Wolfgang Nastainczyk

Serum Antibodies to L-Type Calcium Channels in Patients with Amyotrophic Lateral Sclerosis

BACKGROUND AND METHODS Sporadic amyotrophic lateral sclerosis is a chronic, progressive degenerative disease of the motor neurons of the spinal cord and motor cortex. The cause is unknown. Recent electrophysiologic studies in animals indicate that immunoglobulins from patients with this disease alter presynaptic voltage-dependent calcium currents and calcium-dependent release of neurotransmitters. To determine whether similar interactions might be identified biochemically, we used an enzyme-linked immunosorbent assay (ELISA) to detect the reaction of serum IgG with purified complexes of L-type voltage-gated calcium channels from rabbit skeletal muscle. The results from patients with amyotrophic lateral sclerosis were compared with those obtained from patients with other types of motor neuron disease, patients with autoimmune and non-autoimmune neurologic diseases, and normal subjects. RESULTS Serum samples from 36 of 48 patients with sporadic amyotrophic lateral sclerosis (75 percent) contained IgG that reacted with L-type calcium-channel protein, and serum reactivity on ELISA correlated with the rate of disease progression (Spearman rank-correlation coefficient, 0.62). Reactive serum was present in only 1 of 25 normal subjects and 1 of 35 control patients with no motor neuron disease. Antibodies to L-type voltage-gated calcium channels were identified in 6 of 9 patients with Lambert-Eaton syndrome, and in 3 of 15 patients with Guillain-Barré syndrome. CONCLUSIONS Antibodies to L-type voltage-gated calcium channels are present in the serum of patients with amyotrophic lateral sclerosis, and antibody titers correlate with the rate of disease progression. Together with previous data, these results suggest a role for autoimmune mechanisms in the pathogenesis of sporadic amyotrophic lateral sclerosis.

TRP4 (CCE1) protein is part of native calcium release-activated Ca2+-like channels in adrenal cells.

Mammalian TRP proteins have been implicated to function as ion channel subunits responsible for agonist-induced Ca2+ entry. To date, TRP proteins have been extensively studied by heterologous expression giving rise to diverse channel properties and activation mechanisms including store-operated mechanisms. However, the molecular structure and the functional properties of native TRP channels still remain elusive. Here we analyze the properties of TRP4 (CCE1) channels in their native environment and characterize TRP expression patterns and store-operated calcium currents that are endogenous to bovine adrenal cells. We show by Northern blot analysis, immunoblots, and immunohistochemistry thatTRP4 transcripts and TRP4 protein are present in the adrenal cortex but absent in the medulla. Correspondingly, bovine adrenal cortex cells express TRP4 abundantly. The only otherTRP transcript found at considerable levels wasTRP1, whereas TRP2, TRP3, TRP5(CCE2), andTRP6 were not detectable. Depletion of calcium stores with inositol 1,4,5-trisphosphate or thapsigargin activates store-operated ion channels in adrenal cells. These channels closely resemble calcium release-activated Ca2+ (CRAC) channels. Expression of trp4(CCE1) cDNA in antisense orientation significantly reduces both, the endogenous CRAC-like currents and the amount of native TRP4 protein. These results demonstrate that TRP4 contributes essentially to the formation of native CRAC-like channels in adrenal cells.

Regulation of the L-type calcium channel

Abstract The voltage-operated L-type calcium channel is regulated by protein phosphorylation and G proteins in a variety of tissues and eukaryotes including non-excitable cells. The 165 kDa protein of the dihydropyridine receptor from rabbit skeletal muscle contains all the regulatory sites of an L-type calcium channel and the calcium conducting unit . Franz Hofmann and colleagues suggest that the differences in the regulation observed in various tissues is caused by the interaction of the large conducting protein with different regulatory proteins of approximately 55 kDa .

Cytochrome P450 as an oxene transferase.

