Biomedical subjects

M Ballivet

Publications and source records attributed to M Ballivet.

At least 37 records · Page 2Linked to original sources

PubMed1991

Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

Acetylcholine↗

PubMed1991

Pentameric structure and subunit stoichiometry of a neuronal nicotinic acetylcholine receptor.

Neuronal nicotinic acetylcholine receptors are members of a gene family of ligand-gated transmitter receptors that includes muscle nicotinic receptors, GABAA receptors and glycine receptors. Several lines of evidence indicate that neuronal nicotinic receptors can be made up of only two subunits, an alpha (alpha) subunit which binds ligand, and a non-alpha (n alpha) or beta (beta) subunit. The stoichiometry of each subunit in the functional receptor has been difficult to assess, however. Estimates of the molecular weight of neuronal nicotonic receptor macromolecules suggest that these receptors contain at least four subunits but probably not more than five. We have examined the subunit stoichiometry of the chick neuronal alpha 4/n alpha 1 receptor by first using site-directed mutagenesis to create subunits that confer different single channel properties on the receptor. Co-injection with wild-type and mutant subunits led to the appearance of receptors with wild-type, mutant and hybrid conductances. From the number of hybrid conductances, we could deduce the number of each subunit in the functional receptor.

Acetylcholine↗

PubMed1991

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the alpha subunit in agonist sensitivity and desensitization.

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes alpha 4/n alpha 1 and alpha 3/n alpha 1 were readily distinguishable from one another by ACh sensitivity and desensitization. alpha 3/n alpha 1 receptors showed lower ACh sensitivity and stronger desensitization than alpha 4/n alpha 1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n alpha 1 subunit was unchanged, the alpha subunits must be responsible for these functional differences. Symmetric hybrid alpha cDNAs, alpha 4:alpha 3 and alpha 3:alpha 4, were constructed and functional receptors were obtained by co-injection with n alpha 1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the alpha subunit. In contrast, no part of the alpha subunit was found fully to determine desensitization.

Acetylcholine↗

PubMed1991

Steroids inhibit nicotinic acetylcholine receptors.

Application of progesterone to Xenopus oocytes expressing a cloned neuronal nicotinic acetylcholine (nAChR) revealed two effects. The first effect was a fully reversible reduction of the current induced by acetylcholine (ACh), its onset being nearly instantaneous. The second effect, which developed in a few hours, was an irreversible suppression of ACh-evoked currents. The transient inhibition had an apparent Ki of 7 microM when tested with 50 nM ACh, but the percentage of inhibition was positively correlated to the ACh concentration. A reduction of ACh-induced currents which appeared immediately upon progesterone application was also observed with muscle nAChR expressed in oocytes and with nAChR on membrane patches isolated from ciliary ganglion neurons. Thus nAChRs are modulated by progesterone and steroids may play an important role in nicotinic cholinoception.

Animals↗

PubMed1990

Alpha 5, alpha 3, and non-alpha 3. Three clustered avian genes encoding neuronal nicotinic acetylcholine receptor-related subunits.

In vertebrates, neuronal nicotinic acetylcholine receptors (nAChRs) assemble in an unknown stoichiometry from two homologous subunits, an alpha and a non-alpha. How large is the repertoire of these subunits and how many subtypes of functionally different nAChRs can they constitute? We found in the avian genome a cluster of three closely linked genes spanning 28 kilobase pairs and encoding three proteins, n alpha 3, alpha 3, and alpha 5, that have the features expected of neuronal nAChR subunits. Gene n alpha 3 lies 5' of alpha 3 (whose role in cholinoception has already been established) and is transcribed from the same DNA strand, whereas alpha 5 lies 3' of alpha 3 and is transcribed from the opposite DNA strand. The structure of the n alpha 3 and alpha 5 genes consists of six exons with precisely conserved splice sites and is identical to the structure of the previously characterized avian neuronal receptor subunit genes alpha 2, alpha 3, alpha 4, and n alpha 1. alpha 3, n alpha 3, and alpha 5 transcripts are rare in the central nervous system, but alpha 3 and n alpha 3 are readily detectable in embryonic superior cervical and ciliary ganglia. In order to assay function, the gene encoding n alpha 3 and the cDNAs encoding alpha 3, alpha 4, alpha 5, and n alpha 1 were subcloned into an expression vector, and the constructs were injected into Xenopus oocyte nuclei, either singly or in pairwise combinations of one alpha and one non-alpha. One to five days later, ACh sensitivity of the injected oocytes was examined in voltage clamp. The n alpha 3 gene and n alpha 1 cDNA elicited assembly of nAChRs when coinjected with alpha 3 or alpha 4 cDNA and the electrophysiological properties of the four pairwise combinations were significantly different. alpha 5, however, did not direct the assembly of functional nAChRs when injected alone or in combination with n alpha 1 or n alpha 3.

