Alpha9alpha10 nAChRs Research Articles

Selective inhibition of human heteromeric α9α10 nicotinic acetylcholine receptors at a low agonist concentration by low concentrations of ototoxic organic solvents

Ethylbenzene and para-xylene (p-xylene), but not the chemically closely related organic solvents ortho-xylene (o-xylene) and meta-xylene (m-xylene), are known to cause ototoxicity and irreversible hearing loss, though the underlying mechanisms are still unknown. In this study, effects of ethylbenzene and of p-, o-, and m-xylene on human heteromeric alpha9alpha10 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes were investigated using the two-electrode voltage clamp technique. ACh dose-dependently evoked an alpha9alpha10 nAChR-mediated ion current with an EC(50) of 137 microM. When ACh is applied at a low concentration (10 microM), the nAChR-mediated ion current is inhibited by a low concentration (10 microM) of ethylbenzene and p-xylene, but not by the same concentration of the non-ototoxic solvents. At a high solvent concentration (300 microM), all solvents cause inhibition of the ion currents evoked by 10 microM ACh. Ion currents evoked by a near maximum-effective concentration ACh (1mM) are inhibited by the selected organic solvents only at 300 microM. These results demonstrate that low concentrations of the known ototoxic solvents ethylbenzene and p-xylene inhibit alpha9alpha10 nAChR-mediated ion currents, whereas the structurally related, non-ototoxic solvents m-xylene and o-xylene do not, indicating that the alpha9alpha10 nAChR is a potential target for solvent-induced ototoxicity.

Synthesis and Characterization of125I-α-Conotoxin ArIB[V11L;V16A], a Selective α7 Nicotinic Acetylcholine Receptor Antagonist

The alpha7 nicotinic acetylcholine receptors (nAChRs) are widely expressed both in the central nervous system (CNS) and periphery. In the CNS, 125I-alpha-bungarotoxin is commonly used to identify alpha7 nAChRs specifically. However, alpha-bungarotoxin also interacts potently with alpha1* and alpha9alpha10 nAChRs, two receptor subtypes in peripheral tissues that are colocalized with the alpha7 subtype. [3H]Methyllycaconitine is also frequently used as an alpha7-selective antagonist, but it has significant affinity for alpha6* and alpha9alpha10 nAChR subtypes. In this study, we have developed a highly alpha7-selective alpha-conotoxin radioligand by iodination of a naturally occurring histidine. Both mono- and diiodo derivatives were generated and purified (specific activities were 2200 and 4400 Ci mmol(-1), respectively). The properties of the mono- and diiodo derivatives were very similar to each other, but the diiodo was less stable. For monoidodo peptide, saturation binding to mouse hippocampal membranes demonstrated a K(d) value of 1.15 +/- 0.13 nM, similar to that of 125I-alpha-bungarotoxin in the same preparations (0.52 +/- 0.16 nM). Association and dissociation kinetics were relatively rapid (k(obs) for association at 1 nM was 0.027 +/- 0.007 min(-1); k(off) = 0.020 +/- 0.001 min(-1)). Selectivity was confirmed with autoradiography using alpha7-null mutant tissue: specific binding was abolished in all regions of alpha7(-/-) brains, whereas wild-type mice expressed high levels of labeling and low nonspecific binding. 125I-alpha-conotoxin ArIB[V11L; V16A] should prove useful where alpha7 nAChRs are coexpressed with other subtypes that are also labeled by existing ligands. Furthermore, true equilibrium binding experiments could be performed on alpha7 nAChRs, something that is impossible with 125I-alpha-bungarotoxin.

Are α9α10 Nicotinic Acetylcholine Receptors a Pain Target for α-Conotoxins?

