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Biomedical subjects

S Heinemann

Publications and source records attributed to S Heinemann.

At least 73 records · Page 4Linked to original sources
PubMed1989

Distribution of alpha 2, alpha 3, alpha 4, and beta 2 neuronal nicotinic receptor subunit mRNAs in the central nervous system: a hybridization histochemical study in the rat.

Previous studies have revealed the existence of a gene family that encodes a group of neuronal nicotinic acetylcholine receptor (nAChR) subunits. Four members of this family have been characterized thus far; three of these subunits (alpha 2, alpha 3, and alpha 4) are structurally related to the ligand binding subunit expressed in muscle and form functional nAChRs when combined with the beta 2 gene product in Xenopus oocytes. In addition, the alpha 4 gene appears to encode two different products (alpha 4-1 and alpha 4-2) that have been proposed to arise by alternative mRNA splicing. Nine different [35S]-complementary ribonucleic acid (cRNA) probes were used in the present study to map the distribution of these nAChR subunit mRNAs throughout the central nervous system (CNS) of the rat. It was found that the beta 2 gene is expressed in most regions of the CNS, as are the alpha subunit genes as a group. However, each alpha gene is expressed in a unique, although partly overlapping, set of neuronal structures. Alpha 4 is the most widely expressed alpha gene, and the evidence suggests that mRNAs for the alpha 4-1 and alpha 4-2 products are virtually always found in the same regions, in approximately the same ratios (alpha 4-2 greater than alpha 4-1). In addition, there are several examples of cell groups that express beta 2 but none of the alpha subunit mRNAs examined here (particularly in the hypothalamus), as well as all groups that express the converse, thus suggesting that additional neuronal nAChR subunits remain to be characterized. Finally, the extensive expression of multiple alpha subunits in certain regions, particularly for alpha 3 and alpha 4 in the thalamus, suggests that there is microheterogeneity in a small population of cells or that some neurons may express more than one alpha subunit. This problem needs to be examined directly with double labeling methods but raises the possibility that some neuronal nAChRs may be composed of more than one kind of alpha subunit. The wide expression of these receptor genes suggests that nAChRs constitute major excitatory systems in the CNS.

Animals↗
PubMed1989

Beta 3: a new member of nicotinic acetylcholine receptor gene family is expressed in brain.

Screening of a rat brain cDNA library with a radiolabeled probe made from an alpha 3 cDNA (Boulter, J., Evans, K., Goldman, D., Martin, G., Treco, D., Heinemanns, S., and Patrick, J. (1986) Nature 319, 368-374) resulted in the isolation of a clone whose sequence encodes a protein, beta 3, which is homologous (40-55% amino acid sequence identity) to previously described neuronal nicotinic acetylcholine receptor subunits. The encoded protein has structural features found in other nicotinic acetylcholine receptor (nAChR) subunits. Two cysteine residues that correspond to cysteins 128 and 142 of the Torpedo nAChR alpha subunit are present in beta 3. Absent from beta 3 are 2 adjacent cysteine residues that correspond to cysteines 192 and 193 of the Torpedo subunit. In situ hybridization histochemistry, performed using probes derived from beta 3 cDNAs, demonstrated that the beta 3 gene is expressed in the brain. Thus, beta 3 is the fifth member of the nAChR gene family that is expressed in the brain. The pattern of beta 3 gene expression partially overlaps with that of the neuronal nAChR subunit genes alpha 3, alpha 4, or beta 2. These results lead us to propose that the beta 3 gene encodes a neuronal nAChR subunit.

Amino Acid Sequence↗
PubMed1989

The functional diversity of the neuronal nicotinic acetylcholine receptors is increased by a novel subunit: beta 4.

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.

Amino Acid Sequence↗
PubMed1989

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.

Amino Acid Sequence↗
PubMed1989

Genes encoding neuronal nicotinic acetylcholine receptors.

Four genes (alpha 2, alpha 3, alpha 4, and beta 2), which encode proteins homologous to the Torpedo electric organ and vertebrate muscle nicotinic acetylcholine receptors, have been identified by cloning rat brain cDNAs. Injection of transcripts derived from these cDNAs into Xenopus laevis oocytes results in the formation of three nicotinic acetylcholine receptors. Two of these receptors, alpha 3/beta 2 and alpha 4/beta 2, have the characteristics of ganglionic nicotinic receptors. The third (alpha 2/beta 2) exhibits a previously undescribed pharmacology and thus represents a novel subtype that may be expressed in the brain. The wide distribution of alpha 2, alpha 3, alpha 4, and beta 2 transcripts in the brain indicates that neuronal nicotinic acetylcholine receptors are a major neurotransmitter receptor system.

