Jim Patrick

Autoimmune response to acetylcholine receptor.

Injection of rabbits with acetylcholine receptor highly purified from the electric organ of Electrophorus electricus emulsified in complete Freunds adjuvant resulted in the production of precipitating antibody to acetylcholine receptor. After the second injection of antigen, the animals developed the flaccid paralysis and abnormal electromyographs characteristic of neuromuscular blockade. Treatment with the anticholinesterases edrophonium or neostigmine dramatically alleviated the paralysis and the fatigue seen in electromyography.

Members of a nicotinic acetylcholine receptor gene family are expressed in different regions of the mammalian central nervous system

Nicotinic acetylcholine receptors found in the peripheral and central nervous system differ from those found at the neuromuscular junction. Recently we isolated a cDNA clone encoding the alpha subunit of a neuronal acetylcholine receptor expressed in both the peripheral and central nervous system. In this paper we report the isolation of a cDNA encoding the alpha subunit of a second acetylcholine receptor expressed in the central nervous system. Thus it is clear that there is a family of genes coding for proteins with sequence and structural homology to the alpha subunit of the muscle nicotinic acetylcholine receptor. Members of this gene family are expressed in different regions of the central nervous system and, presumably, code for subtypes of the nicotinic acetylcholine receptor.

The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: β4

A new nicotinic acetylcholine receptor (nAChR) subunit, beta 4, was identified by screening a rat genomic library. In situ hybridization histochemistry revealed expression of the beta 4 gene in the medial habenula of adult rat brains. The primary structure of this subunit was deduced from a cDNA clone isolated from a PC12 cDNA library. Functional nAChRs were detected in Xenopus oocytes injected in pairwise combinations with in vitro synthesized RNAs encoding beta 4 and either the alpha 2, alpha 3, or alpha 4 subunit. Unlike the alpha 3 beta 2 receptor, the alpha 3 beta 4 receptor is not blocked by bungarotoxin 3.1, indicating that the beta subunit can affect the sensitivity of neuronal nAChRs to this toxin. These results extend the functional diversity of nicotinic receptors in the nervous system.

Nerve growth factor mediates phosphorylation of specific proteins

Nerve growth factor (NGF), epidermal growth factor (EGF), insulin, cholera toxin (CT) and cAMP all stimulate the phosphorylation of proteins in the PC12 nerve-like cell line. NGF, CT and cAMP enhance phosphorylation of the same set of proteins including tyrosine hydroxylase, ribosomal protein S6, histones H1 and H3, and the nonhistone chromosomal and cytoplasmic high mobility group (HMG) 17 protein, and reduce phosphorylation of H2A. EGF but not insulin enhances the phosphorylation of tyrosine hydroxylase. Insulin but not EGF enhances the phosphorylation of histone H3 and decreases the phosphorylation of H2A. EGFD and insulin each enhance phosphorylations of both ribosomal protein S6 and histone H1, but neither hormone induces phosphorylation of HMG 17. The extent of these effects depends upon the ligand concentration and is half-maximal at physiological concentrations of the hormones (beta-NGF, 2 ng/ml; EGF, 1 ng/ml. insulin, 0.5 microunits/ml). Maximal effects of NGF are seen within 15 min and persist even after 3 days of culture in the presence of NGF. When phosphorylation of ribosomal protein S6 is maximally stimulated by NGF, no further stimulation can be achieved by adding saturating quantities of either cAMP or CT. However, simultaneous addition of saturating quantities of NGF and either EGF or insulin results in an enhancement of phosphorylation that is equal to the sum of that achieved when the two ligands are added separately. These results suggest that the enhanced phosphorylation of S6 achieved by NGF or cAMP occurs through a common mechanism which differs from those which mediate EGF or insulin-enhanced phosphorylation. The data also provide strong evidence that the action of NGF included protein phosphorylation mediated by cAMP-dependent protein kinase. The phosphorylation of each of these proteins in response to NGF may play an important role in NGF action.

The distribution of mRNA encoded by a new member of the neuronal nicotinic acetylcholine receptor gene family (α5) in the rat central nervous system

The cellular localization of transcripts for a new putative agonist-binding subunit of the neuronal nicotinic acetylcholine receptor (nAChR), alpha 5, was examined using in situ hybridization in the rat central nervous system (CNS), alpha 5 subunit mRNA was localized to a small number of regions when compared with two of the other known agonist-binding subunits, alpha 3 and alpha 4, alpha 5 mRNA is expressed at relatively high levels in neurons of the subiculum (pyramidal layer), presubiculum and parasubiculum (layers IV and VI), which are components of the hippocampal formation, in the substantia nigra pars compacta and ventral tegmental area, in the interpeduncular nucleus, and in the dorsal motor nucleus of the vagus nerve. Moderate hybridization signals were detected in neurons of the isocortex (layer VIb), anterior olfactory nucleus, trigeminal ganglion, superior olivary complex, nucleus of the solitary tract, and area postrema. No hybridization above background levels was seen in the amygdala, septum, thalamus, hypothalamus, or cerebellum. These results suggest that the alpha 5 subunit differs from other known agonist-binding subunits in its distribution.

