Activation by acidic pH of CLC-7 expressed in oocytes from Xenopus laevis

Diewald. L., Rupp, J., Dreger, M., Hucho, F., Gillen, C. and Nawrath, H.
Pharmakologisches Institute der Universitat Mainz, Mainz, Germany.

ClC chloride channels are important in diverse physiological functions such as transepithelial transport, cell volume regulation, excitability, and acidification of intracellular organelles. We have investigated the expression of CLC-7 in oocytes from Xenopus laevis with the two electrode voltage clamp technique and Western blot analysis. Using a specific antibody against CLC-7, we found an approximately 80 kDa protein in oocytes, previously injected with CLC-7-cRNA. In voltage clamp experiments on ClC-7-cRNA-injected oocytes, no current changes were detected at normal pH (7.4). However, acidification of the Ringer solution to pH values between 6 and 4 revealed strong currents which reversed at about -15 mV (30 mV positive to the normal resting potential) and showed strong outward rectification. We therefore suggest that ClC-7 in oocytes is a functional chloride current at acidic pH. Since ClC-7 is also found in neuronal tissues and was upregulated in a rat pain model, we suggest a role of CLC-7 also for nociception and pain. - copyright2002 Elsevier Science (USA).

J Comp Neurol 2002 Mar 12;444(3):245-59

Identification of orcokinins in single neurons in the stomatogastric nervous system of the crayfish, Cherax destructor

Skiebe, P., Dreger, M., Meseke, M., Evers, J.F. and Hucho, F.
Institut fur Biologie, Freie Universitat Berlin, D-14195 Berlin, Germany. skiebe@zedat.fu-berlin.de

The orcokinins are a highly conserved family of crustacean peptides that enhance hindgut contractions in the crayfish Orconectes limosus (Stangier et al. [1992] Peptides 13:859-864). By combining immunocytochemical and mass spectrometrical analysis of the stomatogastric nervous system (STNS) in the crayfish Cherax destructor, we show that multiple orcokinins are synthesized in single neurons. Immunocytochemistry demonstrated orcokinin-like immunoreactivity in all four ganglia of the STNS and in the pericardial organs, a major neurohaemal organ. Identified neurons in the STNS were stained, including a pair of modulatory interneurons (inferior ventricular nerve neuron, IVN), a neuron with its cell body in the stomatogastric ganglion that innervates cardiac muscle c6 via the anterior median nerves (AM-c6), and a sensory neuron (anterior gastric receptor neuron). Five orcokinin-related peptides were identified by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) post source decay fragmentation in samples of either the stomatogastric ganglion or the pericardial organs. Four of these peptides are identical to peptides derived from the cloned Procambarus clarkii precursor (Yasuda-Kamatani and Yasuda [2000] Gen. Comp. Endocrinol.118:161-172), including the original [Asn(13)]-orcokinin (NFDEIDRSGFGFN, [M+H](+) = 1,517.7 Da), [Val(13)]-orcokinin ([M+H](+) = 1,502.7 Da), [Thr(8)-His(13)]-orcokinin ([M+H](+) = 1,554.8 Da), and FDAFTTGFGHS ([M+H](+) = 1,186.5 Da).  The fifth peptide is a hitherto unknown orcokinin variant: [Ala(8)-Ala(13)]-orcokinin ([M+H](+) = 1,458.7 Da). The masses of all five peptides were also detected in the inferior ventricular nerve of C. destructor, which contains the cell bodies and axons of the IVNs as well as the axons of two other orcokinin-like immunoreactive neurons. In the oesophageal nerve, in which all the orcokinin-like immunoreactivity derives from the IVNs, at least two of the orcokinins were detected, indicating that multiple orcokinins are synthesized in these neurons. Similarly, all four orcokinin masses were detected in the anterior median nerves, in which all the orcokinin-like immunoreactivity derives from the AM-c6 neuron. This study therefore lays the groundwork to investigate the function of the orcokinin peptide family using single identified neurons in a well-studied system. Copyright 2002 Wiley-Liss, Inc.

Eur J Biochem 2001 Nov;268(21):5489-96

Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line

Jahnel, R., Dreger, M., Gillen, C., Bender, O., Kurreck, J. and Hucho, F.
Arbeitsgruppe Neurochemie and Arbeitsgruppe Molekulare Medizin, Institut fur Chemie-Biochemie, Freie Universitat Berlin, Germany.

The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia,  hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient  receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.

Proc Natl Acad Sci U S A 2001 Oct 9;98(21):11943-8

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

Dreger, M., Bengtsson, L., Schoneberg, T., Otto, H. and Hucho, F.
Institute for Chemistry/Biochemistry, Free University Berlin, Thielallee 63, D-14195 Berlin, Germany.

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.

Angew. Chem. Int. Ed. 40, 3100-3116 (2001);  German version:  Angew. Chem. 113, 3194-3211 (2001)

Ligand-Gated Ion Channels

Hucho, F. and Weise, C.

Ion channels are at the heart of many biological processes such as nerve activity and muscle contraction. How is their impressive ion selectivity brought about, how their highly specialised gating? In recent years, X-ray crystallography and high-resolution electron microscopy, together with photo-affinity labeling and mutagenesis techniques in combination with patch-clamp electrophysiology, have provided a detailed picture of some channel proteins. Here we summarise the main structural and functional features 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.
 

Farmaco 2001 Jan-Feb;56(1-2):133-5

Binding of polyamine-containing toxins in the vestibule of the nicotinic acetylcholine receptor ion channel

Bixel, M.G., Krauss, M., Weise, C., Bolognesi, M.L., Rosini, M., Usherwood, P.N., Melchiorre, C. and Hucho F.

Several wasp venoms contain philanthotoxins (PhTXs) that act as noncompetitive inhibitors (NCIs) on cation-selective ion channels including the nicotinic acetylcholine receptor (nAChR). In the search for a ligand with high affinity and specificity for the nAChR we tested a series of newly developed PhTX analogues. Modulation of the structural elements of PhTXs can significantly influence their binding affinities. This approach resulted in the development of the photolabile compound MR44. In photoaffinity labelling studies 125I-MR44 was used to map the ligand-binding site at the Torpedo californica nAChR. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was mainly incorporated into the receptor alpha-subunit. Proteolytic mapping and microsequencing identified the site of 125I-MR44 cross-linking within the sequence alphaHis-186 to alphaLeu-199 that in its C-terminal region partially overlaps with the agonist-binding site. Since bound agonists had only minor influence on 125I-MR44 photocrosslinking, the site where the hydrophobic head group of 125I-MR44 binds must be located outside the zone that is sterically influenced by agonists bound at the nAChR. A possible site of interaction of 125I-MR44 would be the N-terminal region of the labelled sequence, in which aromatic amino-acid residues are accumulated. We suggest that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the ion channel, while the aromatic ring of this compound binds in the vestibule of the nAChR to a hydrophobic region on the alpha-subunit that is located close to the agonist binding site.

