Ferdinand Hucho

Nuclear envelope proteomics: novel integral membrane proteins of the inner nuclear 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.

The ion channel of the nicotinic acetylcholine receptor is formed by the homologous helices M II of the receptor subunits.

A binding site for the channel‐blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the α‐, β‐ and δ‐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, α 248, β 254, and δ 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position δ 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.

Snake and snail toxins acting on nicotinic acetylcholine receptors: fundamental aspects and medical applications

This review covers recent data on interactions of nicotinic acetylcholine receptors (AChR) with snake venom proteins (α‐ and κ‐neurotoxins, ‘weak’ toxins recently shown to act on AChRs), as well as with peptide α‐conotoxins from Conus snails. Mutations of AChRs and toxins, X‐ray/nuclear magnetic resonance structures of α‐neurotoxin bound to AChR fragments, and the X‐ray structure of the acetylcholine‐binding protein were used by several groups to build models for the α‐neurotoxin–AChR complexes. Application of snake toxins and α‐conotoxins for pharmacological distinction of muscle, neuronal and neuronal‐like AChR subtypes and for other medical purposes is briefly discussed.

The ion channel of the nicotinic acetylcholine receptor

A binding site for the channel-blocking noncompetitive antagonist [3H]triphenylmethylphosphonium ([3H]TPMP+) was localized in the alpha-, beta- and delta-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, alpha 248, beta 254, and delta 262. The site of the photoreaction appears to not be affected by the functional state of the receptor. [3H]TPMP+ was found in position delta 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.

Identification and characterization of a Ca2+‐sensitive interaction of the vanilloid receptor TRPV1 with tubulin

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 Ca2+‐signalling events. In a proteomic screen for proteins associated with the C‐terminal sequence of TRPV1, we identified β‐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 Ca2+‐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 Ca2+‐sensitivity of this interaction suggests that the microtubule cytoskeleton at the cell membrane may be a downstream effector of TRPV1 activation.

Acetylcholine receptor: SH group reactivity as indicator of conformational changes and functional states.

One of several alternative models of chemical transmission proposes that the conformational state of the acetylcholine receptor (AchR) controls the ion-permeability of the postsynaptic membrane [l-4] This hypothesis is supported by reports of different states of affinity of the receptor in vitro for cholinergic agonists [5-71. At least in solution the different states have been shown to be interconvertible [8,9]. The time scale of the interconversion is of the order of minutes and in this respect similar to the phenomenon of pharmacological desensitization. It was postulated that the desensitized state corresponds to a ‘high affinity state’ of the acetylcholine receptor PI * In addition to binding studies [8,9] the postulated conformational transition was investigated by fluorescence spectroscopy [lo131. In this report we show that the reactivity of -SH groups towards Ellman’s reagent can be used as an intrinsic indicator of conformational changes and functional states of the receptor: incubation with the agonist carbamyl choline slowly reduces the reactivity. Detergent solubilization of the membrane bound receptor exposes further -SH groups. These are rapidly oxidized in Triton X-100 but remain stable in sodium dodecyl sulfate. Since receptor purification is usually performed in Triton X-100 this finding may explain reported differences between membrane bound and isolated receptor. In addition ?o :he reported -SH groups a disulfide bridge cross-linking two &subunits of the receptor was discovered.

Rapid disassembly of dynamic microtubules upon activation of the capsaicin receptor TRPV1

The transmission of pain signalling involves the cytoskeleton, but mechanistically this is poorly understood. We recently demonstrated that the capsaicin receptor TRPV1, a non‐selective cation channel expressed by nociceptors that is capable of detecting multiple pain‐producing stimuli, directly interacts with the tubulin cytoskeleton. We hypothesized that the tubulin cytoskeleton is a downstream effector of TRPV1 activation. Here we show that activation of TRPV1 results in the rapid disassembly of microtubules, but not of the actin or neurofilament cytoskeletons. TRPV1 activation mainly affects dynamic microtubules that contain tyrosinated tubulins, whereas stable microtubules are apparently unaffected. The C‐terminal fragment of TRPV1 exerts a stabilizing effect on microtubules when over‐expressed in F11 cells. These findings suggest that TRPV1 activation may contribute to cytoskeleton remodelling and so influence nociception.

The phospholipid analogue, hexadecylphosphocholine, inhibits protein kinase C in vitro and antagonises phorbol ester-stimulated cell proliferation

The antineoplastic agent, hexadecylphosphocholine, a phospholipid analogue, inhibited phosphatidylserine-activated protein kinase C in vitro at concentrations higher than 40 mumol/l. The half-inhibitory concentration (IC50) was 62 mumol/l. Another alkylphosphocholine, dodecylphosphocholine, did not have an inhibitory effect on protein kinase C. At the same concentrations, hexadecylphosphocholine antagonised the phorbol ester-stimulated proliferation of Madin-Darby canine kidney cells whereas dodecylphosphocholine had no effect. In addition, phorbol ester-induced morphological changes of these epithelial cells were antagonised by hexadecylphosphocholine. Both effects of hexadecylphosphocholine, the inhibition of protein kinase C and the antagonisation of the altered cell morphology induced by phorbol ester, were comparable to those observed after treatment with sphingosine, a known protein kinase C inhibitor. We conclude that one possible mechanism of the antineoplastic action of hexadecylphosphocholine is mediated by inhibition of protein kinase C.

TRPV1 at nerve endings regulates growth cone morphology and movement through cytoskeleton reorganization

While the importance of Ca2+ channel activity in axonal path finding is established, the underlying mechanisms are not clear. Here, we show that transient receptor potential vanilloid receptor 1 (TRPV1), a member of the TRP superfamily of nonspecific ion channels, is physically and functionally present at dynamic neuronal extensions, including growth cones. These nonselective cation channels sense exogenous ligands, such as resenifera toxin, and endogenous ligands, such as N‐arachidonoyl‐dopamine (NADA), and affect the integrity of microtubule cytoskeleton. Using TRPV1‐transiently transfected F11 cells and embryonic dorsal root ganglia explants, we show that activation of TRPV1 results in growth cone retraction, and collapse and formation of varicosities along neurites. These changes were due to TRPV1‐activation‐mediated disassembly of microtubules and are partly Ca2+‐independent. Prolonged activation with very low doses (1 nm) of NADA results in shortening of neurites in the majority of isolectin B4‐positive dorsal root ganglia neurones. We postulate that TRPV1 activation plays an inhibitory role in sensory neuronal extension and motility by regulating the disassembly of microtubules. This might have a role in the chronification of pain.

Ligand-Gated Ion Channels

Ion channels are at the heart of many biological processes such as nerve activity and muscle contraction. How are their impressive ion selectivity and highly specialized gating brought about? In recent years, X-ray crystallography and high-resolution electron microscopy, as well as photo-affinity labeling and site-specific mutagenesis techniques in combination with patch-clamp electrophysiology have provided a detailed picture of some channel proteins. Herein we summarize the main structural and functional properties of channel proteins based on the advances made mainly within the last decade. We integrate these novel insights into a comprehensive description of the class of ligand-gated ion channels.