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H Glossmann

Publications and source records attributed to H Glossmann.

At least 37 records · Page 2Linked to original sources
PubMed1998

Differential effects of Ca2+ channel beta1a and beta2a subunits on complex formation with alpha1S and on current expression in tsA201 cells.

To study the interactions of the alpha1S subunit of the skeletal muscle L-type Ca2+ channel with the skeletal beta1a and the cardiac beta2a, these subunits were expressed alone or in combination in tsA201 cells. Immunofluorescence- and green fluorescent protein-labeling showed that, when expressed alone, beta1a was diffusely distributed throughout the cytoplasm, beta2a was localized in the plasma membrane, and alpha1S was concentrated in a tubular/reticular membrane system, presumably the endoplasmic reticulum (ER). Upon coexpression with alpha1S, beta1a became colocalized with alpha1S in the ER. Upon coexpression with beta2a, alpha1S redistributed to the plasma membrane, where it aggregated in large clusters. Coexpression of alpha1S with beta1a but not with beta2a increased the frequency at which cells expressed L-type currents. A point mutation (alpha1S-Y366S) or deletion (alpha1S-Delta351-380) in the beta interaction domain of alpha1S blocked both translocation of beta1a to the ER and beta2a-induced translocation of the alpha1S mutants to the plasma membrane. However, the point mutation did not interfere with beta1a-induced current stimulation. Thus, beta1a and beta2a are differentially distributed in tsA201 cells and upon coexpression with alpha1S, form alpha1S. beta complexes in different cellular compartments. Complex formation but not current stimulation requires the intact beta interaction domain in the I-II cytoplasmic loop of alpha1S.

Animals↗
PubMed1998

Familial hemiplegic migraine mutations change alpha1A Ca2+ channel kinetics.

Missense mutations in the pore-forming human alpha1A subunit of neuronal P/Q-type Ca2+ channels are associated with familial hemiplegic migraine (FHM). The pathophysiological consequences of these mutations are unknown. We have introduced the four single mutations reported for the human alpha1A subunit into the conserved rabbit alpha1A (R192Q, T666M, V714A, and I1819L) and investigated possible changes in channel function after functional expression of mutant subunits in Xenopus laevis oocytes. Changes in channel gating were observed for mutants T666M, V714A, and I1819L but not for R192Q. Ba2+ current (IBa) inactivation was slightly faster in mutants T666M and V714A than in wild type. The time course of recovery from channel inactivation was slower than in wild type in T666M and accelerated in V714A and I1819L. As a consequence, accumulation of channel inactivation during a train of 1-Hz pulses was more pronounced for mutant T666M and less pronounced for V714A and I1819A. Our data demonstrate that three of the four FHM mutations, located at the putative channel pore, alter inactivation gating and provide a pathophysiological basis for the postulated neuronal instability in patients with FHM.

Animals↗
PubMed1998

Molecular cloning and expression of the human delta7-sterol reductase.

Inhibitors of the last steps of cholesterol biosynthesis such as AY9944 and BM15766 severely impair brain development. Their molecular target is the Delta7-sterol reductase (EC 1.3.1.21), suspected to be defective in the Smith-Lemli-Opitz syndrome, a frequent inborn disorder of sterol metabolism. Molecular cloning of the cDNA revealed that the human enzyme is a membrane-bound protein with a predicted molecular mass of 55 kDa and six to nine putative transmembrane segments. The protein is structurally related to plant and yeast sterol reductases. In adults the ubiquitously transcribed mRNA is most abundant in adrenal gland, liver, testis, and brain. The Delta7-sterol reductase is the ultimate enzyme of cholesterol biosynthesis in vertebrates and is absent from yeast. Microsomes from Saccharomyces cerevisiae strains heterologously expressing the human cDNA remove the C7-8 double bond in 7-dehydrocholesterol. The conversion to cholesterol depends on NADPH and is potently inhibited by AY9944 (IC50 0.013 microM), BM15766 (IC50 1.2 microM), and triparanol (IC50 14 microM). Our work paves the way to clarify whether a defect in the delta7-sterol reductase gene underlies the Smith-Lemli-Opitz syndrome.