Animals↗
PubMed1988

Functional expression of a new pharmacological subtype of brain nicotinic acetylcholine receptor.

A new type of agonist-binding subunit of rat neuronal nicotinic acetylcholine receptors (nAChRs) was identified. Rat genomic DNA and complementary DNA encoding this subunit (alpha 2) were cloned and analyzed. Complementary DNA expression studies in Xenopus oocytes revealed that the injection of messenger RNAs (mRNAs) for alpha 2 and beta 2 (a neuronal nAChR subunit) led to the generation of a functional nAChR. In contrast to the other known neuronal nAChRs, the receptor produced by the injection of alpha 2 and beta 2 mRNAs was resistant to the alpha-neurotoxin Bgt3.1. In situ hybridization histochemistry showed that alpha 2 mRNA was expressed in a small number of regions, in contrast to the wide distribution of the other known agonist-binding subunits (alpha 3 and alpha 4) mRNAs. These results demonstrate that the alpha 2 subunit differs from other known agonist-binding alpha-subunits of nAChRs in its distribution in the brain and in its pharmacology.

Amino Acid Sequence↗
PubMed1988

Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity.

Denervation of adult skeletal muscle results in increased sensitivity to acetylcholine in extrajunctional regions of the muscle fiber. This increase in acetylcholine sensitivity is accompanied by a large increase in the level of mRNAs coding for the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. To determine whether muscle activity is sufficient to regulate expression of extrajunctional acetylcholine receptor mRNA levels, denervated muscles were stimulated with extracellular electrodes. Direct stimulation of denervated muscle suppresses both the increase in extrajunctional acetylcholine sensitivity and the expression of mRNA encoding the alpha-, beta-, gamma-, and delta-subunits of the acetylcholine receptor. These results show that muscle activity regulates the level of extrajunctional acetylcholine receptors by regulating the expression of their mRNAs.

Animals↗
PubMed1988

Primary structure and expression of beta 2: a novel subunit of neuronal nicotinic acetylcholine receptors.

A new subunit, beta 2, of the neuronal nicotinic receptor family has been identified. This subunit has the structural features of a non-agonist-binding subunit. We provide evidence that beta 2 can substitute for the muscle beta 1 subunit to form a functional nicotinic receptor in Xenopus oocytes. Expression studies performed in oocytes have demonstrated that three different neuronal nicotinic acetylcholine receptors can be formed by the pairwise injection of beta 2 mRNA and each of the neuronal alpha subunit mRNAs. The beta 2 gene is expressed in PC12 cells and in areas of the central nervous system where the alpha 2, alpha 3, and alpha 4 genes are expressed. These results lead us to propose that the nervous system expresses diverse forms of neuronal nicotinic acetylcholine receptors by combining beta 2 subunits with different agonist-binding alpha subunits.

Acetylcholine↗
PubMed1987

Muscle acetylcholine receptor biosynthesis. Regulation by transcript availability.

The expression of the muscle nicotinic acetylcholine receptor (AChR) on the cell surface entails a complex biosynthetic pathway, involving the expression and assembly of four subunits. The amount of AChR on the cell surface changes throughout muscle development and upon muscle denervation. We have examined the regulatory role of transcript levels on surface AChR expression by RNA blot analysis. During myogenesis of the fusing mouse muscle cell line C2, which expresses an embryonic type of receptor, changes in the rate of appearance of cell surface AChR have been assayed by 125I-alpha-bungarotoxin binding. The maximal increase in the rate of appearance of cell surface AChR occurs upon cell fusion, closely following the maximal increase in transcript levels for the alpha-, beta-, gamma-, and delta-AChR subunits. AChR alpha-, beta-, gamma-, and delta-subunit gene transcript levels have also been examined in innervated and denervated rat and mouse muscle. Muscle denervation results in an increase of transcripts for all four subunits. However, the amount of beta-subunit transcript in innervated rat skeletal muscle is high relative to the other subunit transcripts, and increases less than the other subunit transcripts upon denervation. Our results indicate that, during myogenesis and upon denervation, the availability of AChR subunit transcripts for translation plays a regulatory role in surface AChR appearance.

Animals↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1987

Transcriptional regulation of nicotinic acetylcholine receptor genes: identification of control elements of a gamma-subunit gene.

