The α9α10 nicotinic acetylcholine receptor is permeable to and is modulated by divalent cations

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Abstract

The native cholinergic receptor that mediates synaptic transmission between olivocochlear fibers and outer hair cells of the cochlea is permeable to Ca2+ and is thought to be composed of both the α9 and the α10 cholinergic nicotinic subunits. The aim of the present work was to study the permeability of the recombinant α9α10 nicotinic acetylcholine receptor to Ca2+, Ba2+ and Mg2+ and its modulation by these divalent cations. Experiments were performed, by the two-electrode voltage-clamp technique, in Xenopus laevis oocytes injected with α9 and α10 cRNA. The relative divalent to monovalent cation permeability was high (∼10) for Ca2+, Ba2+ and Mg2+. Currents evoked by acetylcholine (ACh) were potentiated by either Ca2+ or Ba2+ up to 500 μM but were blocked by higher concentrations of these cations. Potentiation by Ca2+ was voltage-independent, whereas blockage was stronger at hyperpolarized than at depolarized potentials. Mg2+ did not potentiate but it blocked ACh-evoked currents (IC50=0.38 mM). In the absence of Ca2+, the EC50 for ACh was higher (48 μM) than that obtained with 1.8 mM Ca2+ (14.3 μM), suggesting that potentiation by Ca2+ involves changes in the apparent affinity of the α9α10 receptor for ACh.

Introduction

In the vertebrate inner ear, hair cells of the cochlea have the ability to detect sound stimuli and transduce them into electrical signals. In mammals there are two types of hair cells involved in this process, inner hair cells (IHCs) and outer hair cells (OHCs). IHCs are the primary acoustic transducers and receive most of the afferent innervation while OHCs are thought to be involved in sound amplification and receive a prominent efferent innervation from the olivary complex in the brainstem (for reviews see Fuchs, 1996, Hudspeth, 1989, Dallos, 1996). It is hypothesized that OHCs, by virtue of their voltage-driven length changes, feed back mechanical force to the cochlear partition, enhancing its sensitivity and frequency selectivity (see Holley, 1996).

At the synapse between the efferent fibers and OHCs, a non-classical ionotropic cholinergic receptor allows Ca2+ entry to the cell, activating a Ca2+-sensitive K+ current (IKCa) that hyperpolarizes the plasma membrane (Blanchet et al., 1996, Doi and Ohmori, 1993, Dulon et al., 1998, Erostegui et al., 1994a, Erostegui et al., 1994b, Evans, 1996, Housley and Ashmore, 1991, Nenov et al., 1996, Oliver et al., 2000). The physiological consequence of this effect to the mammalian inner ear is a reduction in sensitivity due to suppression of basilar membrane motion, thereby altering the dynamic range of hearing (for review see Guinan, 1996).

The α9 nicotinic acetylcholine subunit is a main component of the cholinergic receptor that mediates synaptic transmission between efferent olivocochlear fibers and OHCs (Elgoyhen et al., 1994, Glowatzki et al., 1995, Morley et al., 1998, Park et al., 1997, Vetter et al., 1999). Expression of α9 cRNA in Xenopus laevis oocytes results in the formation of a homomeric receptor–channel complex with pharmacological properties that greatly differ from other cloned nicotinic acetylcholine receptors (nAChR) (Elgoyhen et al., 1994, Rothlin et al., 1999, Verbitsky et al., 2000), but which are almost identical to those of the cholinergic receptor present at the base of the OHCs (Chen et al., 1996, Erostegui et al., 1994a, Erostegui et al., 1994b). Until the cloning of the α10 subunit (Elgoyhen et al., 2001), our working hypothesis was that the native OHC receptor was a homopentamer composed of α9 subunits. However, there were some biophysical characteristics of the recombinant homomeric α9 receptor that differed from those reported for the native OHC receptor, namely, its Ca2+ sensitivity, its current–voltage relationship and its desensitization kinetics (Blanchet et al., 1996, Dulon and Lenoir, 1996, Evans, 1996, Katz et al., 2000, McNiven et al., 1996). The cloned α10 subunit is not able to form a homomeric functional channel, however, the expression of α9 together with α10 results in the formation of a heteromeric receptor with pharmacological and functional properties very closely resembling those described for the vertebrate native cholinergic receptor (Elgoyhen et al., 2001). The stoichiometry of this heteromeric receptor is so far unknown and its estimation would imply performing site-directed mutagenesis on regions of both the α9 and α10 subunits that could significantly alter channel properties (see Cooper et al., 1991).

