Paul Whiting

Brain α-bungarotoxin binding protein cDNAs and MAbs reveal subtypes of this branch of the ligand-gated ion channel gene superfamily

alpha-Bungarotoxin (alpha Bgt) is a potent, high-affinity antagonist for nicotinic acetylcholine receptors (AChRs) from muscle, but not for AChRs from neurons. Both muscle and neuronal AChRs are thought to be formed from multiple homologous subunits aligned around a central cation channel whose opening is regulated by ACh binding. In contrast, the exact structure and function of high-affinity alpha Bgt binding proteins (alpha BgtBPs) found in avian and mammalian neurons remain unknown. Here we show that cDNA clones encoding alpha BgtBP alpha 1 and alpha 2 subunits define alpha BgtBPs as members of a gene family within the ligand-gated ion channel gene superfamily, but distinct from the gene families of AChRs from muscles and nerves. Subunit-specific monoclonal antibodies raised against bacterially expressed alpha BgtBP alpha 1 and alpha 2 subunit fragments reveal the existence of at least two different alpha BgtBP subtypes in embryonic day 18 chicken brains. More than 75% of all alpha BgtBPs have the alpha 1 subunit, but no alpha 2 subunit, and a minor alpha BgtBP subtype (approximately 15%) has both the alpha 1 and alpha 2 subunits.

Immunohistochemical localization of neuronal nicotinic receptors in the rodent central nervous system

The distribution of nicotinic acetylcholine receptors (AChR) in the rat and mouse central nervous system has been mapped in detail using monoclonal antibodies to receptors purified from chicken and rat brain. Initial studies in the chicken brain indicate that different neuronal AChRs are contained in axonal projections to the optic lobe in the midbrain from neurons in the lateral spiriform nucleus and from retinal ganglion cells. Monoclonal antibodies to the chicken and rat brain AChRs also label apparently identical regions in all major subdivisions of the central nervous system of rats and mice, and this pattern is very similar to previous reports of 3H-nicotine binding, but quite different from that of alpha-bungarotoxin binding. In several instances, the immunohistochemical evidence has strongly indicated that neuronal AChR undergoes axonal transport. The clearest example of this has been in the visual system, where labeling was observed in the retina, the optic nerve and tract, and in all of the major terminal fields of the optic nerve except the ventral suprachiasmatic nucleus. This was confirmed in unilateral enucleation experiments in the rat, where labeling was greatly reduced in the contralateral optic tract, ventral lateral geniculate nucleus, pretectal nuclei receiving direct visual input, superficial layers of the superior colliculus, and medical terminal nucleus, and was significantly reduced in the dorsal lateral geniculate nucleus. Clear neuronal labeling was also observed in dorsal root ganglion cells and in cranial nerve nuclei containing motoneurons that innervate branchial arch-derived muscles, although the possibility that neuronal AChR undergoes axonal transport in the latter cells was not tested experimentally.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a Significant Role of α3-Containing GABAA Receptors in Mediating the Anxiolytic Effects of Benzodiazepines

The GABAA receptor subtypes responsible for the anxiolytic effects of nonselective benzodiazepines (BZs) such as chlordiazepoxide (CDP) and diazepam remain controversial. Hence, molecular genetic data suggest that α2-rather than α3-containing GABAA receptors are responsible for the anxiolytic effects of diazepam, whereas the anxiogenic effects of an α3-selective inverse agonist suggest that an agonist selective for this subtype should be anxiolytic. We have extended this latter pharmacological approach to identify a compound, 4,2′-difluoro-5′-[8-fluoro-7-(1-hydroxy-1-methylethyl)imidazo[1,2-á]pyridin-3-yl]biphenyl-2-carbonitrile (TP003), that is an α3 subtype selective agonist that produced a robust anxiolytic-like effect in both rodent and non-human primate behavioral models of anxiety. Moreover, in mice containing a point mutation that renders α2-containing receptors BZ insensitive (α2H101R mice), TP003 as well as the nonselective agonist CDP retained efficacy in a stress-induced hyperthermia model. Together, these data show that potentiation of α3-containing GABAA receptors is sufficient to produce the anxiolytic effects of BZs and that α2 potentiation may not be necessary.

cDNA clones coding for the structural subunit of a chicken brain nicotinic acetylcholine receptor

Nicotinic acetylcholine receptors (AChRs) immunoaffinity-purified from brains are composed of only two kinds of subunits rather than the four kinds present in muscle-type AChRs. Here we report the N-terminal protein sequences of the structural subunits of AChRs from rat and chicken brains and the cloning of full-length cDNAs for the chicken brain AChR structural subunit. Previously, the N-terminal amino acid sequence of the ACh-binding subunit of AChR immunoaffinity-purified from rat brain was shown to correspond to the cDNA alpha 4. Thus, cDNA sequences are now known for both of the subunits that form one AChR subtype in vivo.

Neuronal nicotinic acetylcholine receptor β‐subunit is coded for by the cDNA clone α4

Acetylcholine receptors (AChRs) with high affinity for nicotine but no affinity for α‐bungarotoxin, which have been purified from rat and chicken brains by immuno‐affinity chromatography, consist of two types of subunits, α and β [1,2]. The β‐subunits form the ACh binding sites [3]. Putative nicotinic AChR subunit cDNAs α3 and α4 have been identified by screening cDNA libraries prepared from rat PC12 cells and rat brain with cDNA probes encoding the mouse muscle AChR α‐subunit. Here we determine the amino‐terminal amino acid sequence of the rat brain AChR β‐subunit by protein microsequencing to be the same as amino acid residues 27–43 of the protein which could be coded by α4. Further, we present evidence consistent with a subunit stoichiometry of α3β2 for this neuronal nicotinic AChR.

