Paola V. Plazas

Stoichiometry of the α9α10 nicotinic cholinergic receptor

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The α10 subunit serves as a “structural” component leading to heteromeric α9α10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant α9α10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner α9 or α10 subunit bearing a reporter mutation of a valine to threonine at position 13′ of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of α9 and α10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17′ of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant α9α10 receptor is a pentamer with a (α9)2(α10)3 stoichiometry.

A Novel α-Conotoxin, PeIA, Cloned from Conus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of α9 and α10 overlaps with that of α7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin α-conotoxin PeIA that discriminates between α9α10 and α7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. α-Conotoxin PeIA displayed a 260-fold higher selectivity for α-bungarotoxin-sensitive α9α10 nAChRs compared with α-bungarotoxin-sensitive α7 receptors. The IC50 of the toxin was 6.9 ± 0.5 nm and 4.4 ± 0.5 nm for recombinant α9α10 and wild-type hair cell nAChRs, respectively. α-Conotoxin PeIA bears high resemblance to α-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither α-conotoxin MII nor α-conotoxin GIC at concentrations of 10 μm blocked acetylcholine responses elicited in Xenopus oocytes injected with the α9 and α10 subunits. Among neuronal non-α-bungarotoxin-sensitive receptors, α-conotoxin PeIA was also active at α3β2 receptors and chimeric α6/α3β2β3 receptors. α-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through α9α10 or α7 nAChRs in those tissues where both receptors are expressed.

A novel alpha-conotoxin, PeIA, cloned from Conus pergrandis, discriminates between rat alpha9alpha10 and alpha7 nicotinic cholinergic receptors.

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of α9 and α10 overlaps with that of α7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin α-conotoxin PeIA that discriminates between α9α10 and α7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. α-Conotoxin PeIA displayed a 260-fold higher selectivity for α-bungarotoxin-sensitive α9α10 nAChRs compared with α-bungarotoxin-sensitive α7 receptors. The IC50 of the toxin was 6.9 ± 0.5 nm and 4.4 ± 0.5 nm for recombinant α9α10 and wild-type hair cell nAChRs, respectively. α-Conotoxin PeIA bears high resemblance to α-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither α-conotoxin MII nor α-conotoxin GIC at concentrations of 10 μm blocked acetylcholine responses elicited in Xenopus oocytes injected with the α9 and α10 subunits. Among neuronal non-α-bungarotoxin-sensitive receptors, α-conotoxin PeIA was also active at α3β2 receptors and chimeric α6/α3β2β3 receptors. α-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through α9α10 or α7 nAChRs in those tissues where both receptors are expressed.

Stoichiometry of the alpha9alpha10 nicotinic cholinergic receptor.

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The α10 subunit serves as a “structural” component leading to heteromeric α9α10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant α9α10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner α9 or α10 subunit bearing a reporter mutation of a valine to threonine at position 13′ of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of α9 and α10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17′ of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant α9α10 receptor is a pentamer with a (α9)2(α10)3 stoichiometry.

The α1 subunit of nicotinic acetylcholine receptors in the inner ear: transcriptional regulation by ATOH1 and co-expression with the γ subunit in hair cells

Acetylcholine is a key neurotransmitter of the inner ear efferent system. In this study, we identify two novel nAChR subunits in the inner ear: α1 and γ, encoded by Chrna1 and Chrng, respectively. In situ hybridization shows that the messages of these two subunits are present in vestibular and cochlear hair cells during early development. Chrna1 and Chrng expression begin at embryonic stage E13.5 in the vestibular system and E17.5 in the organ of Corti. Chrna1 message continues through P7, whereas Chrng is undetectable at post‐natal stage P6. The α1 and γ subunits are known as muscle‐type nAChR subunits and are surprisingly expressed in hair cells which are sensory‐neural cells. We also show that ATOH1/MATH1, a transcription factor essential for hair cell development, directly activates CHRNA1 transcription. Electrophoretic mobility‐shift assays and supershift assays showed that ATOH1/E47 heterodimers selectively bind on two E boxes located in the proximal promoter of CHRNA1. Thus, Chrna1 could be the first transcriptional target of ATOH1 in the inner ear. Co‐expression in Xenopus oocytes of the α1 subunit does not change the electrophysiological properties of the α9α10 receptor. We suggest that hair cells transiently express α1γ‐containing nAChRs in addition to α9α10, and that these may have a role during development of the inner ear innervation.

Differential contribution of subunit interfaces to α9α10 nicotinic acetylcholine receptor function

Nicotinic acetylcholine receptors can be assembled from either homomeric or heteromeric pentameric subunit combinations. At the interface of the extracellular domains of adjacent subunits lies the acetylcholine binding site, composed of a principal component provided by one subunit and a complementary component of the adjacent subunit. Compared with neuronal nicotinic acetylcholine cholinergic receptors (nAChRs) assembled from α and β subunits, the α9α10 receptor is an atypical member of the family. It is a heteromeric receptor composed only of α subunits. Whereas mammalian α9 subunits can form functional homomeric α9 receptors, α10 subunits do not generate functional channels when expressed heterologously. Hence, it has been proposed that α10 might serve as a structural subunit, much like a β subunit of heteromeric nAChRs, providing only complementary components to the agonist binding site. Here, we have made use of site-directed mutagenesis to examine the contribution of subunit interface domains to α9α10 receptors by a combination of electrophysiological and radioligand binding studies. Characterization of receptors containing Y190T mutations revealed unexpectedly that both α9 and α10 subunits equally contribute to the principal components of the α9α10 nAChR. In addition, we have shown that the introduction of a W55T mutation impairs receptor binding and function in the rat α9 subunit but not in the α10 subunit, indicating that the contribution of α9 and α10 subunits to complementary components of the ligand-binding site is nonequivalent. We conclude that this asymmetry, which is supported by molecular docking studies, results from adaptive amino acid changes acquired only during the evolution of mammalian α10 subunits.

Properties of mutated murine α4β2 nicotinic receptors linked to partial epilepsy

We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.

A Novel α-Conotoxin, PeIA, Cloned fromConus pergrandis, Discriminates between Rat α9α10 and α7 Nicotinic Cholinergic Receptors

The α9 and α10 nicotinic cholinergic subunits assemble to form the receptor believed to mediate synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea, one of the few examples of postsynaptic function for a non-muscle nicotinic acetylcholine receptor (nAChR). However, it has been suggested that the expression profile of α9 and α10 overlaps with that of α7 in the cochlea and in sites such as dorsal root ganglion neurons, peripheral blood lymphocytes, developing thymocytes, and skin. We now report the cloning, total synthesis, and characterization of a novel toxin α-conotoxin PeIA that discriminates between α9α10 and α7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. α-Conotoxin PeIA displayed a 260-fold higher selectivity for α-bungarotoxin-sensitive α9α10 nAChRs compared with α-bungarotoxin-sensitive α7 receptors. The IC50 of the toxin was 6.9 ± 0.5 nm and 4.4 ± 0.5 nm for recombinant α9α10 and wild-type hair cell nAChRs, respectively. α-Conotoxin PeIA bears high resemblance to α-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither α-conotoxin MII nor α-conotoxin GIC at concentrations of 10 μm blocked acetylcholine responses elicited in Xenopus oocytes injected with the α9 and α10 subunits. Among neuronal non-α-bungarotoxin-sensitive receptors, α-conotoxin PeIA was also active at α3β2 receptors and chimeric α6/α3β2β3 receptors. α-Conotoxin PeIA represents a novel probe to differentiate responses mediated either through α9α10 or α7 nAChRs in those tissues where both receptors are expressed.