Acetylcholine↗

PubMed1990

Alpha 3, alpha 5, and beta 4: three members of the rat neuronal nicotinic acetylcholine receptor-related gene family form a gene cluster.

We have identified two additional members of the neuronal nicotinic acetylcholine receptor (nAChR)-related gene family. cDNA clones for one new gene, designated alpha 5, were isolated from rat hippocampus and rat PC12 cell line cDNA libraries. The alpha 5 gene encodes a protein of 48,800 daltons (424 amino acids) which exhibits significant overall amino acid sequence identity with the previously cloned rat nAChR subunits alpha 1 (49%), alpha 2 (55%), alpha 3 (52%), and alpha 4 (49%). Features characteristic of other nAChR alpha-subunits are present such as conserved cysteine residues at positions 127, 141, 191, and 192, and four strongly hydrophobic domains. A second addition to the nAChR-related gene family, designated beta 4, is encoded in overlapping rat genomic clones lambda DD15 and lambda RG518A. The beta 4 gene, encoding a mature protein of 53,300 daltons (475 amino acids), consists of 6 exons and has a transcription unit length of approximately 18 kilobase pairs. The beta 4 gene encoded protein shows considerable amino acid sequence identity with nAChR beta 1 (43%), beta 2 (64%), and beta 3 (44%) subunits. Northern blots showed that, along with alpha 3 and beta 2, transcripts for both the alpha 5 and beta 4 genes are present in the PC12 cell line, while in situ hybridization experiments demonstrated expression of the alpha 5 and beta 4 genes in a small number of nuclei in the central nervous system. Finally, the genes that encode the beta 4, alpha 3, and alpha 5 proteins are transcribed with convergent polarities and form a tightly linked gene cluster spanning approximately 60 kilobase pairs.

Amino Acid Sequence↗

PubMed1990

Expression of neuronal nicotinic acetylcholine receptor genes in the developing chick visual system.

Expression of the neuronal non-alpha nicotinic acetylcholine receptor (n alpha nAChR) gene is transiently stimulated in the chick optic tectum between embryonic days 7 and 16 with a peak value reached around embryonic day 12. This stimulation takes place at the time when optic nerve axons are invading this region of the brain and proceeds along a rostral to caudal gradient. Transcripts of the n alpha nAChR gene are localized in the superficial layers of the tectum at the time when cells in these layers are forming synapses with retina axons. The transient expression of n alpha nAChR gene does not take place in the optic tectum of 'eyeless' embryos. The results of our study suggest that the neuronal n alpha nAChR gene may play a role in neurogenesis of retino-tectal connections.

Animals↗

PubMed1990

A neuronal nicotinic acetylcholine receptor subunit (alpha 7) is developmentally regulated and forms a homo-oligomeric channel blocked by alpha-BTX.

cDNA and genomic clones encoding alpha 7, a novel neuronal nicotinic acetylcholine receptor (nAChR) alpha subunit, were isolated and sequenced. The mature alpha 7 protein (479 residues) has moderate homology with all other alpha and non-alpha nAChR subunits and probably assumes the same transmembrane topology. alpha 7 transcripts transiently accumulate in the developing optic tectum between E5 and E16. They are present in both the deep and the superficial layers of E12 tectum. In Xenopus oocytes, the alpha 7 protein assembles into a homo-oligomeric channel responding to acetylcholine and nicotine. The alpha 7 channel desensitizes very rapidly, rectifies strongly above -20 mV, and is blocked by alpha-bungarotoxin. A bacterial fusion protein encompassing residues 124-239 of alpha 7 binds labeled alpha-bungarotoxin. We conclude that alpha-bungarotoxin binding proteins in the vertebrate nervous system can function as nAChRs.

Acetylcholine↗

PubMed1990

Activation and blocking of neuronal nicotinic acetylcholine receptor reconstituted in Xenopus oocytes.