Eur J Biochem 2001 Jan 15;268(2):420-428

Identification of tyrosine-phosphorylated proteins associated with the nuclear envelope

Otto, H., Dreger, M., Bengtsson, L. and Hucho, F.

The nuclear envelope separates the nucleoplasm from the rest of the cell. Throughout the cell cycle, its structural integrity is controlled by reversible protein phosphorylation. Whereas its phosphorylation-dependent disassembly during mitosis is well characterized, little is known about phosphorylation events at this structure during interphase. The few characterized examples cover protein phosphorylation at serine and threonine residues, but not tyrosine phosphorylation at the nuclear envelope. Here, we demonstrate that tyrosine phosphorylation and dephosphorylation occur at the nuclear envelope of intact Neuro2a mouse neuroblastoma cells. Tyrosine kinase and phosphatase activities remain associated with purified nuclear envelopes. A similar pattern of tyrosine-phosphorylated nuclear envelope proteins suggests that the same tyrosine kinases act at the nuclear envelope of intact cells and at the purified nuclear envelope. We have also identified eight tyrosine-phosphorylated nuclear envelope proteins by 2D BAC/SDS/PAGE, immunoblotting with phosphotyrosine-specific antibodies, tryptic in-gel digestion, and MS analysis of tryptic peptides. These proteins are the lamina proteins lamin A, lamin B1, and lamin B2, the inner nuclear membrane protein LAP2beta, the heat shock protein hsc70, and the DNA/RNA-binding proteins PSF, hypothetical 16-kDa protein, and NonO, which copurify with the nuclear envelope.
 

J Biol Chem. 2001 Mar 2;276(9):6151-6160.

Location of the Polyamine Binding Site in the Vestibule of the Nicotinic
Acetylcholine Receptor Ion Channel

Bixel, M.G., Weise, C., Bolognesi, M.L., Rosini, M., Brierly, M., Mellor, I.R., Usherwood, P.N.R., Melchiorre, C. and Hucho, F.

To map the structure of a ligand-gated ion channel we used the photolabile polyamine-containing toxin MR44 as photoaffinity label. MR44 binds with high affinity to the nicotinic acetylcholine receptor in its closed channel conformation. The binding stoichiometry was two molecules of MR44 per receptor monomer. Upon UV irradiation of the receptor-ligand complex, 125I-MR44 was incorporated into the receptor alpha-subunit. From proteolytic mapping studies we conclude that the site of 125I-MR44 cross-linking is contained in the sequence alphaHis-186 to alphaLeu-199, which is part of the extracellular domain of the receptor. This sequence partially overlaps in its C-terminal region with one of the three loops that form the agonist-binding site. The agonist carbachol and the  competitive antagonist alpha-bungarotoxin had only minor influence on the photocross-linking of 125I-MR44. The site where the hydrophobic head group of 125I-MR44 binds must therefore be located outside the zone that is sterically influenced by agonist bound at the nAChR. In binding and photocross-linking experiments the luminal non-competitive  inhibitors ethidium and TPMP+ were found to compete with 125I-MR44. We conclude that the polyamine moiety of 125I-MR44 interacts with the high affinity non-competitive inhibitor site deep in the channel of the nAChR, while the aromatic ring of this compound binds in the upper part of the ion channel, i.e. in the vestibule, to a hydrophobic region on the alpha-subunit that is located in close proximity to the agonist binding site. The region of the alpha-subunit labeled by 125I-MR44 should therefore be accessible from the luminal side of the vestibule.
 

Toxicon. 2001 Jul 1;39(7):921-927.

First tryptophan-containing weak neurotoxin from cobra venom

Utkin Y.N., Kukhtina V.V., Maslennikov I.V., Eletsky A.V, Starkov V.G., Weise C., Franke P., Hucho F. and Tsetlin
V.I.

The weak neurotoxins from Elapidae venom belong to the group of "three-finger toxins" and contain an extra disulfide bridge in the
first loop. Contrary to other types of "three-finger toxins", there are no X-ray or NMR structures for weak neurotoxins, and their
pharmacology is largely unknown. With the purpose of studying structure-function relationships, we have isolated a weak neurotoxin
(WTX) from the venom of cobra Naja kaouthia using a combination of gel-filtration and ion-exchange chromatography. The amino
acid sequence of the isolated toxin was determined by means of Edman degradation and MALDI mass spectrometry, the primary
structure obtained being confirmed by 1H-NMR in the course of spatial structure analysis. The WTX sequence differs slightly from
that of the toxin CM-9a isolated earlier from the same venom (Joubert and Taljaard, 1980). The differences include an extra residue
(Trp36) between Ser35 and Arg37 as well as interchanging of two residues (Tyr52 and Lys50) in the C-terminal part of the toxin
molecule. These changes improve the alignment that can be made with other weak neurotoxin sequences. An extended sequence
comparison reveals that WTX is the first case of a tryptophan-containing weak neurotoxin isolated from cobra venom.
 

Eur J Biochem 2000 Dec 1;267(23):6784-6789.

Muscarinic toxin-like proteins from cobra venom

Kukhtina VV, Weise C, Muranova TA, Starkov VG, Franke P, Hucho F, Wnendt S, Gillen C, Tsetlin VI, Utkin YN

Three new polypeptides were isolated from the venom of the Thailand cobra Naja kaouthia and their amino-acid sequences determined. They consist of 65-amino-acid residues and have four disulfide bridges. A comparison of the amino-acid sequences of the new polypeptides with those of snake toxins shows that two of them (MTLP-1 and MTLP-2) share a high degree of similarity (55-74% sequence identity) with muscarinic toxins from the mamba. The third polypeptide (MTLP-3) is similar to muscarinic toxins with respect to the position of cysteine residues and the size of the disulfide-confined loops, but shows less similarity to these toxins (30-34% sequence identity). It is almost identical with a neurotoxin-like protein from Bungarus multicinctus (TrEMBL accession number Q9W727), the sequence of which has been deduced from cloned cDNA only. The binding affinities of the isolated muscarinic toxin-like proteins towards the different muscarinic acetylcholine receptor (mAChR) subtypes (m1-m5) was determined in competition experiments with N-[3H]methylscopolamine using membrane preparations from CHO-K1 cells, which express these receptors. We found that MTLP-1 competed weakly with radioactive ligand for binding to all mAChR subtypes. The most pronounced effect was observed for the m3 subtype; here an IC50 value of about 3 muM was determined. MTLP-2 had no effect on ligand binding to any of the mAChR subtypes at concentrations up to 1 muM. MTLP-1 showed no inhibitory effect on alpha-cobratoxin binding to the nicotinic acetylcholine receptor from Torpedo californica at concentrations up to 20 muM.