Amino Acid Sequence↗
PubMed1998

Structural basis of drug binding to L Ca2+ channels.

At least five different types of voltage-gated Ca2+ channels exist in electrically excitable mammalian cells. Only one type, the family of L-type Ca2+ channels (L channels), contains high-affinity binding domains within their alpha 1-subunits for different chemical classes of drugs (Ca2+ channel antagonists; exemplified by isradipine, verapamil and diltiazem). Their stereoselective, high-affinity binding induces block of channel-mediated Ca2+ inward currents in heart and smooth muscle, resulting in antihypertensive, cardiodepressive and antiarrhythmic effects. Amino acids involved in drug binding have recently been identified using photoaffinity labelling, chimeric alpha 1-subunits and site-directed mutagenesis. Insertion of the drug-binding amino acids enabled the transfer of drug-sensitivity into Ca2+ channels that are insensitive to Ca2+ channel antagonists ('gain-of-function' approach). In this review, Jörg Striessing and colleagues summarize the present knowledge about the molecular architecture of L channel drug-binding domains and the implications for Ca2+ channel pharmacology and drug development.

Animals↗
PubMed1998

Molecular basis of drug interaction with L-type Ca2+ channels.

Different types of voltage-gated Ca2+ channels exist in the plasma membrane of electrically excitable cells. By controlling depolarization-induced Ca2+ entry into cells they serve important physiological functions, such as excitation-contraction coupling, neurotransmitter and hormone secretion, and neuronal plasticity. Their function is fine-tuned by a variety of modulators, such as enzymes and G-proteins. Block of so-called L-type Ca2+ channels by drugs is exploited as a therapeutic principle to treat cardiovascular disorders, such as hypertension. More recently, block of so-called non-L-type Ca2+ channels was found to exert therapeutic effects in the treatment of severe pain and ischemic stroke. As the subunits of different Ca2+ channel types have been cloned, the modulatory sites for enzymes, G-proteins, and drugs can now be determined using molecular engineering and heterologous expression. Here we summarize recent work that has allowed us to determine the sites of action of L-type Ca2+ channel modulators. Together with previous biochemical, electrophysiological, and drug binding data these results provide exciting insight into the molecular pharmacology of this voltage-gated Ca2+ channel family.

Affinity Labels↗
PubMed1998

Pharmacological analysis of sterol delta8-delta7 isomerase proteins with [3H]ifenprodil.

Sterol Delta8-Delta7 isomerases (SIs) catalyze the shift of the double bond from C8-9 to C7-8 in the B-ring of sterols. Surprisingly, the isoenzymes in fungi (ERG2p) and vertebrates [emopamil binding protein (EBP)] are structurally completely unrelated, whereas the sigma1 receptor, a mammalian protein of unknown function, bears significant similarity with the yeast ERG2p. Here, we compare the drug binding properties of SIs and related proteins with [3H]ifenprodil as a common high affinity radioligand (Kd = 1.4-19 nM), demonstrating an intimate pharmacological relationship among ERG2p, sigma1 receptor, and EBP. This renders SIs a remarkable example for structurally diverse enzymes with similar pharmacological profiles and the propensity to bind drugs from different chemical groups with high affinity. We identified a variety of experimental drugs with nanomolar affinity for the human EBP (Ki = 0.5-14 nM) such as MDL28815, AY9944, triparanol, and U18666A. These compounds, as well as the fungicide tridemorph and the clinically used drugs tamoxifen, clomiphene, amiodarone, and opipramol, inhibit the in vitro activity of the recombinant human EBP (IC50 = 0.015-54 microM). The high affinity of the human EBP for 3H-tamoxifen (Kd = 3 +/- 2 nM) implies that the EBP carries the previously described microsomal antiestrogen binding site. Interactions of the EBP with structurally diverse lipophilic amines suggest that novel compounds of related structure should be counterscreened for inhibition of the enzyme to avoid interference with sterol Delta8-Delta7 isomerization.

