Hans-Georg Breitinger

The Inhibitory Glycine Receptor-Simple Views of a Complicated Channel

The strychnine‐sensitive glycine receptor is the principal mediator of fast inhibitory synaptic transmission in the mammalian spinal cord and brain stem. As a member of the ligand‐gated ion‐channel family, it shares structural homology with the nicotinic acetylcholine, GABAA/C and serotonin 5‐HT3 receptors. Ion‐channel activation and desensitisation are controlled by a variety of factors such as subunit composition, posttranslational modification, absence or presence of modulatory ions or other agents and possibly protein–protein interactions. Glycine‐receptor mutations, either associated with the human motor disorder hyperekplexia or artificially introduced, have helped to define the regulatory domains of the receptor protein. In addition to their effects on glycine‐receptor function, allelic variants of glycine‐receptor genes may also affect biogenesis, assembly and degradation of the receptor.

Synthesis of silica-bound amylose by phosphorolytic elongation of immobilised maltoheptaosyl hydrazides

Maltoheptaoside-alkoxysilane anchor molecules were synthesised by fusing aliphatic ω-Si(OEt3) hydrazide linkers with maltoheptaose. After immobilisation of the primers on porous silica, support-bound amylose was synthesised by phosphorolytic synthesis. The hydrazone linkage as a pre-formed cleavage site allowed removal and subsequent characterisation of immobilised amylose, which showed a broad molecular weight distribution. Under HPLC conditions, amylose assumed a non-helical conformation, making surface interactions and not complexation the primary separation mechanism.

Functional Complementation of Glra1spd-ot, a Glycine Receptor Subunit Mutant, by Independently Expressed C-Terminal Domains

The oscillator mouse (Glra1spd-ot) carries a 9 bp microdeletion plus a 2 bp microinsertion in the glycine receptor α1 subunit gene, resulting in the absence of functional α1 polypeptides from the CNS and lethality 3 weeks after birth. Depending on differential use of two splice acceptor sites in exon 9 of the Glra1 gene, the mutant allele encodes either a truncated α1 subunit (spdot-trc) or a polypeptide with a C-terminal missense sequence (spdot-elg). During recombinant expression, both splice variants fail to form ion channels. In complementation studies, a tail construct, encoding the deleted C-terminal sequence, was coexpressed with both mutants. Coexpression with spdot-trc produced glycine-gated ion channels. Rescue efficiency was increased by inclusion of the wild-type motif RRKRRH. In cultured spinal cord neurons from oscillator homozygotes, viral infection with recombinant C-terminal tail constructs resulted in appearance of endogenous α1 antigen. The functional rescue of α1 mutants by the C-terminal tail polypeptides argues for a modular subunit architecture of members of the Cys-loop receptor family.

Preparation of cyclic ADP-ribose antagonists and caged cyclic ADP- ribose

Publisher Summary Cyclic ADP-ribose (cADPR) is a naturally occurring metabolite of NAD + that is capable of mobilizing calcium from intracellular stores in a variety of biological systems. This chapter presents the procedures for the synthesis of three 8-substituted (8-amino-, 8-azido-, and 8-bromo-) analogs and a caged analog of cyclic ADP-ribose. The use of these analogs demonstrates that the occupation of the cADPR receptor site does not necessarily lead to Ca 2+ release; appropriate interactions between the 8-position of the ligand and receptor are also required. All three compounds are competitive antagonists that bind to the same site as cADPR. The 8-azido-cADPR, with its photoactive azido group, is particularly useful as a photoaffinity probe for the identification and characterization of cADPR-binding proteins. The synthesis of high specific activity 8-azido-[ 32 P]cADPR for use in photoaffinity labeling experiments is also described in the chapter. The use of photoactivable “caged” compounds represents a powerful tool for controlling the release of biologically active molecules in intact cells. Generally, the caging group incorporated into an active molecule is designed to render the molecule inactive. Removal of the caging group by photolysis results in liberation of the active substance, which can be controlled both spatially and temporally.

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor α1-Subunit

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding α- and gephyrin anchoring β-subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (α1-(1–219)) and the large intracellular TM3–4 loop (α1-(309–392)) of the human GlyR α1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested ∼25% α-helix and ∼48% β-sheet for the extracellular domain, while no α-helices were detectable for the TM3–4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of ∼5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of ∼49 kDa indicated oligomerization in a defined native structure. As shown by [3H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.

Overexpression of NMDAR2B in an inflammatory model of Alzheimer's disease: Modulation by NOS inhibitors

BACKGROUND Alzheimers disease (AD) is a common form of age-related dementia, characterized by deposition of amyloid Aβ plaques, neuroinflammation and neurodegeneration. N-methyl-D-aspartate receptors (NMDAR) are postsynaptic glutamate receptors that play a role in memory formation and are targets for memantadine, an anti-AD drug. Nitric oxide (NO) signaling has been involved in both memory development through neuronal NO synthase (nNOS), and neuroinflammation through inducible NO synthase (iNOS) which mediates CNS inflammatory processes. AIM To study the expression of the NMDAR2B subunit in an inflammatory model of AD before and after treatment with NO modulators. MATERIALS AND METHODS AD was induced in mice by a single dose of lipopolysaccharide (LPS). Behavioral tests for spatial and non-spatial memories and locomotor activity were performed to assess disease severity and progression. The effects of L-NAME (general NOS inhibitor), 1400W (iNOS inhibitor), diflunisal (systemic anti-inflammatory drug that does not cross the blood brain barrier), and L-arginine, the substrate for NOS was determined. Immunohistochemistry was done to confirm AD and brain lysates were tested for Aβ formation, levels of NMDAR2B subunits, and brain NO levels. RESULTS Systemic LPS induced AD, as shown by cognitive impairment; increased levels of Aβ and concomitant increase in the brain NO concentrations. This was associated with overexpression of NMDAR2B. All tested drugs improved behavioral dysfunction, prevented Aβ formation and NMDAR overexpression, and lead to decrease in NO concentration in the brain. L-Arginine alone, however, did not produce similar improvements. CONCLUSION NMDAR2B subunits are overexpressed in an inflammatory model of AD and NO inhibitors ameliorate this expression.