Abstract In the presence of iodosobenzene, liver microsomes catalyze the O-dealkylation of 7-ethoxycoumarin. The reaction proceeds in the absence of NADPH and O 2 and is dependent on cytochrome P450. The results indicate that cytochrome P450 acts as an oxene transferase probably involving [FeO] 3+ as the transient intermediate of active oxygen.

The mechanism of reductive dehalogenation of halothane by liver cytochrome P450

The reductive dehalogenation of halothane leading to 2-chloro-1,1,1-trifluoroethane (CTE) and 2-chloro-1,1-difluoroethylene (CDE) has been investigated in vitro using at liver microsomes under anaerobic conditions. The stimulation of NADPH oxidation by halothane as well as the formation of the products were dependent upon cytochrome P450 as indicated by their CO and metyrapone inhibition. After replacement of NADPH by sodium dithionite as a reducing agent CDE was the only product of the enzymatic reaction. The product pattern was influenced by pretreatment with 3-methylcholanthrene, benzo(a)pyrene, phenobarbitone and Arochlor 1254 and by addition of anti-cytochrome P450-PB immunoglobulin. The CTE:CDE ratio was shifted by addition or inhibition of cytochrome b5 and by pH variation indicating a crucial role of the second electron donation to cytochrome P450 in determining the product pattern. The intermediate complex of cytochrome P450 with a Soret band at 470 nm formed with halothane in reduced liver microsomes was shown to decompose spontaneously to give CDE. Therefore we propose the 470 nm peak to represent a cytochrome P450 Fe3+----CHCl-CF3 carbanion complex. From these results a reaction pathway could be derived which includes radical and carbanion intermediates as reactive precursors of CTE and CDE, respectively.

Identification of the site of interaction of the dihydropyridine channel blockers nitrendipine and azidopine with the calcium-channel alpha 1 subunit.

The dihydropyridine binding site of the rabbit skeletal muscle calcium channel alpha 1 subunit was identified using tritiated azidopine and nitrendipine as ligands. The purified receptor complex was incubated either with azidopine or nitrenidpine at an alpha 1 subunit to ligand ratio of 1:1. The samples were then irradiated by a 200 W UV lamp. The ligands were only incorporated into the alpha 1 subunit, which was isolated by size exclusion chromatography and digested either by trypsin (azidopine) or endoproteinase Asp‐N (nitrendipine). Each digest contained two radioactive peptides, which were isolated and sequenced. The azidopine peptides were identical with amino acids 13–18 (minor peak) and 1428–1437 (major peak) of the primary sequence of the skeletal muscle alpha 1 subunit. The nitrendipine peptides were identical with amino acids 1390–1399 (major peak) and 1410–1420 (minor peak). The sequence from amino acids 1390 to 1437 is identical in the alpha 1 subunits of skeletal, cardiac and smooth muscle and follows directly repeat IVS6. These results indicate that dihydropyridines bind to an area that is located at the putative cytosolic domain of the calcium channel.

The mechanism of chloroform and carbon monoxide formation from carbon tetrachloride by microsomal cytochrome P-450.

Abstract [ 14 C]carbon monoxide was a product of the anaerobic incubation of 14 CCl 4 with hepatic microsomes and either NADPH or sodium dithionite. The reaction was dependent upon cytochrome P-450 as indicated by its CO and metyrapone inhibition and by the absence of any product after conversion of cytochrome P-450 to cytochrome P-420. The major metabolite produced in the NADPH-supported reaction was chloroform which, according to isotope experiments, seems to be mainly formed by hydrogen abstraction of the · CCl 3 radical. The rate of CCl 4 -induced NADPH-oxidation could be fully accounted for by the rate of formation of CO, CHCl 3 and covalently bound metabolites. Spectral studies indicated that the immediate precursor of the CO is probably the cytochrome P-450-Fe 2+ dichlorocarbene complex. This was supported by the hydrolytic cleavage of an iron (II)-protoporphyrin IX dichlorocarbene complex to carbon monoxide. A reaction pathway for the formation of CO has been proposed including the possible radical, carbanion and carbene intermediates.