The synthetic alpha-conotoxin Vc1.1 is a small disulfide bonded peptide currently in development as a treatment for neuropathic pain. Unlike Vc1.1, the native post-translationally modified peptide vc1a does not act as an analgesic in vivo in rat models of neuropathic pain. It has recently been proposed that the primary target of Vc1.1 is the alpha9alpha10 nicotinic acetylcholine receptor (nAChR). We show that Vc1.1 and its post-translationally modified analogs vc1a, [P6O]Vc1.1, and [E14gamma]Vc1.1 are equally potent at inhibiting ACh-evoked currents mediated by alpha9alpha10 nAChRs. This suggests that alpha9alpha10 nAChRs are unlikely to be the molecular mechanism or therapeutic target of Vc1.1 for the treatment of neuropathic pain.

Ryanodine is a Positive Modulator of Acetylcholine Receptor Gating in Cochlear Hair Cells

The efferent synaptic specialization of hair cells includes a near-membrane synaptic cistern, whose presence suggests a role for internal calcium stores in cholinergic inhibition. Calcium release channels from internal stores include 'ryanodine receptors', whose participation is usually demonstrated by sensitivity to the eponymous plant alkaloid, ryanodine. However, use of this and other store-active compounds on hair cells could be confounded by the unusual pharmacology of the alpha9alpha10-containing hair cell nicotinic cholinergic receptor (nAChR), which has been shown to be antagonized by a broad spectrum of compounds. Surprisingly, we found that ryanodine, rather than antagonizing, is a positive modulator of the alpha9alpha10 nAChR expressed in Xenopus oocytes, the first such compound to be found. The effect of ryanodine was to increase the apparent affinity and efficacy for acetylcholine (ACh). Correspondingly, ACh-evoked currents through the isolated cholinergic receptors of inner hair cells in excised mouse cochleas were approximately doubled by 200 microM ryanodine, a concentration that inhibits gating of the ryanodine receptor itself. This unusual positive modulation was not unique to the mammalian receptor. The response to ACh of chicken 'short' hair cells likewise was enhanced in the presence of 100 microM ryanodine. This facilitatory effect on current through the AChR could enhance brief ( approximately 1 s) activation of associated calcium-dependent K(+) (SK) channels in both chicken short hair cells and rat outer hair cells. This novel effect of ryanodine provides new opportunities for the design of compounds that potentiate alpha9alpha10-mediated responses and for potential inner ear therapeutics based on this interaction.

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

The alpha9 and alpha10 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 alpha9 and alpha10 overlaps with that of alpha7 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 alpha-conotoxin PeIA that discriminates between alpha9alpha10 and alpha7 nAChRs. This is the first toxin to be identified from Conus pergrandis, a species found in deep waters of the Western Pacific. Alpha-conotoxin PeIA displayed a 260-fold higher selectivity for alpha-bungarotoxin-sensitive alpha9alpha10 nAChRs compared with alpha-bungarotoxin-sensitive alpha7 receptors. The IC50 of the toxin was 6.9 +/- 0.5 nM and 4.4 +/- 0.5 nM for recombinant alpha9alpha10 and wild-type hair cell nAChRs, respectively. Alpha-conotoxin PeIA bears high resemblance to alpha-conotoxins MII and GIC isolated from Conus magus and Conus geographus, respectively. However, neither alpha-conotoxin MII nor alpha-conotoxin GIC at concentrations of 10 microM blocked acetylcholine responses elicited in Xenopus oocytes injected with the alpha9 and alpha10 subunits. Among neuronal non-alpha-bungarotoxin-sensitive receptors, alpha-conotoxin PeIA was also active at alpha3beta2 receptors and chimeric alpha6/alpha3beta2beta3 receptors. Alpha-conotoxin PeIA represents a novel probe to differentiate responses mediated either through alpha9alpha10 or alpha7 nAChRs in those tissues where both receptors are expressed.