Neuronal nicotinic acetylcholine receptor of the alpha 4/non-alpha (alpha 4/n alpha) type was reconstituted in Xenopus oocytes after nuclear injection of cDNA expression vectors. Functional neuronal receptor was only formed when the two subunits alpha 4 and n alpha were coinjected, neither alpha 4 nor n alpha alone being effective. Responses to bath application of acetylcholine (AcCho) have been measured in voltage clamp. AcCho doses as low as 10 nM induce currents of up to 50 nA. Dose-response studies indicate a Kd of about 0.77 x 10(-6) M and a Hill coefficient of 1.5, thus predicting more than one AcCho binding site per receptor molecule. The current-voltage relationship of AcCho-induced currents presents a strong inward rectification. Responses to AcCho were compared to those of three other agonists: L-nicotine, carbachol, and 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Sensitivities to AcCho, nicotine, and DMPP are quite similar. Sensitivity to carbachol is much lower, but the currents are otherwise indistinguishable from those induced by AcCho. Five AcCho antagonists--neuronal bungarotoxin (kappa-bungarotoxin), tubocurarine (TC), hexamethonium bromide (Hex), decamethonium bromide (Dec), and mecamylamine (Mec)--have been tested. Neuronal bungarotoxin has no effect on the alpha 4/n alpha channel, whereas 2.5 microM TC reduces by half the current peak evoked by 1 microM AcCho. The block by TC is independent of membrane voltage. By contrast, the block of AcCho-induced currents by Hex or Dec is strongly voltage dependent, suggesting that these substances enter the channel. The block by Mec is detectable at concentrations as low as 100 nM when applied together with 1 microM AcCho and is voltage independent. Hex, Dec, and Mec are effective only when AcCho is present. While the effects of all other agents are fully reversible, the Mec block is persistent.

Acetylcholine↗

PubMed1989

Autoimmune T lymphocytes in myasthenia gravis. Determination of target epitopes using T lines and recombinant products of the mouse nicotinic acetylcholine receptor gene.

Oligoclonal and cloned T lines from peripheral blood or thymuses of patients with myasthenia gravis (MG) were selected for reactivity against nicotinic acetylcholine receptors (AChR) from Torpedo california, or against a recombinant fusion peptide, X4, representing the extracellular portion of the mouse AChR alpha-chain. All cell lines expressed the CD4 membrane phenotype, and their antigen reactivity was blocked by antibodies against monomorphic HLA DR/DP determinants. Using a panel of fusion proteins of different, overlapping mouse AChR alpha-chain sequences, a major T cell epitope was localized between amino acid positions 85 and 142. This determinant was distinct from the humoral main immunogenic region, which has been identified on the sequence 61-76. The response pattern of uncloned T lines from three patients with different HLA haplotypes suggests, however, that in any one MG patient T lymphocytes may recognize more than one autoantigenic epitope on the AChR alpha-chain, and that the T lymphocyte response profiles vary among individual patients.

Adult↗

PubMed1988

Monoclonal antibodies to the main immunogenic region of the nicotinic acetylcholine receptor bind to residues 61-76 of the alpha subunit.

Monoclonal antibodies (mAbs) to the main immunogenic region (MIR) bind to fusion proteins containing region 37-200 of the alpha chain of Torpedo, mouse, and chicken nicotinic acetylcholine receptor. In the case of the mouse alpha chain, these mAbs react with sequence 61-216 but not with 74-216. A synthetic peptide M1, containing residues 61-76 of the mouse alpha chain, also binds these anti-MIR mAbs, showing that all or part of their binding site is included in this region. The conformational dependence and epitope specificity of the mAbs are discussed.

Amino Acid Sequence↗

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

Genes expressed in the brain define three distinct neuronal nicotinic acetylcholine receptors.

Four genes encode the related protein subunits that assemble to form the nicotinic acetylcholine receptor (nAChR) at the motor endplate of vertebrates. We have isolated from the chicken genome four additional members of the same gene family whose protein products, termed alpha 2, alpha 3, alpha 4 and n alpha (non-alpha) probably define three distinct neuronal nAChR subtypes. The neuronal nAChR genes have identical structures consisting of six protein-coding exons and specify proteins that are best aligned with the chicken endplate alpha subunit, whose gene we have also characterized. mRNA transcripts encoding alpha 4 and n alpha are abundant in embryonic and in adult avian brain, whereas alpha 2 and alpha 3 transcripts are much scarcer. The same set of neuronal genes probably exists in all vertebrates since their counterparts have also been identified in the rat genome.

Age Factors↗

PubMed1988

Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system.

