Marc Ballivet

A neuronal nicotinic acetylcholine receptor subunit (α7) is developmentally regulated and forms a homo-oligomeric channel blocked by α-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.

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.

Physiological properties of neuronal nicotinic receptors reconstituted from the vertebrate beta 2 subunit and Drosophila alpha subunits.

Three cDNAs (ALS, Dα2 and ARD) isolated from the nervous system of Drosophila and encoding putative nicotinic acetylcholine receptor subunits were expressed in Xenopus oocytes in order to study their functional properties. Functional receptors could not be reconstituted from any of these subunits taken singly or in twos and threes. In contrast, large evoked currents (in the μA range) were consistently observed upon agonist application on oocytes co‐injected with ALS or Dα2 in combination with the chick β2 structural subunit. The ALS/β2 and Dα2/β2 receptors are highly sensitive to acetylcholine and nicotine, and their physiological properties resemble those of native or reconstituted receptors from vertebrates. Although the physiological properties of ALS/β2 and Dα2/β2 receptors are quite similar, clear differences appear in their pharmacological profiles. The ALS/β2 receptor is highly sensitive to α‐bungarotoxin while the Dα2/β2 receptor is totally insensitive to this agent. These results demonstrate that the Drosophila ALS and Dα2 cDNAs encode neuronal nicotinic subunits responding to physiological concentrations of the agonists acetylcholine and nicotine.

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.

Pharmacological properties of the homomeric α7 receptor

Abstract The pharmacological properties of the α-bungarotoxin sensitive α7 neuronal nicotinic acetylcholine receptor (nAChR) were studied upon reconstitutio in Xenopus oocytes. Channels formed by α7 are about 10-fold more sensitive to nicotine and cytisine than to ACh but are little, if at all, activated by the ganglionic agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP). Tubocurarine (TC) was found to act as a non-competitive inhibitor, whereas dihydro-β-erythroidine (DHβE) behaves as a pure competitive inhibitor whose blockade is fast and fully reversible. In addition, the α7 receptor displays a poor sensitivity to methonium salts. The pharmacological properties of the α7 channels are readily distinguishable from those of other identified neuronal nicotinic receptors.

Early steps in the production of sensory neurons by the neurogenic placodes.

Neurogenic placodes are specialized regions of the embryonic ectoderm that generate the majority of the neurons of the cranial sensory ganglia. Here we have accurately determined the onset of neurogenesis in each of the placodes in the chick, and we have also analyzed the expression profiles of genes that are believed to be involved in determining the types of sensory neurons produced by each placode. Interestingly, we find that there is a major difference in the expression domains of neurogenin-1 and neurogenin-2 in the chick, when compared with those reported for the mouse. We do find, however, that Brn-3a and Phox-2a and Phox-2b which are also associated with the specification of neuronal type are expressed in the same domains in the chick as they are in the mouse. These results suggest that neurogenin-1 and neurogenin-2 are functionally interchangeable in neurogenic placodes. We have also found major differences between the ophthalmic and maxillomandibular trigeminal placodes, and while all of the other placodes generate mitotically active cells the ophthalmic trigeminal placode seems to throw off postmitotic neuronal cells.

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.

Electrophysiology of Neuronal Nicotinic Acetylcholine Receptors Expressed in Xenopus Oocytes following Nuclear Injection of Genes or cDNAs

Publisher Summary This chapter presents an overview of electrophysiology of neuronal nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes following nuclear injection of genes or cDNAs. The Xenopus oocyte system allows testing the functions of the many neuronal nAChR-related subunits isolated by molecular cloning. In vivo , some brain nuclei and peripheral nervous system ganglia contain many different species of nAChR-related subunit mRNAs and may, therefore, simultaneously express several neuronal nAChR subtypes. Moreover, there is molecular and physiological evidence that some of the neuronal subunits that have been isolated do not assemble into conventional nAChRs at all. Thus, injecting oocytes with defined pairs of cloned subunit cRNAs or cDNAs is the only direct and rapid way to determine which pairs of subunits assemble into functional receptors and which do not, and what the detailed electrophysiological properties of the various reconstituted receptors are.

Neuronal specificity of the alpha 7 nicotinic acetylcholine receptor promoter develops during morphogenesis of the central nervous system.

A transient transfection assay has been developed to analyse promoter activity in neuronal cells freshly dissociated from the chick central nervous system. The assay enabled us to identify cis‐acting regulatory elements within the 5′‐flanking region of the alpha 7 nicotinic acetylcholine receptor gene. In differentiated retina, regulatory elements direct reporter gene expression to a small subset of neurons which has been identified as ganglion cells, i.e. to the population of neurons in which alpha 7 transcripts were localized by in situ hybridization. However, these promoter elements exhibit ubiquitous activity in undifferentiated neural cells and in mesodermal stem cells. Our study supports the idea that alpha 7 regulatory elements acquire their neuronal specificity in the course of embryogenesis.

The neuronal α6 subunit forms functional heteromeric acetylcholine receptors in human transfected cells

We examine some of the biological and physiological properties of the avian α6 neuronal nicotinic acetylcholine receptor (nAChR) subunit. We show here that, beginning at embryonic day 5, α6 mRNA is abundantly expressed in the developing chick neuroretina, where it coexists with other nicotinic receptor subunit mRNAs such as α3, β2 and β4. In contrast, α6 mRNA is absent from the optic tectum and from the peripheral ganglia. Despite numerous efforts, the α6 subunit has long failed the critical test of functional reconstitution. Here we use patch‐clamp techniques and confocal laser microscopy to measure ACh‐activated currents and nicotine‐elicited Ca2+ transients in human BOSC 23 cells transfected with chick α6 in combination with other chick nAChR neuronal subunits. Heterologously expressed α6 and β4 subunits form functional heteromeric nAChRs, which are permeable to Ca2+ ions and blocked by the nicotinic antagonist methyllycaconitine (10 μm). Likewise, ACh elicits measurable currents in cells transfected with α6 and β2. Hill analysis of the dose–response curves in cells transfected with α3, β4 and α6 cDNAs, suggests the assembly of functional α3β4α6 receptor, with an apparent affinity for ACh threefold lower than α3β4. Our results indicate that α6‐containing nAChRs assemble in heterologous expression systems and are probably present in retinal cells.