Effects of Quinine, Quinidine, and Chloroquine on 9 10 Nicotinic Cholinergic Receptors

In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on 9 10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in 9 10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 0.39 M) quinine (IC50 0.97 M) quinidine (IC50 1.37 M). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 M, 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 [H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 M 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 9 10 nAChRs. The present results add to the pharmacological characterization of 9 10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds. Quinoline derivatives such as quinine, quinidine, and chloroquine are well known for their use in the treatment of malaria. Their side effects on the auditory system have long been recognized and include reversible (but sometimes permanent) sensorineural hearing loss, tinnitus, and vertigo (Jung et al., 1993). The mechanism of ototoxicity may involve different levels of the auditory system (Eggermont and Kenmochi, 1998; Jarboe and Hallworth, 1999). However, there is considerable evidence showing that the auditory periphery is the primary location underlying the reversible hearing loss induced by quinine (Puel et al., 1990; Lin et al., 1998). Perfusion of quinine into the perilymphatic space of guinea pig cochlea can result in a reduction of the compound action potential, cochlear microphonic, and summating potential (Puel et al., 1990). In addition, it can affect the electromotility of outer hair cells (Zheng et al., 2001). Moreover, it can inhibit the K current of outer hair cells and both the K and Na currents of the spiral ganglion cells (Lin et al., 1998), thus indicating a variety of effects of this compound on different ion channels. It has been reported that quinine and quinidine can also block acetylcholine (ACh)-induced K currents in outer hair cells and influence the effect of ACh on the compound action potential, suggesting a putative effect on the olivocochlear efferent system physiology (Daigneault et al., 1970; Yamamoto et al., 1997). Pharmacological and biophysical studies performed with the native cholinergic receptors present in mammalian and chicken hair cells (Fuchs, 1996) and cellular localization data (Elgoyhen et al., 1994, 2001; Lustig et al., 2001; Sgard et al., 2002) strongly suggest that the native receptor present at the efferent cholinergic olivocochlear-outer and developing inner hair cell synapse is asThis work was supported by an International Research Scholar grant from the Howard Hughes Medical Institute, the Agencia Nacional de Promoción Cientı́fica y Tecnológica, the University of Buenos Aires (to A.B.E.), and the Welcome Trust (to N.S.M.) J.A.B. is supported by an undergraduate fellowship from the University of Buenos Aires, P.V.P. and M.E.G.-C. by a CONICET predoctoral fellowship, J.T. by a fellowship from ANPCyT, and S.K. by a Welcome Trust studentship in Neuroscience. 1 Current address: The Salk Institute for Biological Studies, La Jolla, California. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.105.014431. ABBREVIATIONS: ACh, acetylcholine; nAChR, nicotinic acetylcholine receptors; BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetic acid; I-V, current-voltage relationship; MLA, methyllycaconitine; nAChR, nicotinic acetylcholine receptor; 5-HT, 5-hydroxytryptamine. 0026-895X/05/6803-822–829$20.00 MOLECULAR PHARMACOLOGY Vol. 68, No. 3 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 14431/3048551 Mol Pharmacol 68:822–829, 2005 Printed in U.S.A. 822 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from sembled from both the 9 and 10 nicotinic subunits (Elgoyhen et al., 1994, 2001). Thus, 9 10-containing nicotinic acetylcholine receptors (nAChRs) might be targets of the effects of quinoline compounds within the auditory system. In this regard, aminoglycosides, ototoxic drugs not related in structure to the quinoline compounds, have been reported as blockers of the 9 10 nAChRs (Rothlin et al., 2000), pinpointing this receptor as a possible site of interaction of ototoxic drugs. Moreover, the interaction of quinine concentrations greater than 50 M with nAChRs has been reported for receptors present at the neuromuscular junction, in which it produces long-lived open-channel as well as a closed-channel block and can normalize the open duration of channel events in the slow-channel congenital myasthenic syndrome (Sieb et al., 1996; Fukudome et al., 1998). We have examined the effects of the quinoline derivatives quinine, quinidine, and chloroquine (Fig. 1) on recombinant 9 10 nAChRs, reconstituted in Xenopus laevis oocytes. We show evidence that these compounds block the 9 10 nAChRs. The underlying mechanisms range from competitive in the case of chloroquine to mixed competitive and noncompetitive in the case of quinine. Moreover, we demonstrate that chloroquine blocks the native 9 10-containing nAChRs of inner hair cells. The present results indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a direct target of these ototoxic antimalarial compounds. Materials and Methods Expression of Recombinant Receptors in X. laevis Oocytes. For expression studies, 9 and 10 rat nAChR subunits were subcloned into a modified pGEMHE vector (Liman et al., 1992). Capped cRNAs were in vitro-transcribed from linearized plasmid DNA templates using the mMessage mMachine T7 Transcription Kit (Ambion, Austin, TX). The maintenance of X. laevis and the preparation and cRNA injection of stage V and VI oocytes have been described in detail elsewhere (Katz et al., 2000). Oocytes were typically injected with 50 nl of RNase-free water containing 0.01 to 1.0 ng of cRNAs (at a 1:1 M ratio) and maintained in Barth’s solution at 17°C. Electrophysiological recordings were performed 2 to 6 days after cRNA injection under two-electrode voltage-clamp with a Geneclamp 500 amplifier (Axon Instruments Inc., Union City, CA). Both voltage and current electrodes were filled with 3 M KCl and had resistances of 1 to 2 M . Data acquisition was performed using a Digidata 1200 and the pClamp 7.0 software (Axon Instruments). Data were analyzed using ClampFit from the pClamp 6.1 software. During electrophysiological recordings, oocytes were continuously superfused ( 10 ml/min) with normal frog saline composed of 115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES buffer, pH 7.2. Unless otherwise indicated, the membrane potential was clamped to 70 mV. Drugs were applied in the perfusion solution of the oocyte chamber. To minimize activation of the endogenous Ca -sensitive chloride current (Elgoyhen et al., 2001), all experiments were performed in oocytes incubated with the Ca chelator BAPTA-acetoxymethyl ester (100 M) for 3 to 4 h before electrophysiological recordings. Concentration-response curves were normalized to the maximal agonist response in each oocyte. For the inhibition curves, antagonists were added to the perfusion solution for 2 min before the addition of 10 M ACh and then were coapplied with this agonist. Responses were referred to as a percentage of the response to ACh. The mean and S.E.M. values for peak current responses are represented. Agonist concentration-response curves were iteratively fitted with the equation I/Imax A/ A EC50) (1) where I is the peak inward current evoked by agonist at concentration A, Imax is the current evoked by the concentration of agonist eliciting a maximal response, EC50 is the concentration of agonist inducing half-maximal current response, and n is the Hill coefficient. An equation of the same form was used to analyze the concentrationdependence of antagonist-induced blockage. The parameters derived were the concentration of antagonist producing a 50% block of the control response to ACh (IC50) and the associated interaction coefficient (n). Analysis of competitive inhibition was performed by the Schild plot (Arunlakshana and Schild, 1959), with the following