Amino Acid Sequence↗

PubMed1988

Acetylcholine receptor synthesis rate and levels of receptor subunit messenger RNAs in chick muscle.

Levels of mRNAs specific for the alpha-, gamma- and delta-subunit of the nicotinic acetylcholine receptor were measured in chick skeletal muscle by solution hybridization, using a genomic DNA probe containing the intramembrane segments M2 and M3 of the alpha-subunit and probes comprising exons 2-6 and exons 4-8, respectively, of the gamma- and delta-subunit. In the innervated calf musculature of adult chickens, receptor-specific messages were detected in approx. 100-fold excess over the amount required to account for the observed synthesis rate. Within 1 week after section of the sciatic nerve, alpha-, gamma- and delta-subunit message levels rose 112-, 42- and 24-fold, respectively, while receptor expression rate increased about 150-fold. The rise in message levels preceded the denervation-induced increase in receptor concentration. In differentiating myogenic cells all three messages were found in excess over the amounts required for the observed rate of receptor synthesis. Treatment of differentiated myotubes with drugs that change receptor synthesis rate selectively affects alpha-subunit mRNA. In all situations in vitro and in vivo the alpha-subunit mRNA was found to reach final levels faster, and to be from 3 to over 30 times more abundant, than the other messages. These observations corroborate earlier evidence for a regulatory mechanism in which the supply of mRNA determines acetylcholine receptor synthesis rate. They also suggest that receptor expression is not simply proportional to acetylcholine receptor subunit mRNA concentrations, but rather is controlled, to a considerable extent, by the efficiency with which the receptor-specific mRNAs and/or the subunits they code for are subsequently utilized.

Animals↗

PubMed1988

Electrophysiology of a chick neuronal nicotinic acetylcholine receptor expressed in Xenopus oocytes after cDNA injection.

Brain nicotinic acetylcholine receptors (nAChRs) are made up of protein subunits that differ from those constituting muscle nAChRs. To characterize the physiological properties of one class of avian brain nicotinic receptor, we injected the nuclei of Xenopus oocytes with full-length cDNAs for the ligand binding (alpha 4) and structural (n alpha) subunits. Injected oocytes had large ACh-induced currents in the microampere range that were insensitive to alpha-bungarotoxin, as expected for neuronal nAChRs. We found that these brain nAChRs incorporate at least two alpha 4 subunits and that their functional properties differ from muscle nAChRs in at least two respects: the elementary conductance is considerably smaller (20 pS), and channels in outside out patches stop functioning within a few minutes.

Animals↗

PubMed1988

Expression and regulation of neuronal acetylcholine receptor mRNA in chick ciliary ganglia.

A chicken genomic clone encoding a portion of the neuronal acetylcholine receptor (AChR) alpha 3 subunit was used to identify homologous mRNA in embryonic chick ciliary ganglia. In situ hybridization indicated that the mRNA was neuronal. Northern blot analysis revealed a major hybridizing species of 3.5 kb. Protection experiments confirmed that ganglionic RNA contained material indistinguishable by RNAase digestion from the 300 nucleotide probe used. No transcripts were detected by in situ hybridization or Northern blot analysis for chick neuronal AChR alpha 2 or alpha 4 genes. alpha 3 transcripts were present at all times examined (E6 to 1 year posthatch). Both postganglionic axotomy and preganglionic denervation of ciliary ganglia in newly hatched chicks produced declines in alpha 3 mRNA levels, implying regulation of neuronal AChR mRNA by cell-cell interactions.

Animals↗

PubMed1988

A cell type-specific enhancer drives expression of the chick muscle acetylcholine receptor alpha-subunit gene.

The regulation of acetylcholine receptor alpha-subunit gene expression was analyzed by transient expression assays. Using rabbit beta-globin cDNA as a reporter gene, we have confirmed that the 5'-flanking sequence of the chicken acetylcholine receptor alpha-subunit gene directs specific expression in differentiated C2C12 cells, a mouse muscle cell line, but not in undifferentiated C2C12 cells and mouse 3T3 fibroblasts. Testing chimeric plasmids containing Bal31 deletion mutants of the alpha-subunit gene upstream sequence, we found the -116 to -81 region of the alpha-subunit to be responsible for tissue- and stage-specific expression. This 36 bp fragment stimulates the activity of both alpha-subunit and SV40 promoters in a distance- and orientation-independent manner, thus fulfilling the criteria of an enhancer.

Animals↗