Biophys Chem 2000 Jul 15;85(2-3):141-52

Functional immobilisation of the nicotinic acetylcholine receptor in tethered lipid membranes

Sevin-Landais A, Rigler P, Tzartos S, Hucho F, Hovius R, Vogel H

The nicotinic acetylcholine receptor from Torpedo was immobilised in tethered membranes. Surface plasmon resonance was used to quantify the binding of ligands and antibodies to the receptor. The orientation and structural integrity of the surface-reconstituted receptor was probed using monoclonal antibodies, demonstrating that approximately 65% of the receptors present their ligand-binding site towards the lumen of the flow cell and that at least 85% of these receptors are structurally intact. The conformation of the receptor in tethered membranes was investigated with Fourier transform infrared spectroscopy and found to be practically identical to that of receptors reconstituted in lipid vesicles. The affinity of small receptor ligands was determined in a competition assay against a monoclonal antibody directed against the ligand-binding site which yielded dissociation constants in agreement with radioligand binding assays. The presented method for the functional immobilisation of the nicotinic acetylcholine receptor in tethered membranes might be generally applicable to other membrane proteins.

J Biol Chem. 2000 Nov 3;275(44):34359-64.

CBP/p300 activates MyoD by acetylation

Polesskaya A, Duquet A, Naguibneva I, Weise C, Vervisch A, Bengal E, Hucho F, Robin P, Harel-Bellan A

The myogenic protein MyoD requires two nuclear histone acetyl-transferases (HATs), CBP/p300 and PCAF, to transactivate muscle promoters. MyoD is acetylated by PCAF in vitro, which seems to increase its affinity for DNA. We here show that MyoD is constitutively acetylated in muscle cells. In vitro, MyoD is acetylated both by CBP/p300 and by PCAF on two lysines located at the boundary of the DNA binding domain. MyoD acetylation by CBP/p300 (as well as by PCAF) increases its activity on a muscle specific promoter, as assessed by microinjection experiments. MyoD mutants that cannot be acetylated in vitro are not activated in the functional assay. Our results provide direct evidence that MyoD acetylation functionally activates the protein and show that both PCAF and CBP/p300 are candidate enzymes for MyoD acetylation in vivo.

Exp Cell Res 2000 Jul 10;258(1):204-14

Analysis of the subcellular distribution of protein kinase Calpha using PKC-GFP fusion proteins

Wagner S, Harteneck C, Hucho F, Buchner K

One important factor for the determination of the specific functions of protein kinase C (PKC) isoforms is their specific subcellular localization. In NIH 3T3 fibroblasts phorbol esters induce translocation of PKCalpha to the plasma membrane and the nucleus. In order to investigate PKCalpha's subcellular distribution and especially its nuclear accumulation in more detail we used fusion proteins consisting of PKCalpha and the green fluorescent protein (GFP). Purified GFP-PKCalpha from baculovirus-infected insect cells undergoes nuclear accumulation without any further stimuli in digitonin-permeabilized cells. Interestingly, permeabilization appears to be a trigger for PKCalpha's nuclear translocation, since the fusion protein also translocates to the nucleus in transiently transfected cells following permeabilization. This suggests that PKCalpha has a high nuclear binding capacity even in the case of large protein amounts.
In contrast to endogenous PKCalpha, overexpressed GFP-PKCalpha as well as overexpressed PKCalpha itself translocates mainly to the plasma membrane and only to a smaller extent to the nucleus following stimulation with phorbol ester. Use of fusion proteins of GFP and different mutants of PKCalpha enabled determination of motifs involved PKCalpha's subcellular distribution: A25E and K368R point mutations of PKCalpha showed enhanced affinity for the plasma membrane, whereas sequences within the regulatory domain probably confer PKCalpha's nuclear accumulation.

J Biol Chem 2000 Jul 18

Binding Properties of Agonists and Antagonists to Distinct Allosteric States of the Nicotinic Acetylcholine Receptor are Incompatible with a Concerted Model

Krauss M, Korr D, Herrmann A, Hucho F

Recent work has shown that the nicotinic acetylcholine receptor (nAChR) can be fixed in distinct conformations by chemical cross-linking with glutardialdehyde which abolishes allosteric transitions in the protein. Here, two conformations that resemble the desensitized and the resting states were compared with respect to their affinities for different classes of ligands. The same ligands were tested for their ability to convert the nAChR from a conformation with low-affinity to a conformation with high-affinity for acetylcholine. As expected, agonists were found to bind with higher affinity to the desensitized state-like conformation and to induce a shift of the nAChR to this high-affinity state. In contrast, although most antagonists tested bound preferentially to the desensitized receptor as well they failed to induce a change of the affinity for acetylcholine. These observations sharply contradict basic predictions of the concerted model including the postulate of a pre-formed equilibrium between the different states of the nAChR in absence of agonist. With a similar approach we could show that the non-competitive inhibitor ethidium is displaced in a non-allosteric manner by other well-characterized channel blockers from the cross-linked nAChR. These results require revision of current models for the mechanisms underlying non-competitive antagonism at the nAChR.