The muscle nicotinic acetylcholine receptor undergoes profound changes in abundance and distribution in response to innervation and denervation. As a start towards understanding the detailed mechanisms of acetylcholine receptor gene regulation, we have identified transcriptional regulatory regions of a receptor subunit gene. A region of genomic DNA at the 5' end of the mouse acetylcholine receptor gamma-subunit gene has been shown to promote transcription of the bacterial chloramphenicol acetyltransferase gene in a cell-type specific manner. In addition, the transcriptional activity of this fragment is developmentally regulated in mouse muscle C2C12 cells. We propose that this fragment contains transcriptional control elements of the mouse muscle acetylcholine receptor gamma-subunit gene.

Acetyltransferases↗
PubMed1987

Functional expression of two neuronal nicotinic acetylcholine receptors from cDNA clones identifies a gene family.

A family of genes coding for proteins homologous to the alpha subunit of the muscle nicotinic acetylcholine receptor has been identified in the rat genome. These genes are transcribed in the central and peripheral nervous systems in areas known to contain functional nicotinic receptors. In this paper, we demonstrate that three of these genes, which we call alpha 3, alpha 4, and beta 2, encode proteins that form functional nicotinic acetylcholine receptors when expressed in Xenopus oocytes. Oocytes expressing either alpha 3 or alpha 4 protein in combination with the beta 2 protein produced a strong response to acetylcholine. Oocytes expressing only the alpha 4 protein gave a weak response to acetylcholine. These receptors are activated by acetylcholine and nicotine and are blocked by Bungarus toxin 3.1. They are not blocked by alpha-bungarotoxin, which blocks the muscle nicotinic acetylcholine receptor. Thus, the receptors formed by the alpha 3, alpha 4, and beta 2 subunits are pharmacologically similar to the ganglionic-type neuronal nicotinic acetylcholine receptor. These results indicate that the alpha 3, alpha 4, and beta 2 genes encode functional nicotinic acetylcholine receptor subunits that are expressed in the brain and peripheral nervous system.

Amino Acid Sequence↗
PubMed1986

Isolation and sequence of cDNA clones coding for the precursor to the gamma subunit of mouse muscle nicotinic acetylcholine receptor.

cDNA libraries have been constructed in plasmid (pBR322) and bacteriophage lambda gammagt10) vectors with poly (A+) RNA isolated from the nonfusing mouse muscle cell line BC3H-1. The libraries were screened with a restriction fragment derived from a genomic clone coding for a human acetylcholine receptor gamma subunit. Several clones were obtained whose cDNA inserts possessed nucleotide and deduced amino acid sequence homology with acetylcholine receptor gamma subunits from Torpedo californica, chick, calf, and human. One isolate, lambda BMG419, has 88 nucleotides of 5'-untranslated sequence, an open reading frame of 1,557 nucleotides coding for the precursor to the mouse acetylcholine receptor gamma subunit, and 144 nucleotides of 3'-untranslated sequence. Alignment of the lambda BMG419-deduced amino acid sequence with homologs from other species predicts a precursor peptide of 519 amino acids and a mature protein of 497 amino acids, with nonglycosylated molecular weights of 58,744 and 56,424 daltons, respectively. Comparison of the deduced amino acid sequence of the mouse gamma subunit with Torpedo, chick, calf, and human sequences showed overall homologies of 54%, 67%, 90%, and 90%, respectively; however, significantly higher homologies were found in several putative functional domains. Radiolabeled lambda BMG419 has been used to identify homologous RNA species, one of approximately 2 kb and one of about 3.5 kb, in poly (A+) RNA prepared from BC3H-1 cells and denervated mouse limb muscle. gamma Subunit-coding RNA species are considerably more abundant in denervated than in innervated muscle, suggesting that neural regulation of the abundance of the gamma subunit is exerted through regulation of the amount of its mRNA.

Animals↗
PubMed1986

Mapping of brain areas expressing RNA homologous to two different acetylcholine receptor alpha-subunit cDNAs.

We have used an in situ RNA X RNA hybridization technique to determine, in the central nervous systems of the mouse and rat, the distribution of RNA homologous to cDNA clones encoding the alpha subunit of a putative neural nicotinic acetylcholine receptor and the alpha subunit of the muscle nicotinic acetylcholine receptor. Hybridization of the neural alpha-subunit probe was strongest in the medial habenula but was also detected consistently in the compact part of the substantia nigra and ventral tegmental area, in the neocortex, and in certain parts of the thalamus and hypothalamus. The in situ hybridization technique makes it possible to compile a map of brain regions containing cell bodies expressing RNA coding for a specific receptor type and subsequently to apply the techniques of molecular biology to study these brain receptors.

Animals↗