Behavioral and Neurochemical Alterations in Mice Deficient in Anaplastic Lymphoma Kinase Suggest Therapeutic Potential for Psychiatric Indications

The receptor tyrosine kinase product of the anaplastic lymphoma kinase (ALK) gene has been implicated in oncogenesis as a product of several chromosomal translocations, although its endogeneous role in the hematopoietic and neural systems has remained poorly understood. We describe that the generation of animals homozygous for a deletion of the ALK tyrosine kinase domain leads to alterations in adult brain function. Evaluation of adult ALK homozygotes (HOs) revealed an age-dependent increase in basal hippocampal progenitor proliferation and alterations in behavioral tests consistent with a role for this receptor in the adult brain. ALK HO animals displayed an increased struggle time in the tail suspension test and the Porsolt swim test and enhanced performance in a novel object-recognition test. Neurochemical analysis demonstrates an increase in basal dopaminergic signalling selectively within the frontal cortex. Altogether, these results suggest that ALK functions in the adult brain to regulate the function of the frontal cortex and hippocampus and identifies ALK as a new target for psychiatric indications, such as schizophrenia and depression, with an underlying deregulated monoaminergic signalling.

Affinity labelling of neuronal acetylcholine receptors localizes acetylcholine-binding sites to their β-subunits

Neuronal nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats consist of two types of subunits, α and β, of which a shares some antigenic determinants with α‐subunits from AChRs of electric organ and muscle [(1986) Biochemistry 25, 2082‐2093; (1986) J. Neurosci. G, 3061‐3069; (1986) Proc. Natl. Acad. Sci. USA, in press]. Here we demonstrate that after reduction with dithiothreitol (DTT) the AChRs can be specifically labelled with the acetylcholine‐binding site directed reagent 4‐(N‐maleimido)benzyltri [3H]methylammonium iodide. Labelling of the β‐subunits of neuronal nicotinic AChRs indicates that the acetylcholine‐binding site, and amino acids which may be homologous to Cys 192–193 of the α‐subunits of AChRs from electric organ and muscle, are located on the β‐subunit of neuronal AChRs. These results suggest that although neuronal nicotinic AChRs have some structural homologies to AChRs from muscle and electric organs, the AChRs from these sources are quite distant relatives in an extended gene family.

Antibodies in sera from patients with myasthenia gravis do not bind to nicotinic acetylcholine receptors from human brain

Nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats have recently been purified and characterized (Whiting and Lindstrom, Biochemistry, 25 (1986) 2082-2093; J. Neurosci., 6 (1986) 3061-3069; Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 595-599). Using both antisera and monoclonal antibodies prepared to AChRs from rat brain, we have demonstrated the existence of a homologous AChR in human brain. Here we report that antibodies to muscle AChRs in the sera of patients with myasthenia gravis (MG) do not bind to AChRs from human brain. Similarly, there was no binding of sera from patients with Guillain-Barré, amyotrophic lateral sclerosis, multiple sclerosis, or Lambert-Eaton myasthenic syndrome. Additionally, no binding of any of these sera to the alpha-bungarotoxin (alpha-Bgt) binding protein from human brain could be detected. This data is consistent with other data using antibodies to AChRs from muscle and nerve in demonstrating that the AChR in brain is antigenically distinct from the AChR in skeletal muscle AChR, and, together with the lack of central neurological symptoms in MG, suggests that the low concentrations of anti-AChR antibodies in the cerebrospinal fluid of MG patients do not bind to AChRs in brain.

Functional blockade of neuronal acetylcholine receptors by antisera to a putative receptor from brain.

Antisera to a putative acetylcholine receptor purified from chick brain specifically inhibit the acetylcholine response of chick ciliary ganglion neurons in cell culture. The putative brain receptor and a similar membrane component previously identified on ciliary ganglion neurons appear to be functional nicotinic acetylcholine receptors in the nervous system and are clearly distinct from membrane components in the tissues that bind alpha-bungarotoxin.

Antisera against an acetylcholine receptor α3 fusion protein bind to ganglionic but not to brain nicotinic acetylcholine receptors

Neuronal nicotinic acetylcholine receptor (AChR) subtypes have been defined pharmacologically, immunologically, and by DNA cloning, but the correlations between these approaches are incomplete. Vertebrate neuronal AChRs that have been isolated are composed of structural subunits and ACh‐binding subunits. A single kind of subunit can be used in more than one AChR subtype. Monoclonal antibody (mAb) 35 binds to structural subunits of subtypes of AChRs from both chicken brain and ganglia. By using antisera to a unique sequence of α3 ACh‐binding subunits expressed in bacteria, we show that ganglionic AChRs contain α3 ACh‐binding subunits, whereas the brain AChR subtype that binds mAb 35 does not. Subunit‐specific antisera raised against recombinant proteins should be a valuable approach for identifying the subunit composition of receptors in multigene, multisubunit families.