Biophysical and pharmacological characterization of nicotinic cholinergic receptors in rat cochlear inner hair cells

Before the onset of hearing, a transient efferent innervation is found on inner hair cells (IHCs). This synapse is inhibitory and mediated by a nicotinic cholinergic receptor (nAChR) probably formed by the alpha9 and alpha10 subunits. We analysed the pharmacological and biophysical characteristics of the native nAChR using whole-cell recordings from IHCs in acutely excised apical turns of the rat organ of Corti. Nicotine did not activate but rather blocked the acetylcholine (ACh)-evoked currents with an IC50 of 1 +/- 0.1 microM. Antagonists of non-cholinergic receptors such as strychnine, bicuculline and ICS-205930 blocked ACh-evoked responses with an IC50 of 8.6 +/- 0.8 nM, 59 +/- 4 nM and 0.30 +/- 0.02 microM, respectively. The IHC nAChR was both permeable to (P(Ca)/P(Na) = 8 +/- 0.9) and modulated by external Ca2+. ACh-evoked currents were potentiated by Ca2+ up to 500 microM but were reduced by higher concentrations of this cation. Ba2+ mimicked the effects of Ca2+ whereas Mg2+ only blocked these currents. In addition, elevation of extracellular Ca2+ reduced the amplitude of spontaneous synaptic currents without affecting their time course. The receptor had an EC50 for ACh of 60.7 +/- 2.8 microM in 0.5 mM Ca2+. In the absence of Ca2+, the EC50 for ACh increased, suggesting that potentiation by Ca2+ involves changes in the apparent affinity for the agonist. These pharmacological and biophysical characteristics of the IHC nAChR closely resemble those of the recombinant alpha9alpha10 nAChR, reinforcing the hypothesis that the functional nAChR at the olivocochlear efferent-IHC synapse is composed of both the alpha9 and alpha10 subunits.

Effects of Quinine, Quinidine, and Chloroquine on α9α10 Nicotinic Cholinergic Receptors

In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on alpha9alpha10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in alpha9alpha10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 = 0.39 microM) > quinine (IC50 = 0.97 microM) approximately quinidine (IC50= 1.37 microM). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 microM, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [3H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 microM for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with alpha9alpha10 nAChRs. The present results add to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.

Pharmacological properties of alpha 9 alpha 10 nicotinic acetylcholine receptors revealed by heterologous expression of subunit chimeras.

The nicotinic acetylcholine receptor (nAChR) alpha9 and alpha10 subunits are expressed primarily within hair cells of the inner ear and have been implicated in auditory processing. Although functional recombinant nAChRs generated by the coexpression of alpha9 and alpha10 in Xenopus laevis oocytes have been described previously, there have been no reports of the successful heterologous expression of alpha9alpha10 nAChRs in cultured cell lines. In this study, subunit chimeras (alpha9chi and alpha10chi) have been constructed that contain the extracellular, ligand binding domain of the alpha9 or alpha10 subunits fused to the C-terminal domain of the 5-hydroxytryptamine type 3A (5HT3A) subunit. Specific high-affinity binding of the nicotinic radioligand [3H]methyllycaconitine was detected in membrane preparations of mammalian cells transfected with alpha9chi or alpha10chi alone, but significantly higher levels of binding were detected when alpha9chi and alpha10chi were cotransfected, providing evidence of a requirement for coassembly of alpha9 and alpha10 for the efficient formation of a nicotinic binding site. The pharmacological profile of alpha9chialpha10chi receptors, determined by equilibrium radioligand binding studies, is broadly similar to that determined previously by electrophysiological studies conducted with native and recombinant alpha9alpha10 nAChRs. In agreement with evidence that alpha9alpha10 nAChRs exhibit an atypical pharmacological profile, we have identified specific high-affinity binding of several non-nicotinic ligands including strychnine (a glycine receptor antagonist), bicuculline (a GABAA receptor antagonist), and atropine (a muscarinic acetylcholine receptor antagonist). Results have also been compared with radioligand binding data conducted with a previously described alpha7/5HT3A (alpha7chi) subunit chimera.