Single-channel currents of rat neuronal nicotinic acetylcholine receptors expressed in xenopus oocytes

The neuronal nicotinic acetylcholine receptor subunits alpha 2, alpha 3, and alpha 4 form functional receptors with the beta 2 subunit. Each of these subunit combinations shows two distinct open states (referred to as primary and secondary). The primary open states of alpha 2 beta 2, alpha 3 beta 2, and alpha 4 beta 2 receptors were 33.6 +/- 1.8 pS, 15.4 +/- 0.8 pS, and 13.3 +/- 1.5 pS, respectively. The open times of the alpha 3 beta 2 primary open state were significantly longer than the open times of the other primary conductance states. The secondary open states of alpha 2 beta 2 and alpha 3 beta 2 were 15.5 +/- 1.3 pS and 5.1 +/- 0.4 pS, respectively. Secondary open states were seen infrequently with alpha 4 beta 2. Oocytes injected with alpha 2 RNA and a 9-fold excess of beta 2 RNA showed an enhanced expression of the secondary open state.

Isolation of a clone coding for the alpha-subunit of a mouse acetylcholine receptor

The mouse cell line BC3H-I synthesizes an acetylcholine receptor (AChR) with the pharmacological properties of a muscle nicotinic cholinergic receptor. We have purified mRNA from this cell line and used the size- fractionated poly(A)+RNA to produce a cDNA library of approximately 50,000 clones. The library was screened with a subclone containing genomic sequences coding for the putative acetylcholine-binding site of the alpha-subunit of chicken AChR. We obtained a plasmid, pMAR alpha 15, with a 1,717-base pair insert. The insert cDNA has 26 nucleotides at the 5′-end which code for a portion of the signal peptide followed by a single open reading frame of 1,311 nucleotides which code for a protein of 49,896 daltons. The insert has 377 bases of 3′-untranslated sequence with 3 polyadenylation sites. Radiolabeled plasmid DNA has been used to identify homologous RNA species of about 2 kilobases in Northern blot analyses of poly(A)+ selected RNA from BC3H-I cells. A similar size mRNA is seen in innervated mouse diaphragm and leg muscle, and both mouse and rat brain. Comparisons of the deduced amino acid sequence of the mouse AChR alpha-subunit with Torpedo marmorata, T. californica, chicken, human, and calf sequences show overall homologies of 80%, 80%, 86%, 96%, and 95%, respectively. More detailed analyses reveal a non-random distribution of amino acid substitutions in several structural domains. Based on the absolute conservation of cysteine residues, a new model for the arrangement of the disulfide bonds in the extracellular portion of the alpha-subunit is proposed.

The brain nicotinic acetylcholine receptor gene family

Publisher Summary During the past decade, the availability of radiolabeled nicotine with high specific activity has led to the discovery and mapping of nicotine binding sites in the mammalian brain. These data suggested that the mammalian brain contains an important nicotinic receptor system. This chapter discusses the family of brain nicotinic acetylcholine receptors that have discovered in the past few years through the use of the molecular genetic approach. It identifies seven genes in the rat or mouse genome that code for proteins with homology to the nicotinic acetylcholine receptor. These genes are expressed in the mammalian brain and in some peripheral neurons. The primary structures of the brain nicotinic receptor subunits expressed in the brain have been deduced from the sequences of the cDNA clones. Analysis of the hydrophobicity profiles of the brain nicotinic receptor subunits suggests that they fold through the membrane in an identical manner to the Torpedo fish nicotinic receptor.

The nicotinic receptor genes.

Summary: The causative factor(s) of Alzheimers disease (AD) are presently unknown. However, it has been shown that the number as well as the fraction of high‐ to low‐affinity nicotine binding sites is altered in patients suffering from this disease. This finding, along with the identification of seven genes which code for nicotinic receptors expressed in the mammalian brain, has led to the idea that one nicotinic receptor subtype may be specifically altered in AD. The present article reviews how, through a molecular genetic approach, a family of genes coding for nicotinic acetylcholine receptor subtypes was uncovered. Also discussed is the use of in situ hybridization to determine the distribution of expression of the mRNA encoding for each receptor subtype and the patch clamp technique to characterize their biophysical properties. Determination of the promoters of these genes, as well as the properties of the expressed receptor subtypes, may make it possible to design new specific nicotinic receptor subtype drugs that will treat not only the symptoms of AD but the progression of the disease process as well.

Molecular Biology of Nicotinic Acetylcholine Receptors

Acetlylcholine is the neurotransmitter at the vertebrate neuromuscular junction, sympathetic ganglia, and in the central nervous system. Although in each location the presynaptic neuron releases acetylcholine, the receptor on the postsynaptic cell may differ. The nicotinic acetylcholine receptors at the neuromuscular junction are pharmacologically distinguishable from those in the sympathetic ganglia, and the nicotinic receptors in the central nervous system may further be distinguished from both the muscle and ganglionic type receptors. We know much more about the nicotinic acetylcholine receptors on muscle than about their counterparts on neurons. The reasons for this are twofold: (1) the identification of sources rich in nicotinic receptors, such as the electric organs of various rays and fishes; and (2) the identification of aneurotoxins, such as a-bungarotoxin, which bind with very high affinity to the muscle nicotinic acetylcholine receptor. The availability of a specific ligand and a rich source of receptor made possible biochemical studies of the receptor and led to our current understanding of this molecule. However, unlike its muscle counterpart, a rich source of neuronal nicotinic acetylcholine receptors has not been found. Furthermore, abungarotoxin has not provided the access to the neuronal nicotinic acetylcholine receptor that it did to the muscle-type nicotinic receptor. Consequently, our appreciation of its structure and regulation is less well developed. However, molecular biological approaches, which have been so valuable for studies of the muscle-type nicotinic acetylcholine receptors, now also provide access to the neuronal nicotinic acetylcholine receptor. In the following paragraphs we discuss our studies of the mouse muscle nicotinic acetylcholine receptor and describe a clone that, we propose, encodes a neuronal nicotinic acetylcholine receptor a-subunit.