast cells depleted of Nup82p stop cell growth and concomitantly show a defect in poly(A) + RNA export, but no major alterations of nuclear envelope structure and nuclear pore density are seen by EM. This shows that Nsplp participates in multiple interactions at the NPC and thus has the capability to physically interact with different NPC structures.

The vanilloid receptor TRPV1 plays a well-established functional role in the detection of a range of chemical and thermal noxious stimuli, such as those associated with tissue inflammation and the resulting pain. TRPV1 activation results in membrane depolarization, but may also trigger intracellular Ca 2+ -signalling events. In a proteomic screen for proteins associated with the C-terminal sequence of TRPV1, we identified b-tubulin as a specific TRPV1-interacting protein.We demonstrate that the TRPV1 C-terminal tail is capable of binding tubulin dimers, as well as of binding polymerized microtubules. The interaction is Ca 2+ -sensitive, and affects microtubule properties, such as microtubule sensitivity towards low temperatures and nocodazole. Our data thus provide compelling evidence for the interaction of TRPV1 with the cytoskeleton. The Ca 2+ -sensitivity of this interaction suggests that the microtubule cytoskeleton at the cell membrane may be a downstream effector of TRPV1 activation.