TMP21 AND P24A, TWO TYPE I PROTEINS ENRICHED IN PANCREATIC MICROSOMAL MEMBRANES, ARE MEMBERS OF A PROTEIN FAMILY INVOLVED IN VESICULAR TRAFFICKING

We report here on the isolation, cloning, and expression of two Mr 21,000 proteins from rat pancreatic acinar cells, the rat-Tmp21 (ransembrane rotein, Mr 21,000) and the rat-p24A. Both proteins are transmembrane proteins with type I topology and share weak but significant homology to one another (23% identity). We further show the cloning and characterization of the human homologs, hum-Tmp21, which is expressed in two variants (Tmp21-I and Tmp21-II), and hum-p24A. Tmp21 proteins and p24A have highly conserved COOH-terminal tails, which contain motifs related to the endoplasmic reticulum retention and retrieval consensus sequence KKXX. The rat-p24 sequence is identical to the hamster CHOp24, a recently characterized component of coatomer-coated transport vesicles, which defines a family of proteins (called the p24 family) proposed to be involved in vesicular transport processes (Stamnes, M. A., Craighead, M. W., Hoe, M. H., Lampen, N., Geromanos, S., Tempst, P., and Rothman, J. E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8011-8015). Sequence alignment and structural features identify the Tmp21 protein as a new member of this p24 family. Northern analysis of various tissues indicates that the Tmp21 proteins and the p24A protein are ubiquitously expressed. The integral membrane components Tmp21 and p24A are localized in microsomal membranes, zymogen granule membranes, and the plasma membrane and are absent from the cytosol. Both p24A and Tmp21 show weak homology to the yeast protein Emp24p, which recently has been shown to be involved in secretory protein transport from the endoplasmic reticulum to the Golgi apparatus. This leads us to conclude that the receptor-like Tmp21 and p24A are involved in vesicular targeting and protein transport.

Allosteric activation of the protein kinase PDK1 with low molecular weight compounds

Organisms rely heavily on protein phosphorylation to transduce intracellular signals. The phosphorylation of a protein often induces conformational changes, which are responsible for triggering downstream cellular events. Protein kinases are themselves frequently regulated by phosphorylation. Recently, we and others proposed the molecular mechanism by which phosphorylation at a hydrophobic motif (HM) regulates the conformation and activity of many members of the AGC group of protein kinases. Here we have developed specific, low molecular weight compounds, which target the HM/PIF‐pocket and have the ability to allosterically activate phosphoinositide‐dependent protein kinase 1 (PDK1) by modulating the phosphorylation‐dependent conformational transition. The mechanism of action of these compounds was characterized by mutagenesis of PDK1, synthesis of compound analogs, interaction‐displacement studies and isothermal titration calorimetry experiments. Our results raise the possibility of developing drugs that target the AGC kinases via a novel mode of action and may inspire future rational development of compounds with the ability to modulate phosphorylation‐dependent conformational transitions in other proteins.

Effect of oxygen concentration on the reaction of halothane with cytochrome P450 in liver microsomes and isolated perfused rat liver

Abstract The influence of oxygen on the complex formation of reduced cytochrome P450 with halothane has been investigated with liver microsomes and perfused livers from phenobarbital-pretreated rats. The reductive formation of the trifluoro carbene complex from halothane in liver microsomes was inhibited at high oxygen concentrations but started to appear below 50 μM oxygen and was maximal under anaerobic conditions. Metyrapone was an efficient inhibitor of the carbene complex formation. Organ spectrophotometry of isolated perfused livers established that the complex appeared already under slightly hypoxic conditions and that metyrapone addition to the perfusion medium abolished its formation. The results indicate the possibility of a reductive in vivo -metabolism of halothane to reactive intermediates when the oxygen concentration of the cell becomes lower than about 50 μM.