Research reportTetraethylammonium ions block the nicotinic cholinergic receptors of cochlear outer hair cells
Introduction
Outer hair cells (OHCs) of the mammalian cochlea are unique sensory cells which can contract and elongate at high frequencies in response to changes in membrane potential [7], [8], [18]. This electromechanical property of OHCs is believed to be at the origin of the cochlear amplifier, which gives the exquisite sensitivity and frequency resolution of the mammalian hearing organ. This electromotile process involves a voltage-sensitive intramembranous motor protein, which has recently been proposed to be prestin, a newly discovered protein displaying some similarities with pendrin and related sulfate/anion transport proteins [32]. This active mechanical property of OHCs is believed to be under the control of cholinergic efferent fibers arising from brainstem neurons located in the superior olivary complex [6], [17], [24]. Indeed, OHCs express nicotinic acetylcholine receptors (nAChRs) composed of nicotinic α9 [11], [19] and α10 subunits [12]. The OHC cholinergic receptors are ligand-gated channels permeable to Ca2+ ions [4], [15], [16]. The activation of these nAChRs results in a fast and strong hyperpolarization due to the activation of co-localized (SK-like) Ca2+-activated K+ (KCaACh) channels [10], [20], [25], [27].
The large cation tetraethylammonium (TEA) has been demonstrated to completely block at millimolar concentration the ACh-induced K+ current in mammalian cochlear OHCs [20], [31] and also the efferent inhibitory post-synaptic potential in turtle hair cells [3]. This mechanism of block by TEA has not yet been elucidated. TEA is well recognized to block Ca2+-activated K+ channels and in particular SK-type channels [22] but also nAChRs of various tissues [2], [23], [26], [28]. The aim of our study was to determine in isolated guinea pig OHCs, using the whole-cell patch-clamp technique, whether the primary site of block by TEA was at the nAChR itself or at the associated KCaACh channels.
Section snippets
Preparation of isolated cochlear outer hair cells
Guinea-pig outer hair cells were isolated as previously described [4], [9]. Briefly, young guinea-pigs (weight 200–300 g) were deeply anaesthetized with an intramuscular injection of 0.3 ml of a mixed solution of 2/3 ketamine hydrochlorate (50 mg/ml; Ketalar, Parke-Davis, France) and 1/3 xylazine (2%; Rompum, Bayer, Germany) and decapitated. Animal handling and sacrifice were done in accordance with the French Ministry of Agriculture in agreement with EEC regulation. The cochleae were separated
TEA does not block the cholinergic currents from the cytoplasmic side
In whole-cell patch-clamp recording condition, the presence of 10 mM TEA in the internal recording solution (solution 1) did not prevent the activation by 100 μM ACh of large K+ currents (Fig. 1) in four out of five cells tested. In the presence of intracellular TEA, the ACh-induced currents displayed a bell- (or N-) shaped I–V curve peaking near −30 mV and reversing near the Nernst equilibrium potential for K+ ions. These currents were comparable in amplitude and voltage-dependence to those
Discussion
The main finding of our study is that micro-molar concentration of extracellular TEA blocked the ACh-induced currents in OHCs by acting at the nicotinic receptor itself. The twofold differences in the IC50 (as well as the differences in Hill coefficients) measured at the ACh-induced K+ current compared to the one measured at the ionotropic current may easily be explained by the differences we have previously reported for the ACh EC50 of both currents [5]: 100 μM ACh is close to the EC50 for the
Acknowledgements
This work was also supported by grants from Fondation Pour la Recherche Médicale (Paris, France) and Conseil Régional d’Aquitaine.
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2022, Frontiers in Cellular Neuroscience
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Present address: Department of Physiology, CMU, 1 rue M. Servet, CH-1211 Geneve 4, Switzerland.