Membr Cell Biol 2000;13(2):143-64

Structural organization of nicotinic acetylcholine receptors

Utkin YuN, Tsetlin VI, Hucho F

Nicotinic acetylcholine receptor of the electric ray Torpedo is the most comprehensively characterized neurotransmitter receptor. It consists of five subunits (alpha2beta gammadelta) amino acid sequences of which were determined by cDNA cloning and sequencing. The shape and size of the receptor were determined by electron cryomicroscopy. It has two agonist/competitive antagonist binding sites which are located between subunits near the membrane surface. The receptor ion channel is formed by five transmembrane helices (M2) of all five subunits. The position of the binding site for noncompetitive ion channel blockers was found by photoaffinity labelling and site-directed mutagenesis. The intrinsic feature of the receptor structure is the position of the agonist/competitive antagonist binding sites in close vicinity to the ion channel spanning the bilayer membrane. This peculiarity may substantially enhance allosteric transitions transforming the ligand binding into the channel opening and physiological response. Muscle nicotinic acetylcholine receptors from birds and mammals are also pentaoligomers consisting of four different subunits (alpha2beta gammadelta or alpha2beta epsilondelta) with high homology to the Torpedo receptor. Apparently, the pentaoligomeric structure is the main feature of all nicotinic, both muscle and neuronal, receptors. However, the neuronal receptors are formed only by two subunit types (alpha and beta) or are even pentahomomers (alpha7 neuronal receptors). All nicotinic receptors are ligand-gated ion channel, the properties of the channels being essentially determined by amino acid residues forming M2 transmembrane fragments.

Eur J Biochem 2000 Jan;267(1):110-20

Structure-activity relationship and site of binding of polyamine derivatives at the
nicotinic acetylcholine receptor

Bixel MG, Krauss M, Liu Y, Bolognesi ML, Rosini M, Mellor IS, Usherwood PN, Melchiorre C, Nakanishi K, Hucho F

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic
acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been
investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that
an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost
completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity,
whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues. The
stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of
nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well-characterized luminal NCIs. To
elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross-linked to the nAChR in its closed channel
conformation resulting in labeling yields for the two alpha and the beta, gamma and delta subunits of 10.4, 11.1, 4.0 and 7.4%, respectively.
Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The
hydrophobic head groups most likely bind to the high-affinity NCI site, while the positively charged polyamine chains presumably interact with
the negatively charged selectivity filter located deep in the channel lumen.

J Med Chem 1999 Dec 16;42(25):5212-23

Design, synthesis, and biological evaluation of symmetrically and unsymmetrically substituted  methoctramine-related polyamines as muscular nicotinic receptor noncompetitive antagonists

Rosini M, Budriesi R, Bixel MG, Bolognesi ML, Chiarini A, Hucho F, Krogsgaard-Larsen P, Mellor IR, Minarini A, Tumiatti V, Usherwood PN, Melchiorre C

The universal template approach to drug design foresees that a polyamine can be modified in such a way to recognize any neurotransmitter receptor. Thus, hybrids of polymethylene tetraamines and philanthotoxins, exemplified by methoctramine (1) and PhTX-343 (2), respectively, were synthesized to produce novel inhibitors of muscular nicotinic acetylcholine receptors. Polyamines 3-25 were synthesized and their biological profiles were evaluated at frog rectus abdominis muscle nicotinic receptors and guinea pig left atria (M(2)) and ileum longitudinal muscle (M(3)) muscarinic acetylcholine receptors.
All of the compounds, like prototypes 1 and 2, were noncompetitive antagonists of nicotinic receptors while being, like 1, competitive antagonists at muscarinic M(2) and M(3) receptor subtypes. Interestingly, polyamines bearing a low number of methylenes between the nitrogen atoms, as in 3, 6, and 7, displayed a biological profile similar to that of 2: a noncompetitive antagonism at nicotinic receptors in the 7-25 microM range while not showing any antagonism for muscarinic receptors up to 10 microM. Increasing the number of methylenes separating these nitrogen atoms in methoctramine-related tetraamines resulted in a significant improvement in potency at nicotinic receptors. The most potent tetraamine was 19, bearing a 12methylene spacer between the nitrogen atoms, which was 12-fold and 250-fold more potent than prototypes 1 and 2, respectively. Tetraamines 9-11, bearing a rather rigid spacer between the nitrogen atoms instead of the very flexible polymethylene chain, displayed a profile similar to that of 1 at nicotinic receptors, whereas a significant decrease in potency was observed at muscarinic M(2) receptors. This finding may have relevance in understanding the mode of interaction with these receptors. Similarly, the constrained analogue 12 of methoctramine showed a decrease in potency at nicotinic and muscarinic M(2) receptors, revealing that the tricyclic system, which incorporates the 2-methoxybenzylamine moiety of 1, does not represent a good pharmacophore for activity at these sites. A most intriguing finding was the observation that the photolabile tetraamine 22 was more potent than methoctramine at nicotinic receptors and, what is more important, it inhibited a closed state of the receptor.

Eur J Biochem 1999 Nov 1;265(3):902-910

How do acetylcholine receptor ligands reach their binding sites?

Saez-Briones P, Krauss M, Dreger M, Herrmann A, Tsetlin VI, Hucho F

The access pathway to the binding sites for large competitive antagonists of the nicotinic acetylcholine receptor from Torpedo californica electric tissue was analyzed by binding and photolabeling experiments with alpha-neurotoxins. Binding assays with [125I]alpha-bungarotoxin showed an increase in the number of accessible binding sites upon stepwise solubilization of the receptor-rich membranes. Similarily, ligand binding is facilitated upon fluidization of the membrane by increasing the temperature. The access to the binding sites seems to be sterically 'hindered' in the densely packed membrane state. Using a novel series of large biotinylated photoactivatable derivatives of neurotoxin II, we observed that the accessibility to the alpha/gamma- but not to the alpha/delta-binding site was considerably decreased for some derivatives under native conditions. This effect was less apparent at higher temperatures and could be abolished by complete solubilization. These observations support the nonequivalence of the receptor's binding sites. Together, our data suggest (a) that alpha-neurotoxins approach their binding sites from the membrane-facing periphery of the receptor's extramembrane domain rather than through the channel mouth and (b) that different entrance pathways to each binding site exist which vary in their sensitivity to the physical state of the plasma membrane.
 

Biochemistry 1999 Jul 20;38(29):9426-34

Identification of phosphorylation sites in native lamina-associated polypeptide 2 beta.

