RESUMO
Humans can recognize differences in sound intensity of up to 6 orders of magnitude. However, it is not clear how this is achieved and what enables our auditory systems to encode such a gradient. Özçete & Moser (2021) report in this issue that the key to this lies in the synaptic heterogeneity within individual sensory cells in the inner ear.
Assuntos
Acústica , HumanosRESUMO
In this study we report the effects of neramexane, a novel amino-alkyl-cyclohexane derivative that is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, on recombinant rat alpha9alpha10 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. We compared its effects with those of memantine, a well-studied pore blocker of NMDA receptors, currently used in therapeutics for the treatment of Alzheimer's disease. Our results indicate that both compounds block acetylcholine-evoked responses at micromolar concentrations with a rank order of potency of neramexane>memantine, P<0.05. Block by neramexane of acetylcholine responses was not overcome at high concentrations of the agonist, indicative of a non-competitive inhibition. The lack of interaction of neramexane with the ligand binding domain was confirmed by radioligand binding experiments in transfected tsA201 cells. Moreover, block did not involve an increase in desensitization kinetics, it was independent of the resting potential of the membrane at low concentrations of neramexane and slightly voltage-dependent at concentrations higher than 1 microM. Finally, clinically-relevant concentrations of neramexane blocked native alpha9alpha10-containing nicotinic acetylcholine receptors of rat inner hair cells, thus demonstrating a possible in vivo relevance in potentially unexplored therapeutic areas.
Assuntos
Ciclopentanos/farmacologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Linhagem Celular , Células Ciliadas Auditivas Internas/fisiologia , Humanos , Memantina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
The alpha9 and alpha10 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 alpha10 subunit serves as a "structural" component leading to heteromeric alpha9alpha10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant alpha9alpha10 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 alpha9 or alpha10 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 alpha9 and alpha10 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 alpha9alpha10 receptor is a pentamer with a (alpha9)2(alpha10)3 stoichiometry.
Assuntos
Subunidades Proteicas/química , Receptores Nicotínicos/química , Acetilcolina/farmacologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Feminino , Matemática , Oócitos , Concentração Osmolar , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina , Valina , Xenopus laevisRESUMO
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.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Quinidina/farmacologia , Quinina/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Antimaláricos/toxicidade , Cloroquina/toxicidade , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Quinidina/toxicidade , Quinina/toxicidade , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/efeitos dos fármacos , Xenopus laevisRESUMO
We have performed a systematic mutagenesis of three hydrophobic rings (17', 13' and 9') within transmembrane region (TM) 2 of the alpha9alpha10 nicotinic cholinergic receptor (nAChR) to a hydrophilic (threonine) residue and compared the properties of mutant receptors reconstituted in Xenopus laevis oocytes. Phenotypic changes in alpha9alpha10 mutant receptors were evidenced by a decrease in the desensitization rate, an increase in both the EC(50) for ACh as well as the efficacy of partial agonists and the reduction of the allosteric modulation by extracellular Ca(2+). Mutated receptors exhibited spontaneous openings and, at the single-channel level, an increased apparent mean open time with no major changes in channel conductance, thus suggesting an increase in gating of the channel as the underlying mechanism. Overall, the degrees of the phenotypes of mutant receptors were more overt in the case of the centrally located V13'T mutant. Based on the atomic model of the pore of the electric organ of the Torpedo ray, we can propose that the interactions of side chains at positions 13' and 9' are key ones in creating an energetic barrier to ion permeation. In spite of the fact that the roles of the TM2 residues are mostly conserved in the distant alpha9alpha10 member of the nAChR family, their mechanistic contributions to channel gating show significant differences when compared to other nAChRs. These differences might be originated from slight differential intramolecular rearrangements during gating for the different receptors and might lead each nAChR to be in tune with their physiological roles.
Assuntos
Ativação do Canal Iônico/genética , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Acetilcolina/farmacologia , Regulação Alostérica , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transfecção , Xenopus laevisRESUMO
Studies of the electrophysiological response to acetylcholine (ACh) in mammalian outer hair cells (OHCs) are hindered by the presence of a large potassium current, I(K,n), most likely mediated by channels containing the KCNQ4 subunit. Since I(K,n) can be blocked by linopirdine, cholinergic effects might be better revealed in the presence of this compound. The aim of the present work was to study the effects of linopirdine on the ACh-evoked responses through alpha9alpha10-containing native and recombinant nicotinic cholinergic receptors. Responses to ACh were blocked by linopirdine in both OHCs and inner hair cells (IHCs) of rats at postnatal days 21-27 (OHCs) and 9-11 (IHCs). In addition, linopirdine blocked responses of recombinant alpha9alpha10 nicotinic cholinergic receptors (nAChRs) in a concentration-dependent manner with an IC(50) of 5.2 microM. Block by linopirdine was readily reversible, voltage independent, and surmountable at high concentrations of ACh, thus suggestive of a competitive type of interaction with the receptor. The present results contribute to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that linopirdine should be used with caution when analyzing the cholinergic sensitivity of cochlear hair cells.
Assuntos
Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/fisiologia , Indóis/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Subunidades Proteicas/metabolismo , Piridinas/farmacologia , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Expressão Gênica , Potenciais da Membrana/efeitos dos fármacos , Oócitos/fisiologia , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
In the mature cochlea, inner hair cells (IHCs) transduce acoustic signals into receptor potentials, communicating to the brain by synaptic contacts with afferent fibers. Before the onset of hearing, a transient efferent innervation is found on IHCs, mediated by a nicotinic cholinergic receptor that may contain both alpha9 and alpha10 subunits. Calcium influx through that receptor activates calcium-dependent (SK2-containing) potassium channels. This inhibitory synapse is thought to disappear after the onset of hearing [after postnatal day 12 (P12)]. We documented this developmental transition using whole-cell recordings from IHCs in apical turns of the rat organ of Corti. Acetylcholine elicited ionic currents in 88-100% of IHCs between P3 and P14, but in only 1 of 11 IHCs at P16-P22. Potassium depolarization of efferent terminals caused IPSCs in 67% of IHCs at P3, in 100% at P7-P9, in 93% at P10-P12, but in only 40% at P13-P14 and in none of the IHCs tested between P16 and P22. Earlier work had shown by in situ hybridization that alpha9 mRNA is expressed in adult IHCs but that alpha10 mRNA disappears after the onset of hearing. In the present study, antibodies to alpha10 and to the associated calcium-dependent (SK2) potassium channel showed a similar developmental loss. The correlated expression of these gene products with functional innervation suggests that Alpha10 and SK2, but not Alpha9, are regulated by synaptic activity. Furthermore, this developmental knock-out of alpha10, but not alpha9, supports the hypothesis that functional nicotinic acetylcholine receptors in hair cells are heteromers containing both these subunits.