Edward Hawrot

Long-term culture of dissociated sympathetic neurons

This chapter discusses long-term primary culture of dissociated neurons, a technique that proved to be a valuable tool in studies of neuronal development and function. Such cultures offer the potential for systematic manipulation of the fluid medium surrounding the cells. These neuron-alone cultures are particularly well suited to biochemical analysis as the properties of any function under study are directly attributable to the neurons being cultured, without complicating contamination from other cell types. In dissociated cell culture under appropriate conditions, sympathetic neurons exhibit a normal developmental differentiation and maturation into adrenergic neurons. Medium conditioned by incubation on cultures of appropriate non-neuronal cells, when added to neuron-alone cultures produces an increase in neuronal cholinergic characteristics with a concomitant decrease in adrenergic characteristics. The extracellular environment is critically important in determining the differentiated fate of sympathetically derived neurons.

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.

Cultured sympathetic neurons: Effects of cell-derived and synthetic substrata on survival and development☆

The long-term culture of dissociated rat sympathetic neurons requires strong adhesion of the neuronal processes to the culture substratum. A variety of artificial and cell-derived substrata was examined for their effects on the survival, neurite outgrowth, and neurotransmitter development of these neurons. Compared to dried collagen films, both three-dimensional hydrated collagen gels and surfaces coated with basic polymers provided a substratum highly adherent for developing neurons. Polylysine and polyornithine were most suitable for long-term culture when covalently linked with glutaraldehyde to an underlying layer of dried gelatin. Dissociated neurons also attached strongly to a substratum of killed nonneuronal cells fixed by paraformaldehyde, heat, ethanol, or trichloroacetic acid. In addition, an extracellular, substrate-associated material apparently produced by nonneuronal cells (rat cardiac myocytes and associated fibroblasts) promoted the long-term adhesion of growing neurites. The adhesive property of this microexudate was sensitive to trypsin, periodate, and alkali, but resistant to hyaluronidase, chondroitinase, 8 M urea, and 0.5 M acetic acid. Similar characteristics have been reported for fibronectin, an extracellular glycoprotein produced by many cells and cell lines. This protein, or one with similar features, may function in vivo in the extension and guidance of neuronal fibers. The choice and development of neurotransmitter function were unaffected by the various substrata tested, with one exception. Nonneuronal cells fixed with paraformaldehyde caused a significant induction of cholinergic properties similar to that seen with nonneuronal conditioned medium.

Demonstration of a tandem pair of complement protein modules in GABAB receptor 1a

We have subcloned and expressed the N‐terminal portion of the recently sequenced metabotropic GABA receptor, GABABR1a. This region of the receptor contains a complement protein‐like amino acid sequence. The purified 140‐residue recombinant protein fragment was soluble and stable. Mass spectrometry indicated formation of four disulfide bonds, as expected if two complement protein modules (CPs, also known as SCRs, Sushi domains) are formed. The circular dichroism spectrum was unusual and characteristic of CPs. Differential scanning calorimetry demonstrated a melting point (64°C), and total enthalpy commensurate with two fully folded domains. We thus conclude that the 1a subtype of the GABAB receptor, but not the 1b subtype, contains a pair of CPs and we present a three‐dimensional model of this region.

Proteomic Analysis of an α7 Nicotinic Acetylcholine Receptor Interactome

The alpha7 nicotinic acetylcholine receptor (nAChR) is well established as the principal high-affinity alpha-bungarotoxin-binding protein in the mammalian brain. We isolated carbachol-sensitive alpha-bungarotoxin-binding complexes from total mouse brain tissue by affinity immobilization followed by selective elution, and these proteins were fractionated by SDS-PAGE. The proteins in subdivided gel lane segments were tryptically digested, and the resulting peptides were analyzed by standard mass spectrometry. We identified 55 proteins in wild-type samples that were not present in comparable brain samples from alpha7 nAChR knockout mice that had been processed in a parallel fashion. Many of these 55 proteins are novel proteomic candidates for interaction partners of the alpha7 nAChR, and many are associated with multiple signaling pathways that may be implicated in alpha7 function in the central nervous system. The newly identified potential protein interactions, together with the general methodology that we introduce for alpha-bungarotoxin-binding protein complexes, form a new platform for many interesting follow-up studies aimed at elucidating the physiological role of neuronal alpha7 nAChRs.