Dreger M, Otto H, Neubauer G, Mann M, Hucho F

Lamina-associated polypeptide 2 beta (LAP 2 beta), an integral protein of the inner nuclear membrane, appears to be involved in the spatial organization of the interface between nucleoplasma, lamina, and nuclear envelope. Its ability to interact with other proteins and the structural integrity of the nuclear envelope is probably regulated by phosphorylation. Here, we report nonmitotic LAP 2 beta phosphorylation sites that are phosphorylated in the native protein when purified from nuclear envelopes of mouse neuroblastoma Neuro2a cells. Five phosphorylation sites were detected by nano-electrospray mass spectrometric analysis of tryptic LAP 2 beta
peptides using parent ion scans specific for phosphopeptides. By mass spectrometric sequencing of these peptides, we identified as phosphorylated residues Thr 74, Thr 159, Ser 176, and Ser 179. Two of the phosphorylation sites, Thr 74 (within a region known to bind chromatin) and Thr 159, are part of consensus sequences of proline-directed kinases. Ser 179 is part of a consensus site for protein kinase C which is able to highly phosphorylate LAP 2 beta in vitro. Three phosphorylation sites, Thr 159, Ser 176, and Ser 179, are located within a stretch of 20 amino acids, thereby forming a highly phosphorylated protein domain which may integrate signaling by multiple protein kinases. Additionally, we identified for the first time at the protein level the LAP 2 splice variant LAP 2 epsilon in nuclear envelopes.
 

Eur J Biochem 1999 Jun;262(2):501-6

Analysis of ectatomin action on cell membranes.

Pluzhnikov K, Nosyreva E, Shevchenko L, Kokoz Y, Schmalz D, Hucho F, Grishin E

Laboratory of neuroreceptors and neuroregulators, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, RAS, Moscow, Russia.

Ectatomin (m = 7928 Da) is a toxic component from the Ectatomma tuberculatum ant venom containing two homologous polypeptide chains (37 and 34 residues) linked to each other by a disulfide bond. In aqueous solution it forms a four alpha-helix bundle. At concentrations of 0.05-0.1 microm, ectatomin forms channels in
cellular and artificial bilayer membranes. Immunochemical analysis of the intracellular distribution of ectatomin showed that the toxin gets efficiently inserted into the plasma membrane at a concentration of 5 x 10-7 m and does not penetrate inside the cell. The effect of ectatomin on cardiac L-type calcium current was studied.
Calcium currents (ICa) in isolated rat cardiac ventricular myocytes were measured using the whole-cell perforated patch-clamp technique. It was shown that ectatomin at concentrations of 0.01-10 nm inhibited ICa after a latency of few seconds. ICa was decreased twofold by 10 nm ectatomin. However, the most prominent effect of ectatomin was observed after stimulation of ICa by isoproterenol, an agonist of beta-adrenoreceptors, for forskolin, a stimulator of adenylate cyclase. At a concentration of 1 nm, ectatomin abolished the isoproterenol- and forskolin-sensitive components of ICa. The inhibitory effect of ectatomin was partially reversed by subsequent application of 2 &mgr;m of forskolin, whereas subsequent isoproterenol application did not produce the same effect.
 

J Protein Chem 1999 Feb;18(2):205-14

Butyrylcholinesterase is complexed with transferrin in chicken serum.

Weitnauer E, Ebert C, Hucho F, Robitzki A, Weise C, Layer PG

Department of Developmental Biology and Neurogenetics, Darmstadt University of Technology, Germany.

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.
 

J Recept Signal Transduct Res 1999 Jan-Jul;19(1-4):559-71

Benzophenone-type photoactivatable derivatives of alpha-neurotoxins and alpha-conotoxins in studies on Torpedo nicotinic acetylcholine receptor.

Kasheverov I, Zhmak M, Chivilyov E, Saez-Brionez P, Utkin Yu, Hucho F, Tsetlin V

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russian Federation.

By chemical modification of different lysine residues, benzoylbenzoyl (BzBz) groups were introduced into neurotoxin II Naja naja oxiana (NT-II), a short-chain snake venom alpha-neurotoxin, while p-benzoylphenylalanyl (Bpa) residue was incorporated in the course of peptide synthesis at position 11 of alpha-conotoxin G1, a neurotoxic peptide from marine snails. Although the crosslinking yields for iodinated BzBz derivatives of NT-II and for Bpa analogue of G1 to the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR) are relatively low, the subunit labeling patterns confirm the earlier conclusions,
derived from arylazide or diazirine photolabels, that alpha-neurotoxins and alpha-conotoxins bind at the subunit interfaces. Detecting the labeled alpha-subunit with iodinated Bpa analogue of G1 provided a direct proof for the contact between this subunit and alpha-conotoxin molecule.
 

J Physiol Paris 1998 Apr;92(2):85-88

The role of subunit interfaces for the nicotinic acetylcholine receptor's allosterism.

Hucho F, Methfessel C, Watty A

Cross-linking of nicotinic acetylcholine receptors, combined with binding studies and patch-clamp electrophysiology, has proven the existence of a 'pre-existing equilibrium' of functional states and the functional role of subunit interfaces, two key postulates of the allosteric model.
 

J Cell Sci 1998 Jul;111 ( Pt 13):1823-30

Nuclear import of protein kinase C occurs by a mechanism distinct from the mechanism used by proteins with a classical nuclear localization signal.

Schmalz D, Hucho F, Buchner K

Protein kinase C does not have any known nuclear localization signal but, nevertheless, is redistributed from the cytoplasm to the nucleus upon various stimuli. In NIH 3T3 fibroblasts stimulation with phorbol ester leads to a translocation of protein kinase C alpha to the plasma membrane and into the cell nucleus. We compared the mechanism of protein kinase C alpha's transport into the nucleus with the transport mechanism of a protein with a classical nuclear localization signal at several steps. To this end, we co-microinjected fluorescently labeled bovine serum albumin to which a nuclear localization signal peptide was coupled, together with substances interfering with conventional nuclear protein import. Thereafter, the distribution of both the nuclear localization signal-bearing reporter protein and protein kinase C alpha was analyzed in the same cells. We can show that, in contrast to the nuclear localization signal-dependent transport, the phorbol ester-induced transport of protein kinase C alpha is not affected by microinjection of antibodies against the nuclear import factor p97/importin/karyopherin beta or microinjection of non-hydrolyzable GTP-analogs. This suggests that nuclear import of protein kinase C alpha is independent of p97/importin/karyopherin beta and independent of GTP. At the nuclear pore there are differences between the mechanisms too, since nuclear transport of protein kinase C alpha cannot be inhibited by wheat germ agglutinin or an antibody against nuclear pore complex proteins. Together these findings demonstrate that the nuclear import of protein kinase C alpha occurs by a mechanism distinct from the one used by classical nuclear localization signal-bearing proteins at several stages.
 

Eur J Biochem 1998 Mar 1;252(2):222-228

The accessible surface of the nicotinic acetylcholine receptor. Identification by chemical modification and cross-linking with 14C-dimethyl suberimidate.