The α9 and α10 subunits form part of the nAChR family of ligand-gated ion channels, which includes receptors that are distributed throughout the nervous system and at the neuromuscular junction. Muscle and neuronal nAChRs are permeable to monovalent cations like Na+ and K+ and also to Ca2+ and other divalent cations (Bertrand et al., 1993, Decker and Dani, 1990, Mulle et al., 1992a, Mulle et al., 1992b, Séguéla et al., 1993, Vernino et al., 1992, Vernino et al., 1994). Neuronal nAChRs have a high Ca2+ permeability and agonist-evoked currents through these receptors are potentiated by Ca2+ (Galzi et al., 1996, Mulle et al., 1992a, Mulle et al., 1992b, Vernino et al., 1992). The recombinant homomeric α9 receptor is, like the neuronal α7 receptor (Séguéla et al., 1993), highly permeable to Ca2+ (Katz et al., 2000). However, acetylcholine (ACh)-evoked currents through the α9 receptor are not potentiated but strongly blocked by this cation (IC50 100 μM) in a voltage-dependent manner (Katz et al., 2000). Preliminary studies have suggested that the recombinant α9α10 receptor is permeable to Ca2+ and that it is tightly modulated by divalent cations (Elgoyhen et al., 2001).

Since there is strong evidence supporting the hypothesis that the functional native cholinergic receptor present at the base of OHCs is composed of both α9 and α10 subunits and given the key role Ca2+ plays at that sensory inhibitory synapse, the aim of the present study was to perform an extensive characterization of the permeability to and the modulation by Ca2+ of the recombinant α9α10 nAChR. This work provides evidence that the α9α10 receptor is highly permeable to Ca2+ and that it is also potentiated and blocked by physiological external Ca2+ concentrations through different mechanisms.

Section snippets

Expression of the α9α10 receptor in X. laevis oocytes and electrophysiological procedures

Full length rat α9 and α10 cDNAs constructed in a modified pGEMHE vector suitable for X. laevis oocyte expression studies were used as described previously (Elgoyhen et al., 2001). cRNA was synthesized using the mMessage mMachine T7 transcription kit (Ambion, Austin, TX, USA) with plasmid linearized with NheI. The isolation and maintenance of oocytes was carried out as described previously (Elgoyhen et al., 1994). Briefly, oocytes were surgically removed from the ovaries of X. laevis frogs

Permeability of the recombinant α9α10 nAChR to divalent cations

Ionic currents through the α9α10 receptor were studied in X. laevis oocytes that had been injected with a mixture of α9 and α10 cRNAs (molar ratio 1:1). The ability of ACh to activate the native IClCa in oocytes expressing recombinant nAChRs can be used to evaluate whether the expressed receptor is permeable to Ca2+. As we have previously shown (Elgoyhen et al., 2001), currents elicited by bath application of 100 μM ACh to α9α10-expressing oocytes superfused with NFS (1.8 mM Ca2+) were reduced

Discussion

In this work we report three major functional characteristics of the recombinant α9α10 nAChR receptor. First, it is highly permeable to Ca2+ and also to other divalent cations like Ba2+ and Mg2+. Second, it is potentiated and blocked, through different mechanisms, by external Ca2+ in the physiological range. Third, both Ba2+ and Mg2+ are able to block this receptor, whereas only Ba2+ is able to substitute for Ca2+ in its potentiating effect.

Acknowledgements

This work was supported by an International Research Scholar grant from the Howard Hughes Medical Institute, a grant from Agencia Nacional de Promociones Cientı́ficas y Técnicas Argentina and the Research Fellowship Ramón Carrillo-Arturo Oñativia (Argentina) to A.B.E.

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