NMR Structural Analysis of α-Bungarotoxin and Its Complex with the Principal α-Neurotoxin-binding Sequence on the α7 Subunit of a Neuronal Nicotinic Acetylcholine Receptor

We report a new, higher resolution NMR structure of α-bungarotoxin that defines the structure-determining disulfide core and β-sheet regions. We further report the NMR structure of the stoichiometric complex formed between α-bungarotoxin and a recombinantly expressed 19-mer peptide (178IPGKRTESFYECCKEPYPD196) derived from the α7 subunit of the chick neuronal nicotinic acetylcholine receptor. A comparison of these two structures reveals binding-induced stabilization of the flexible tip of finger II in α-bungarotoxin. The conformational rearrangements in the toxin create an extensive binding surface involving both sides of the α7 19-mer hairpin-like structure. At the contact zone, Ala7, Ser9, and Ile11 in finger I and Arg36, Lys38, Val39, and Val40 in finger II of α-bungarotoxin interface with Phe186, Tyr187, Glu188, and Tyr194 in the α7 19-mer underscoring the importance of receptor aromatic residues as critical neurotoxin-binding determinants. Superimposing the structure of the complex onto that of the acetylcholine-binding protein (1I9B), a soluble homologue of the extracellular domain of the α7 receptor, places α-bungarotoxin at the peripheral surface of the inter-subunit interface occluding the agonist-binding site. The disulfide-rich core of α-bungarotoxin is suggested to be tilted in the direction of the membrane surface with finger II extending into the proposed ligand-binding cavity.

Chimeric analysis of a neuronal nicotinic acetylcholine receptor reveals amino acids conferring sensitivity to alpha-bungarotoxin.

We have investigated the molecular determinants responsible for α-bungarotoxin (αBgtx) binding to nicotinic acetylcholine receptors through chimeric analysis of two homologous α subunits, one highly sensitive to αBgtx block (α1) and the other, αBgtx-insensitive (α3). By replacing rat α3 residues 184–191 with the corresponding region from the Torpedo α1 subunit, we introduced a cluster of five α1 residues (Trp-184, Trp-187, Val-188, Tyr-189, and Thr-191) into the α3 subunit. Functional activity and αBgtx sensitivity were assessed following co-expression in Xenopus oocytes of the chimeric α3 subunit (α3/α1[5]) with either rat β2 or β4 subunits. Agonist-evoked responses of α3/α1[5]-containing receptors were blocked by αBgtx with nanomolar affinity (IC50 values: 41 nm for α3/α1[5]β2 and 19 nm for α3/α1[5]β4). Furthermore, receptors containing the single point mutation α3K189Y acquire significant sensitivity to αBgtx block (IC50 values: 186 nm for α3K189Yβ2 and 179 nm for α3K189Yβ4). Another α3 chimeric subunit, α3/α7[6], similar to α3/α1[5] but incorporating the corresponding residues from the αBgtx-sensitive α7 subunit, also conferred potent αBgtx sensitivity to chimeric receptors when co-expressed with the β4 subunit (IC50 value = 31 nm). Our findings demonstrate that the residues between positions 184 and 191 of the αBgtx-sensitive subunits α1 and α7 play a critical functional role in the interaction of αBgtx with nicotinic acetylcholine receptors sensitive to this toxin.