Watty A, Weise C, Dreger M, Franke P, Hucho F

To obtain structural information on the nicotinic acetylcholine receptor from Torpedo electric tissue we modified and cross-linked lysine residues with the agonistic bifunctional reagent [14C]dimethyl suberimidate. This reagent labels exposed lysine residues, especially those located near the ligand-binding site, and cross-links lysine residues located not more than 11 A, the length of the cross-linker, apart. Using this method, we identified a cross-link located between betaLys177 and betaLys191 showing that the 13 amino acids in between form a loop with these two residues located at the surface. Cross-linking also occurred between the vicinal lysine residues alphaLys76 and alphaLys77, indicating that these neighbouring lysine residues are not involved in a beta-sheet structure. A total of 21 out of 97 lysine residues present in the receptor were modified by [14C]dimethyl suberimidate. Thus these residues are located on the accessible extramembrane surface. The two lysine residues alphaLys76 and alphaLys179 were predominantly labelled. Because of the agonistic property of [14C]dimethyl suberimidate [Watty, A., Methfessel, C. & Hucho, F. (1997) Proc. Natl Acad. Sci. USA 94, 8202-8209] this might be due to their close proximity to the ligand binding site.
 

Eur J Biochem 1998, 253,:229-35.

Labeling of Torpedo californica nicotinic acetylcholine receptor subunits by cobratoxin derivatives with photoactivatable groups of different chemical nature at Lys23.

Utkin YN, Krivoshein AV, Davydov VL, Kasheverov IE, Franke P, Maslennikov IV, Arseniev AS, Hucho F, Tsetlin VI

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
utkin@ibch.siobc.ras.ru

Different photoactivatable derivatives of toxin 3 (CTX) Naja naja siamensis were obtained after CTX reaction with N-hydroxysuccinimide esters of p-azidobenzoic, p-azidotetraflourobenzoic, p-benzoylbenzoic and p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoic acids. The ion-exchange HPLC profiles for the reaction products were very similar in four cases, with one predominant peak corresponding to the derivative containing the label at Lys23. After [125I]iodination, CTX photoactivatable derivatives were cross-linked to the nicotinic acetylcholine receptor from Torpedo californica under optimized conditions. The highest cross-linking yield (up to 16% of the bound toxin) was observed for azidobenzoyl-Lys23-CTX. Different receptor subunits were found to be labelled depending on the nature of the photoactivatable group: the azido derivatives labelled the gamma and delta subunits, benzoylbenzoyl derivative labelled the alpha and delta subunits, while
p-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl derivative reacted with alpha, gamma and delta subunits. The cross-linking experiments in the presence of varying concentrations of (+)-tubocurarine demonstrated that the Lys23-attached diazirinyl group contacts the delta and alpha subunits in one ligand-binding site, whereas at the other site, for another CTX molecule, the contacts of the Lys23-diazirinyl are with gamma and alpha subunits. This means that the central loop in the two CTX molecules binds at the alpha/gamma and alpha/delta interfaces. Calculation of the sterically possible displacement of diazirinyl nitrogen, basing on the known X-ray structure of CTX, showed that this value does not exceed 13 A. The results obtained favor the disposition of the ligand-binding sites at the subunit interfaces, with the distance between alpha and delta, or alpha and gamma subunits at these sites being not more than 13 A.

Naturwissenschaften 1997 Jul;84(7):281-290

Signal transduction and protein kinases: the long way from the plasma membrane into the nucleus.

Hucho F, Buchner K

All living cells must be able to receive information from the extracellular space and to react to it by processing and converting it into intracellular effects. If the properties of cells are to change in the long term, some signals must reach the nucleus in order to bring about changes in gene transcription. Three of the pathways, beginning with an extracellular signal and ending with the nucleus, serve to illustrate some principles of signal transduction such as signal conversion, signal cascade, cross-talk, and on/off switch. One element common to most of the pathways is the activation of protein kinases. One example of these kinases, the protein kinase C, is discussed as a vehicle of signal transport toward the nucleus and as a means of cross-talk between different signaling pathways.

Publication Type: REVIEW

Proc Natl Acad Sci U S A 1997 Jul 22;94(15):8202-8207

Fixation of allosteric states of the nicotinic acetylcholine receptor by chemical cross-linking.

Watty A, Methfessel C, Hucho F

Receptor activity can be described in terms of ligand-induced transitions between functional states. The nicotinic acetylcholine receptor (nAChR), a prototypic ligand-gated ion channel, is an "unconventional allosteric protein" which exists in at least three interconvertible conformations, referred to as resting (low agonist affinity, closed channel), activated (open channel), and desensitized (high agonist affinity, closed channel). Here we show that 3,3'-dimethyl suberimidate (DMS) is an agonistic bifunctional cross-linking reagent, which irreversibly "freezes" the nAChR in a high agonist affinity/closed-channel state. The monofunctional homologue methyl acetoimidate, which is also a weak cholinergic agonist, has no such irreversible effect. Glutardialdehyde, a cross-linker that is not a cholinergic effector, fixes the receptor in a low-affinity state in the absence of carbamoylcholine, but, like DMS, in a high-affinity state in its presence. Covalent cross-linking thus allows us to arrest the nAChR in defined conformational states.

Biochemistry 1997 Jan 28;36(4):839-847

Interactions of the nicotinic acetylcholine receptor transmembrane segments with the lipid bilayer in native receptor-rich membranes.

Dreger M, Krauss M, Herrmann A, Hucho F

Proper ion channel function of the nicotinic acetylcholine receptor (nAChR) requires the interaction of the protein with distinct lipid species present in the receptor's membrane microenvironment. Two classes of lipid binding sites present at the protein-membrane interface have been postulated: annular binding sites primarily occupied by phospholipids and non-annular binding sites mainly occupied by cholesterol [Jones & McNamee (1988) Biochemistry 27, 2364-2374]. We investigated the binding of these lipids to the nAChR and potential dynamics of these interactions during events associated with signal transduction by electron spin resonance spectroscopy (ESR) using spin-labeled analogues of phospholipids, androstane, and stearic acid. Protein-lipid interactions were characterized in receptor-rich membranes prepared from Torpedo californica electric tissue preserving the native lipid environment of the nAChR. We found a strong preference of the receptor for the phosphatidylserine (PS) analogue as compared to the other probes. Up to 57% of PS were perturbed by the membrane protein, while the fraction of motionally restricted lipid for the other analogues was on the order of 30%. After removal of the extramembrane portions of the membrane-bound receptor, we observed a loss of binding sites for the spin-labeled analogue of androstane and for stearic acid, but not for phospholipids and sphingomyelin analogues. Our results demonstrate the existence of topologically distinct lipid binding sites for different lipid species. In the case of cholesterol, extramembrane portions of the receptor are involved, whereas the transmembrane segments meet the requirements for the binding of phospholipids. Tyrosine phosphorylation of the nAChR did not affect protein-lipid interactions in samples of intact nAChR. Similarly, no significant changes were observed in the presence of carbamoylcholine at concentrations that caused rapid and quantitative desensitization of the nAChR.