Adrenergic alpha-Antagonists↗
PubMed1997

Nine L-type amino acid residues confer full 1,4-dihydropyridine sensitivity to the neuronal calcium channel alpha1A subunit. Role of L-type Met1188.

Pharmacological modulation by 1,4-dihydropyridines is a central feature of L-type calcium channels. Recently, eight L-type amino acid residues in transmembrane segments IIIS5, IIIS6, and IVS6 of the calcium channel alpha1 subunit were identified to substantially contribute to 1,4-dihydropyridine sensitivity. To determine whether these eight L-type residues (Thr1066, Gln1070, Ile1180, Ile1183, Tyr1490, Met1491, Ile1497, and Ile1498; alpha1C-a numbering) are sufficient to form a high affinity 1,4-dihydropyridine binding site in a non-L-type calcium channel, we transferred them to the 1, 4-dihydropyridine-insensitive alpha1A subunit using site-directed mutagenesis. 1,4-Dihydropyridine agonist and antagonist modulation of barium inward currents mediated by the mutant alpha1A subunits, coexpressed with alpha2delta and beta1a subunits in Xenopus laevis oocytes, was investigated with the two-microelectrode voltage clamp technique. The resulting mutant alpha1A-DHPi displayed low sensitivity for 1,4-dihydropyridines. Analysis of the 1,4-dihydropyridine binding region of an ancestral L-type alpha1 subunit previously cloned from Musca domestica body wall muscle led to the identification of Met1188 (alpha1C-a numbering) as an additional critical constituent of the L-type 1,4-dihydropyridine binding domain. The introduction of this residue into alpha1A-DHPi restored full sensitivity for 1,4-dihydropyridines. It also transferred functional properties considered hallmarks of 1, 4-dihydropyridine agonist and antagonist effects (i.e. stereoselectivity, voltage dependence of drug modulation, and agonist-induced shift in the voltage-dependence of activation). Our gain-of-function mutants provide an excellent model for future studies of the structure-activity relationship of 1, 4-dihydropyridines to obtain critical structural information for the development of drugs for neuronal, non-L-type calcium channels.

Amino Acid Sequence↗
PubMed1997

Analysis of membrane protein self-association in lipid systems by fluorescence particle counting: application to the dihydropyridine receptor.

Fluorescence particle counting (FPC) is employed to analyze the distribution of a purified membrane protein, the dihydropyridine receptor (DHP-R), in detergent micelles, in lipid vesicles, and in lipid monolayers generated from the vesicles. The method was used to identify conditions for which DHP-Rs occur singly distributed in micelles and in vesicles. In monolayers, the DHP-R showed self-association, starting from monomeric distribution at concentrations (c) of typically 10 DHP-R/microm2. The average cluster size [m(t)] of associates was followed by FPC in time and the dependence of the lateral diffusion constant [D(lat)(m,pi)] of the associates on the surface pressure (pi) was determined. By studying the dependence of m(t) on c, pi, D(lat)(pi), and salt concentration (c(s)), we derived an empirical expression for the association rate constant (k(a)) and for m(t) that fits the experimental m(t) relations. Theoretical justification for these dependencies is obtained from collision theory, leading to a mechanistic picture of the aggregation process. DHP-R association is irreversible. Its rate is not diffusion-limited. A large number of collisions is required to overcome an interaction energy barrier of about 6-11 kT, depending on m and c(s) but not on pi. The increase in association rate with increasing average cluster size m is related to increasing van der Waals attraction, while the increase in rate with increasing c(s) relates to decreasing electrostatic repulsion. Van der Waals and electrostatic forces represent, however, only part of the interaction energy. The main contribution was not dependent on the variables studied and, most likely, reflects hydration forces which need to be overcome for association.

Calcium Channels↗
PubMed1997

L-type calcium channels: binding domains for dihydropyridines and benzothiazepines are located in close proximity to each other.