Novel Regulatory Site within the TM3–4 Loop of Human Recombinant α3 Glycine Receptors Determines Channel Gating and Domain Structure

Glycine receptors are Cys loop ligand-gated ion channels that mediate fast inhibitory synaptic transmission in the mammalian central nervous system. The functionally distinct splice variants α3L and α3K of the human glycine receptor differ by a 15-amino acid insert within the long intracellular TM3–4 loop, a region of high intersubunit diversity. In a mutational study, effects of the insert on ion channel function and secondary structure of the TM3–4 loop were investigated. Whole cell current responses and protein surface expression data indicated that the major effect of mutations within the insert was on channel gating. Changes in channel gating correlated with the distribution of charged residues about the splice region. Analysis of complex molecular weight indicated that recombinant TM3–4 loops of α3L and α3K associated into oligomers of different stoichiometry. Secondary structure analysis suggested that the insert stabilized the overall fold of the large cytoplasmic domain of α3L subunits. The absence of the insert resulted in a channel that was still functional, but the TM3–4 cytoplasmic domain appeared not stably folded. Thus, our data identified the spliced insert within the large TM 3–4 loop of α3 Gly receptors as a novel regulatory motif that serves a 2-fold role: (i) the presence of the insert stabilizes the overall spatial structure of the domain, and (ii) the insert presents a control unit that regulates gating of the receptor ion channel.

Synergistic inhibition of glycinergic transmission in vitro and in vivo by flavonoids and strychnine.

The inhibitory glycine receptor (GlyR) is a key mediator of synaptic signaling in spinal cord, brain stem, and higher central nervous system regions. The flavonoids quercetin and genistein have been identified previously as promising GlyR antagonists in vitro, but their detailed mechanism of action was not known. Here, inhibition of recombinant human α1 GlyRs in HEK 293 cells by genistein, quercetin, and strychnine was studied using whole-cell recording techniques. The interaction of several inhibitors applied alone or in combination was analyzed using a minimum mechanism of receptor activation and inhibition. Receptor inhibition in vivo was studied in a mouse model of strychnine toxicity. Genistein, quercetin, and strychnine were noncompetitive GlyR inhibitors. The inhibitory potency of one flavonoid (either genistein or quercetin) was not affected by simultaneous application of the other, suggesting that both flavonoids target the same site on the receptor. In combination with strychnine, flavonoid inhibition was augmented, indicating that strychnine binds to a position on the receptor physically distant from the flavonoid site. Potentiation of strychnine inhibition by flavonoids was also observed in vivo, where harmless doses of flavonoids enhanced strychnine toxicity in mice. Thus, in vitro and in vivo studies demonstrated a true synergism between flavonoids and strychnine for GlyR inhibition. The mechanism-based approach used here allows a rapid analysis of the effects of single drugs versus drug combinations.

Hydroxylated residues influence desensitization behaviour of recombinant α3 glycine receptor channels

The human glycine receptor subunit α3 exists in two splice variants (α3K/L), with α3L bearing an additional segment of 15 amino acids within the cytoplasmic TM3‐4 loop. Homomeric α3K glycine receptors show faster desensitization than α3L receptors. Ion channel properties were compared of α3L, α3K, and of the triple mutant α3LΔOH = α3L(T358A/Y367F/S370A), where hydroxyl functions of the spliced insert had been removed by site‐directed mutagenesis. Upon recombinant expression in HEK 293 cells, patch‐clamp recording experiments revealed that removal of hydroxyl functions primarily affected receptor desensitization. The fraction of non‐desensitizing current was 68 ± 13% for α3L, 21 ± 13% for α3K, and 48 ± 16% for α3LΔOH. Desensitization time constants at saturating glycine concentration were 8.4 ± 2.8 s, 1.9 ± 2.3 s, and 2.8 ± 0.4 s, for α3L, α3K, and the triple mutant α3LΔOH, respectively. In contrast, single‐channel and whole‐cell properties were similar for all three constructs. Thus, ion channel activation, desensitization, and conductance properties are independently controlled by distinct structural elements. Hydroxyl functions within the M3‐4 loop of the glycine receptor α3 subunit are crucial, but not exclusive, determinants of receptor desensitization.

The novel hyperekplexia allele GLRA1(S267N) affects the ethanol site of the glycine receptor

Mutations in the GLRA1 gene, which encodes the α1-subunit of the inhibitory glycine receptor (GlyR), are the underlying causes in the majority of cases of hereditary startle disease (OMIM no. 149400). GlyRs are modulated by alcohols and volatile anesthetics, where a specific amino acid at position 267 has been implicated in receptor modulation. We describe a hyperekplexia family carrying the novel dominant missense allele GLRA1(S267N), that affects agonist responses and ethanol modulation of the mutant receptor. This study implies that a disease-related receptor allele carries the potential to alter drug responses in affected patients.