J. Cell Sci. 1996 Sep;109( Pt 9):2401-2406

Transport of protein kinase C alpha into the nucleus requires intact cytoskeleton while the transport of a protein containing a canonical nuclear localization signal does not.

Schmalz D, Kalkbrenner F, Hucho F, Buchner K

Protein kinase C undergoes a redistribution from the cytosol into the nucleus upon various stimuli. Since protein kinase C does not contain any known nuclear localization signal, the exact pathway and mechanism of the translocation into the nucleus is not known. We used immunofluorescence microscopy to investigate the role of the cytoskeleton in this process, and to detect the subcellular distribution of protein kinase C alpha in NIH 3T3 fibroblasts. In these cells protein kinase C alpha is translocated into the nucleus after stimulation with phorbol ester. We observed that cells treated with the cytoskeleton disrupting agents cytochalasin B or colchicine do not show the nuclear translocation of protein kinase C alpha after stimulation. In contrast, the nuclear accumulation of a nuclear localization signal containing reporter protein in an in vitro nuclear transport assay is not affected by these drugs. This observation has been confirmed for intact cells by microinjection experiments: cells which have been incubated with cytochalasin B or colchicine prior to microinjection of the reporter protein show the same accumulation in the nucleus as untreated cells. Our data show that intact cytoskeleton plays an important role in the translocation of protein kinase C alpha into the nucleus but not in the nuclear import of a karyophilic reporter protein.

Eur J Biochem 1996 Aug 1;239(3):539-557

The emerging three-dimensional structure of a receptor. The nicotinic acetylcholine receptor.

Hucho F, Tsetlin VI, Machold J

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 nm x 8 nm) were deduced from electron-microscopy studies. Its subunits are arranged around a five-fold axis of pseudosymmetry in the order (clockwise) alpha H gamma alpha L delta beta. Its two agonist/competitive-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.

J Neurochem 1996 May;66(5):1781-1792

Structural biology of key nervous system proteins.

Hucho F, Tsetlin V

Publication Types: REVIEW - No MEDLINE abstract available

Eur J Biochem 1995 Dec 1;234(2):427-430

The handedness of the subunit arrangement of the nicotinic acetylcholine receptor from Torpedo californica.

Machold J, Weise C, Utkin Y, Tsetlin V, Hucho F

Cross-linking an alpha-neurotoxin with a known three-dimensional structure and with photoactivatable groups in known positions to native membrane-bound acetylcholine receptor reveals its quaternary structure, including the handedness of its circular subunit arrangement. Photolabelling with alpha-neurotoxin carrying the photoactivatable group at position Lys46 is inhibited by the competitive antagonist (+)-tubocurarine in a biphasic manner, indicating that it reacts with both alpha-subunits that were shown to have different affinities for this antagonist [Neubig, R. R. & Cohen, J. B. (1979) Biochemistry 18, 5464-5475]. Lys46 is located on loop III of the neurotoxin. The other information necessary for the elucidation of the handedness was provided by the recent finding that the central loop of the toxin (loop II) is oriented towards the central pore of the receptor, securing the overall orientation of the bound toxin [Machold, J., Utkin, Y. N., Kirsch, D., Kaufmann, R., Tsetlin, V. & Hucho, F. (1995b) Proc. Natl Acad. Sci. USA 92, 7282-7286]. Looking at the receptor from the synaptic side of the postsynaptic membrane, it was concluded that the clockwise subunit arrangement is alpha H-gamma-alpha L-delta-beta (alpha H and alpha L are the alpha-subunits binding (+)-tubocurarine with high and low affinity, respectively). Its mirror image alpha alpha L-gamma-alpha H-beta-delta could thus be excluded.

Proc Natl Acad Sci U S A 1995 Aug 1;92(16):7282-7286

Photolabeling reveals the proximity of the alpha-neurotoxin binding site to the M2 helix of the ion channel in the nicotinic acetylcholine receptor.

Machold J, Utkin Y, Kirsch D, Kaufmann R, Tsetlin V, Hucho F

A photoactivatable derivative of neurotoxin II from Naja naja oxiana containing a 125I-labeled p-azidosalicylamidoethyl-1,3'-dithiopropyl label at Lys-25 forms a photo-induced cross-link with the delta subunit of the membrane-bound Torpedo californica nicotinic acetylcholine receptor (AChR). The cross-linked radioactive receptor peptide was isolated by reverse-phase HPLC after tryptic digestion of the labeled delta subunit. The sequence of this peptide, delta-(260-277), and the position of the label at Ala-268 were established by matrix-assisted laser-desorption-ionization mass spectrometry based on the molecular mass and on post-source decay fragment analysis. With the known dimensions of the AChR molecule, of the photolabel, and of alpha-neurotoxin, finding the cross-link at delta Ala-268 (located in the upper part of the channel-forming transmembrane helix M2) means that the center of the alpha-neurotoxin binding site is situated at least approximately 40 A from the extracellular surface of the AChR, proximal to the channel axis.

J Biol Chem 1995 Jun 9;270(23):13787-13793

The nuclear pore complex protein p62 is one of several sialic acid-containing proteins of the nuclear envelope.

Emig S, Schmalz D, Shakibaei M, Buchner K

While investigating the glycosylation of nuclear envelope proteins of neuroblastoma cells, we found several proteins that bound the sialic acid-specific Sambucus nigra agglutinin. The strongest signals were obtained for proteins with apparent molecular masses of 66 and 180 kDa. The specificity of the lectin binding was checked by acylneuraminyl hydrolase treatment of nuclear envelope proteins, which prohibited S. nigra agglutinin binding. Digestion of nuclear envelope proteins with the N-glycosidase F revealed that sialic acid was N-glycosidically linked to the 180-kDa protein and very probably O-glycosidically linked to the 66-kDa protein. Upon extraction, the latter behaved like the nucleoporin p62 in that it was partly extracted by high ionic strength buffers, could not be solubilized by nonionic detergent, and was completely removed from the nuclear envelope with urea. Two-dimensional gel electrophoretic comparison showed that the S. nigra agglutinin-binding protein and p62 have an identical isoelectric point of about 5.0 and an identical apparent molecular mass of 66 kDa. This, together with the binding of the anti-nucleoporin antibody, demonstrated the identity of the 66-kDa sialoprotein and p62. S. nigra agglutinin inhibits nuclear protein transport in neuroblastoma cells, strongly suggesting a functional significance of sialylation of p62.