We investigated the binding of a fluorescent diltiazem analogue (3R,4S)-cis-1-[2-[[3-[[3-[4,4-difluoro-3a,4-dihydro-5,7-dimethyl-4-bo ra-3a,4a-diaza-s-indacen-3-yl]propionyl]amino]propyl]amin o]ethy]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(triflu oromethyl)-2H-1-benzazepin-2-one (DMBODIPY-BAZ) to L-type Ca2+ channels in the presence of different 1,4-dihydropyridines (DHPs) by using fluorescence resonance energy transfer (FRET) [Brauns, T., Cai, Z.-W., Kimball, S. D., Kang, H.-C., Haugland, R. P., Berger, W., Berjukov, S., Hering, S., Glossmann, H., & Striessnig, J. (1995) Biochemistry 34, 3461]. When channels are occupied with DMBODIPY-BAZ, a rapid fluorescence change occurred upon addition of different DHPs. The direction of this intensity modulation was found to be only dependent on the chemical composition of the dihydropyridine employed. DHPs containing a nitro group decreased, whereas others (e.g., isradipine) enhanced the fluorescence signal. In addition, all DHPs markedly decreased the association rate constant for DMBODIPY-BAZ without affecting equilibrium binding. Both observations together are best explained by a steric model where the DHP binding site is located in close proximity to the accession pathway of DMBODIPY-BAZ.

Animals↗
PubMed1997

High affinity of sigma 1-binding sites for sterol isomerization inhibitors: evidence for a pharmacological relationship with the yeast sterol C8-C7 isomerase.

1. The sigma-drug binding site of guinea-pig liver is carried by a protein which shares significant amino acid sequence similarities with the yeast sterol C8-C7 isomerase (ERG2 protein). Pharmacologically-but not structurally-the sigma 1-site is also related to the emopamil binding protein, the mammalian sterol C8-C7 isomerase. We therefore investigated if sterol C8-C7 isomerase inhibitors are high affinity ligands for the (+)-[3H]-pentazocine labelled sigma 1-binding site. 2. Among the compounds which bound with high affinity to native hepatic and cerebral as well as to yeast expressed sigma 1-binding sites were the agricultural fungicide fenpropimorph (Ki 0.005 nM), the antihypocholesterinaemic drugs triparanol (Ki 7.0 nM), AY-9944 (Ki, 0.46 nM) and MDL28,815 (Ki 0.16 nM), the enantiomers of the ovulation inducer clomiphene (Ki 5.5 and 12 nM, respectively) and the antioestrogene tamoxifen (Ki 26 nM). 3. Except for tamoxifen these affinities are essentially identical with those for the [3H]-ifenprodil labelled sterol C8-C7 isomerase of S. cerevisiae. This demonstrates that sigma 1-binding protein and yeast isomerase are not only structurally but also pharmacologically related. Because of its affiliations with yeast and mammalian sterol isomerases we propose that the sigma 1-binding site is localized on a sterol isomerase related protein, involved in postsqualene sterol biosynthesis.

Animals↗
PubMed1996

Yeast sterol C8-C7 isomerase: identification and characterization of a high-affinity binding site for enzyme inhibitors.