Receptor Binding Activities of Biotinylated Derivatives of β‐Nerve Growth Factor

Abstract: β‐nerve growth factor (NGF) was modified by biotinylation via carboxyl group substitution (C‐bio‐NGF) using biotin hydrazide and the coupling reagent 1‐ethyl‐3‐(3‐dimethylaminopropyl)‐carbodiimide, under reaction conditions that yielded an average of 3 biotin additions per NGF subunit. NGF was also biotinylated through amino group substitution, using N‐hydroxysuc‐cinimidyl biotin, to produce derivatives with ratios of one, two, and four biotin moieties per NGF subunit (N‐bio‐NGF). The various biotinylated NGF derivatives were compared with native NGF for their capacity to compete with 125I‐NGF for binding to NGF receptors on rat pheochromocytoma (PC12) cells at 4°C. On the basis of such radioreceptor assays, C‐bio‐NGF was as effective as native NGF in binding to NGF receptors. C‐bio‐NGF was also as effective as native NGF in promoting neunte outgrowth from PC12 cells. In contrast, N‐bio‐NGF containing one biotin per NGF subunit was only 28% as active in binding as native NGF. Increasing the biotin:NGF ratio to 2 or 4 further decreased receptor binding to 13% and 6%, respectively, as compared to native NGF. Once bound to cells, C‐bio‐NGF had the capacity to mediate the specific binding of 125I‐streptavidin to PC12 cells. This binding of streptavidin was prevented by excess native NGF and by antiserum to NGF, but not by RNase A, insulin, cytochrome c, or nonimmune serum. In addition, a variant PC 12 line lacking functional NGF receptors was not labeled by 125I‐streptavidin after prior incubation with C‐bio‐NGF.

The metabotropic GABA receptor: molecular insights and their functional consequences.

Abstract. Recent years have seen rapid and significant advances in our understanding of the G-protein-coupled γ-amino butyric acid, B-type (GABAB) receptor, which could be a therapeutic target in conditions as diverse as epilepsy and hypertension. This progress originated with the ground-breaking work of Bernhard Bettler’s team at Novartis who cloned the DNA encoding a GABAB receptor in 1997. Currently, the receptor is thought to be an unusual, possibly unique, example of a heterodimer composed of homologous, seven-transmembrane-domain (7TMD) subunits (named GABAB R1 and GABAB R2), neither of which is fully functional when expressed alone. The large N-terminal domain of the GABAB R1 subunit projects extracellularly and contains a ligand binding site. The similarity of the amino acid sequence of this region to some bacterial periplasmic amino acid-binding proteins of known structure has enabled structural and functional modelling of the N-terminal domain, and the identification of residues whose substitution modulates agonist/antagonist binding affinities. The intracellular C-terminal domains of the R1 and R2 subunits appear to constitute an important means of contact between the two subunits. Alternative splice variants, a common and functionally important feature of 7TMD proteins, have been demonstrated for the R1 subunit. Notably GABAB R1a differs from GABAB R1b by the possession of an N-terminal extension containing two complement protein modules (also called SCRs, or sushi domains) of unknown function. The levels at which each of the respective variants is expressed are not equal to one another, with variations occurring over the course of development and throughout the central nervous system. It is not yet clear, however, whether one variant is predominantly presynaptically located and the other postsynaptically located. The existence of as yet unidentified splice variants, additional receptor subtypes and alternative quaternary composition has not been ruled out as a source of receptor heterogeneity.

Binding of rabies virus to purified Torpedo acetylcholine receptor

The binding of 125I- and 35S-labeled rabies virus (CVS strain) to affinity-purified acetylcholine receptor from Torpedo electric organ was demonstrated. The binding of rabies virus to the acetylcholine receptor increased with increasing receptor concentration, was dependent on the pH of the incubation medium, and was saturable with increasing virus concentration. Binding of radioactively labeled virus was effectively competed by unlabeled homologous virus particles. Binding of 35S-labeled rabies virus to the AChR was inhibited up to 50% by alpha-bungarotoxin and up to 30% by (+)-tubocurarine but was not affected by atropine. These results demonstrate direct binding of rabies virus to a well-defined neurotransmitter receptor, namely the acetylcholine receptor and indicate that at least a portion of the virus interaction occurs near the acetylcholine binding site on the receptor. These findings support the hypothesis that the acetylcholine receptor may serve as a rabies virus receptor in vivo.