Eur J Biochem 1995 Mar 1;228(2):211-221

Protein kinase C in the transduction of signals toward and within the cell nucleus.

Buchner K

Publication Types: REVIEW - No MEDLINE abstract available

Trends Biochem Sci 1994 Sep;19(9):383-387

Beta-structure in the membrane-spanning part of the nicotinic acetylcholine receptor (or how helical are transmembrane helices?).

Hucho F, Gorne-Tschelnokow U, Strecker A

The 'four-transmembrane-helix receptors' transmit their signals from the extracellular space to the cytoplasm via an intramembrane domain. In the case of the nicotinic acetylcholine receptor this domain comprises an ion channel formed by homologous secondary structure elements in the receptor subunits. It was believed to be exclusively alpha-helical, but recent experimental evidence questions the widely accepted model: beta-strands seem to be part of the membrane-spanning domain.

EMBO J 1994 Jan 15;13(2):338-341

The transmembrane domains of the nicotinic acetylcholine receptor contain alpha-helical and beta structures.

Gorne-Tschelnokow U, Strecker A, Kaduk C, Naumann D, Hucho F

The transmembrane domain of the nicotinic acetylcholine receptor (nAChR) from Torpedo californica electric tissue contains both alpha-helical and beta structures. The secondary structure was investigated by Fourier transform infrared (FTIR) spectroscopy after the extramembrane moieties of the protein from the extracellular and intracellular sides of the membrane were removed by proteolysis using proteinase K. The secondary structure composition of this membrane structure was: alpha-helical 50%, beta structure and turns 40%, random 10%. The alpha-helices are shown to be oriented with respect to the membrane plane in a way allowing them to span the membrane, while no unidirectional structure for the beta structures was observed. These findings contradict previous secondary structure models based on hydropathy plots alone.

Eur J Biochem 1992 Nov 15;210(1):45-51

Nuclear substrates of protein kinase C.

Beckmann R, Buchner K, Jungblut PR, Eckerskorn C, Weise C, Hilbert R, Hucho F

Starting from the finding that, for neuronal cells, the nuclear-membrane-associated protein kinase C (PKC) is the so-called 'membrane inserted', constitutively active form, we attempted to identify substrates of this nuclear PKC. For this purpose, nuclear membranes and other subcellular fractions were prepared from bovine brain, and in-vitro phosphorylation was performed. Several nuclear membrane proteins were found, the phosphorylation of which was inhibited by specific PKC inhibitors and effectively catalyzed by added PKC. Combining the methods of two-dimensional gel electrophoresis, in-situ digestion, reverse-phase HPLC and microsequencing, two of these nuclear PKC substrates were identified; the known PKC substrate Lamin B2, which serves as a control of the approach and the nucleolar protein B23. Our data suggest, that, for B23, Ser225 is a site of phosphorylation by PKC.

Biochemistry 1992 Sep 8;31(35):8239-8244

Investigation of ligand-binding sites of the acetylcholine receptor using photoactivatable derivatives of neurotoxin II from Naja naja oxiana.

Kreienkamp HJ, Utkin YN, Weise C, Machold J, Tsetlin VI, Hucho F

Several photoaffinity derivatives of neurotoxin II from the venom of the central Asian cobra Naja naja oxiana have been prepared. After reaction of the 125I-labeled derivatives with the nicotinic acetylcholine receptor from electric organ, the alpha-subunit of the nAChR is almost exclusively labeled by the derivative carrying the photoactivatable group in position Lys46. In contrast to this, a reactive group at Lys26 predominantly labels the gamma- and delta-subunits, while the alpha- and beta-subunits incorporate much less radioactivity. Competition experiments with d-tubocurarine show that the gamma-subunit is labeled when this derivative occupies the high affinity d-tubocurarine-binding site, while the delta-subunit is labeled by the toxin bound at the low-affinity d-tubocurarine site. A model is discussed for the orientation of different loops of the toxin molecules in the binding site for agonists and competitive antagonists.

Proc Natl Acad Sci U S A 1991 Jul 15;88(14):6117-6121

Anionic subsites of the catalytic center of acetylcholinesterase from Torpedo and from cobra venom.

Kreienkamp HJ, Weise C, Raba R, Aaviksaar A, Hucho F

A peptide of acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) from the venom of the cobra Naja naja oxiana labeled by the affinity reagent N,N-dimethyl-2-phenylaziridinium (DPA) has been identified. The sequence is Gly-Ala-Glu-Met-Trp-Asn-Pro-Asn. In AcChoEase from Torpedo californica, a homologous peptide was labeled and isolated. Its sequence is Ser-Gly-Ser-Glu-Met-Trp-Asn-Pro-Asn, representing positions 79 through 87. In both cases labeling can be prevented by 0.1 mM edrophonium, indicating that the respective peptides form part of the anionic subsite of the catalytic center. The modified residue was tryptophan (Trp-84 in Torpedo AcChoEase) in both enzymes. In contrast to AcChoEase from Torpedo, the enzyme from cobra venom does not contain a peripheral anionic binding site.

EMBO J 1990 Dec;9(12):3885-3888

Anionic subsites of the acetylcholinesterase from Torpedo californica: affinity labelling with the cationic reagent N,N-dimethyl-2-phenyl-aziridinium.

Weise C, Kreienkamp HJ, Raba R, Pedak A, Aaviksaar A, Hucho F

Several peptides of acetylcholinesterase of Torpedo californica labelled with the alkylating reagent [3H]N,N-dimethyl-2-phenyl-aziridinium (DPA) were localized within the primary structure. One peptide had the sequence KPQELIDVE (positions 270-278); the incorporation of DPA into this peptide could be specifically suppressed by propidium, which suggests that it is part of the peripheral anionic site. The incorporation of DPA into two other peptides was insensitive to propidium but could be prevented by edrophonium; the sequence of one of the peptides assumed to be part of the anionic site in the catalytic centre was found to be DLFR (positions 217-220). Decamethonium efficiently blocked alkylation by DPA in all three investigated peptides.