The yeast gene ERG2 encodes a sterol C8-C7 isomerase and is essential for ergosterol synthesis and cell proliferation. Its striking homology with the so-called sigma1 receptor of guinea pig brain, a polyvalent steroid and drug binding protein, suggested that the yeast sterol C8-C7 isomerase (ERG2) carries a similar high affinity drug binding domain. Indeed the sigma ligands [3H]haloperidol (Kd = 0.3 nM) and [3H]ifenprodil (Kd = 1.4 nM) bound to a single population of sites in ERG2 wild type yeast microsomes (Bmax values of 77 and 61 pmol/mg of protein, respectively), whereas binding activity was absent in strains carrying ERG2 gene mutations or disruptions. [3H]Ifenprodil binding was inhibited by sterol isomerase inhibitors such as fenpropimorph (Ki = 0.05 nM), tridemorph (Ki = 0.09 nM), MDL28,815 (Ki = 0.44 nM), triparanol (Ki = 1.5 nM), and AY-9944 (Ki = 5.8 nM). [3H]Haloperidol specifically photoaffinity-labeled a protein with an apparent molecular weight of 27400, in agreement with the molecular mass of the sterol C8-C7 isomerase (24900 Da). 9E10 c-myc antibodies specifically immunoprecipitated the c-myc tagged protein after [3H]haloperidol photolabeling, unequivocally proving that the drug binding site is localized on the ERG2 gene product. Haloperidol, trifluperidol, and ifenprodil inhibited the growth of Saccharomyces cerevisiae and reduced the ergosterol content of cells grown in their presence. Our results demonstrate that the yeast sterol C8-C7 isomerase has a polyvalent high-affinity drug binding site similar to mammalian sigma receptors and that in yeast sigma ligands inhibit sterol biosynthesis.

Animals↗
PubMed1996

Two amino acid residues in the IIIS5 segment of L-type calcium channels differentially contribute to 1,4-dihydropyridine sensitivity.

The transmembrane segment IIIS5 of the L-type calcium channel alpha1 subunit participates in the formation of the 1,4-dihydropyridine (DHP) interaction domain (Grabner, M., Wang, Z., Hering, S., Striessnig, J., and Glossmann, H. (1996) Neuron 16, 207-218). We applied mutational analysis to identify amino acid residues within this segment that contribute to DHP sensitivity. DHP agonist and antagonist modulation of Ba2+ inward currents was assessed after coexpression of chimeric and mutant calcium channel alpha1 subunits with alpha2delta and beta1a subunits in Xenopus oocytes. Whereas DHP antagonists required Thr-1066, DHP agonist modulation crucially depended on the additional presence of Gln-1070 (numbering according to alpha1C-a), which also further increased the sensitivity to DHP antagonists. Asp-955, which is found at the corresponding position in the calcium channel alpha1S subunit from carp skeletal muscle, displayed functional similarity to Gln-1070 with respect to DHP interaction. We conclude that these residues (Thr-1066 plus Gln-1070 or Asp-955), which are located in close vicinity on the same side of the putative alpha-helix of transmembrane segment IIIS5, form a crucial DHP binding motif.

Amino Acid Sequence↗
PubMed1996

Block of P/Q-type calcium channels by therapeutic concentrations of aminoglycoside antibiotics.

Aminoglycoside antibiotics can cause neuromuscular block by inhibiting Ca2+ influx into motor nerve terminals. P/Q-type Ca2+ channels, which are formed by alpha 1A subunits, are mainly responsible for depolarization-dependent presynaptic Ca2+ entry in motor neurons. We therefore investigated the possibility that aminoglycosides function as P/Q-type channel blockers. They inhibited [125I]-omega-CTx-MVIIC binding to P/Q-type channels in guinea pig cerebellum membranes with nanomolar IC50 values (e.g., 8 nM for neomycin). Divalent cations decreased the apparent affinity of neomycin. Barium inward currents through alpha 1A subunits expressed in Xenopus oocytes were partially blocked by therapeutic concentrations of aminoglycosides. This explains that therapeutically relevant concentrations of these drugs decrease the reserve of neuromuscular transmission, which can lead to neuromuscular block. We conclude that micromolar concentrations of aminoglycosides block not only N-type but also P/Q-type channels in mammalian neurons.

Animals↗
PubMed1996

Transfer of high sensitivity for benzothiazepines from L-type to class A (BI) calcium channels.

To investigate the molecular basis of the calcium channel block by diltiazem, we transferred amino acids of the highly sensitive and stereoselective L-type (alpha1S or alpha1C) to a weakly sensitive, nonstereoselective class A (alpha1A) calcium channel. Transfer of three amino acids of transmembrane segment IVS6 of L-type alpha1 into the alpha1A subunit (I1804Y, S1808A, and M1811I) was sufficient to support a use-dependent block by diltiazem and by the phenylalkylamine (-)-gallopamil after expression in Xenopus oocytes. An additional mutation F1805M increased the sensitivity for (-)-gallopamil but not for diltiazem. Our data suggest that the receptor domains for diltiazem and gallopamil have common but not identical molecular determinants in transmembrane segment IVS6. These mutations also identified single amino acid residues in segment IVS6 that are important for class A channel inactivation.

Amino Acid Sequence↗
PubMed1996

Identification of PK-A phosphorylation sites in the carboxyl terminus of L-type calcium channel alpha 1 subunits.

Full length L-type calcium channel alpha 1 subunits are rapidly phosphorylated by protein kinase A (PK-A) in vitro and in vivo at sites located in their long carboxyl terminal tails. In skeletal muscle, heart, and brain the majority of biochemically isolated alpha 1 subunits lacks these phosphorylation sites due to posttranslational proteolytic processing. Truncation may therefore modify the regulation of channel activity by PK-A. We combined site-directed mutagenesis and heterologous expression to investigate the extent to which putative cAMP-dependent phosphorylation sites in the C-terminus of alpha 1 subunits from skeletal muscle, heart, and brain are phosphorylated in vitro. The full length size form of wild-type and mutant calcium channel alpha 1 subunits was obtained at high yield after heterologous expression in Saccharomyces cerevisiae. Like in fetal rabbit myotubes [Rotman, E.I., et al. (1995) J. Biol. Chem. 270, 16371-16377], the rabbit skeletal muscle alpha 1 C-terminus was phosphorylated at serine residues 1757 and 1854. In the carboxyl terminus of alpha 1S from carp skeletal muscle and alpha 1C from rabbit heart a single serine residue was phosphorylated by PK-A in vitro. The C-terminus of alpha 1D was phosphorylated at more than one site. Employing deletion mutants, most of the phosphorylation ( > 70%) was found to occur between amino acid residues 1805 and 2072. Serine 1743 was identified as additional phosphorylation site in alpha 1D. We conclude that in class S and C calcium channels the most C-terminal phosphorylation sites are substrate for PK-A in vitro, whereas in class D calcium channels phosphorylation also occurs at a site which is likely to be retained even after posttranslational truncation.

Animals↗
PubMed1996

Purification, molecular cloning, and expression of the mammalian sigma1-binding site.

Sigma-ligands comprise several chemically unrelated drugs such as haloperidol, pentazocine, and ditolylguanidine, which bind to a family of low molecular mass proteins in the endoplasmic reticulum. These so-called sigma-receptors are believed to mediate various pharmacological effects of sigma-ligands by as yet unknown mechanisms. Based on their opposite enantioselectivity for benzomorphans and different molecular masses, two subtypes are differentiated. We purified the sigma1-binding site as a single 30-kDa protein from guinea pig liver employing the benzomorphan(+)[3H]pentazocine and the arylazide (-)[3H]azidopamil as specific probes. The purified (+)[3H]pentazocine-binding protein retained its high affinity for haloperidol, pentazocine, and ditolylguanidine. Partial amino acid sequence obtained after trypsinolysis revealed no homology to known proteins. Radiation inactivation of the pentazocine-labeled sigma1-binding site yielded a molecular mass of 24 +/- 2 kDa. The corresponding cDNA was cloned using degenerate oligonucleotides and cDNA library screening. Its open reading frame encoded a 25.3-kDa protein with at least one putative transmembrane segment. The protein expressed in yeast cells transformed with the cDNA showed the pharmacological characteristics of the brain and liver sigma1-binding site. The deduced amino acid sequence was structurally unrelated to known mammalian proteins but it shared homology with fungal proteins involved in sterol synthesis. Northern blots showed high densities of the sigma1-binding site mRNA in sterol-producing tissues. This is also in agreement with the known ability of sigma1-binding sites to interact with steroids, such as progesterone.

Amino Acid Sequence↗