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Ligand-binding properties of an unusual nicotinic acetylcholine receptor subtype on isolated outer hair cells from guinea pig cochlea
ELSEVIER
Neuroscience Letters 195 (1995) 64-68
N[UKgSCI[HC[ LETTERS
Ligand-binding properties of an unusual nicotinic acetylcholine receptor subtype on isolated outer hair cells from guinea pig cochlea G. Lawoko a,b, L. J~lebark,, E. H e i l b r o n n a,* aDepartment ~'Neurochemistry and Neurotoxicology, Stockholm University, S-106 91 Stockholm, Sweden bDepartment of Pharmacology, University ~Lund, Lund, Sweden
Received 30 March 1995;accepted 15 June 1995
Abstract Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [125I]ct-bungarotoxin revealed a KD of 62 _+2 nM, Bmax = 7.2 -+ 1.8 × 10 7 binding sites/OHC, and a slowly reversible dissociation rate constant, k_1 = 2.2_+ 0.01 × 10-4 min-l. L-[3H]Nicotine bound reversibly (estimated KD~ 230 nM and Bmax ~ 5 X 10 7) with kinetic rate constants of association kj = 6.2 _+0.06 × 10 4 min-I nM-1 and dissociation k_1 = 0.23 ± 0.003 min-1. [3H]Strychnine bound to OHC with a KD of 35 +_6 nM and Bma x = 2.6 + 0.5 × 10 7, and binding increased 3-4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > n M concentrations, of [3H]3quinuclidinyl benzilate (KD = 11.5 ± 5 nM; Bma x = 2.5 + 0.6 × 106) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 ± 0.5 and 6.5 ± 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.
Keywords: Auditory
system; Outer hair cell; Efferent synapse; Acetylcholine receptor; Radioligand binding; Nicotinic ligands; Strychnine; Muscarinic ligands
Two types of mechanoreceptors in the mammalian inner ear convert sound from mechanical to electrical signals. Inner hair cells are the primary transducers of incoming acoustic information in the cochlea, i.e. deflection of hair cell stereocilia regulates neurotransmitter release to afferents of the eighth cranial nerve. Outer hair cells (OHC) are innervated by medial olivocochlear efferent neurons and form a modulatory feedback mechanism by virtue of their electromotile properties, thereby affecting frequency selectivity and sensitivity in the organ of Corti [1]. A number of data indicate that acetylcholine (ACh) is the main neurotransmitter at the efferent synapse [9]. The crossed olivocochlear bundle (an efferent inhibitory tract of nerve fibers which forms synaptic junctions at the base of the scala tympani of mammalia) has been shown to be blocked by both cholinergic and noncholinergic drugs introduced into the medial efferent system. Distortion product otoacoustic emissions * Corresponding author, Tel.: +46 8 164269; Fax: +46 8 161371; E-mail: [email protected].
(DPOAEs), thought to reflect OHC activity and mediated by the efferent cholinergic system, were blocked by selective antagonists of nicotinic and muscarinic receptors [18] as well as by the glycine receptor (GIyR) antagonist strychnine, suggesting that these receptors may be present in the cochlea. Muscarinic AChR, although present at the efferent presynapse [4], have, however, not been demonstrated on OHC, thus block of transmitter release from the cholinergic presynapse was probably involved. The presence of GIyR at the efferent synapse was ruled out [5] as was the presence of strychnine binding sites on rat cochlea mAChR, since neither binding of the mAChR antagonist 3-quinuclidinyl benzilate (3-QNB) nor carbachol-activated inositol phosphate formation were inhibited by strychnine (1-50/~M) [3]. Results from molecular cloning of cochlear nAChR subunits have suggested that an a 5 (rat) [15] and/or a 7 (chick) [2] subunit is present. Furthermore, recent cloning of a novel ct subunit, a 9 [7], from olfactory epithelium, and subsequent amplification of rat cochlear cDNA (polymerase chain reaction) using a9-specific primers revealed ex-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95)11774-N
G. Lawoko et al. /Neuroscience Letters 195 (1995) 64-68
pression of this subunit gene in the inner ear. In situ hybridization analysis confirmed expression in the hair cell area. Previous binding studies in this laboratory have identified the guinea pig OHC AChR as primarily nicotinic, but not identical with the 'classical' AChR [21]. The present paper further characterizes this AChR and approximates the number of a-bungarotoxin (a-BTX), nicotine, strychnine and 3-QNB-binding sites/OHC. Binding of the muscarinic antagonist 3-QNB, observed at very high concentrations, was competitively reduced by other muscarinic antagonists (atropine and 4-diphenylacetoxy-Nmethylpiperidine methiodide; 4-DAMP) and the possibility of strychnine binding to sites other than extracellular nAChR domains is discussed. OHC were non-enzymatically isolated (to avoid receptor modification) from pigmented guinea pigs of either sex (Duncin Hartley, body weight 300-400 g) after anaesthesia with CO2 prior to decapitation [24]. In short, the temporal bones were taken out, stored in ice-cold buffer and the lateral wall of the bony cochlea removed under a dissection microscope. The organ of Corti with the modiolus bone was transferred to a balanced salt solution (125 mM NaCI, 5 mM KCI, 1.2 mM MgSO 4, 1.2 mM KH2PO4, 1.0mM CaCI 2, 25 mM Tris-HCl, pH 7.4, osmolarity 300___ 10mosm/l), also used for all binding assays. OHC were microdissected from all turns of the cochlea and separated from membranes and supporting cells (mostly Deiters cells) by gentle pipetting and transferring to a Petri dish. An assay utilizing release of cytosolic enzyme lactate dehydrogenase (LDH) [22] was used to check the integrity of isolated OHC; no LDH activity was detected outside the cells 1 h after incubation at 21°C. Binding assays (modified from Ref. [16]) were performed in Eppendorf tubes (silanized for a-BTX) at 21°C (pH 7.4), final incubation volume 500~1, containing 1600 _+ 200 OHC. After centrifugation (5 min, 4°C, 14 500 x g) the supernatant was discarded, pellets were washed three times with ice-cold buffer and radioactivity was counted, using a y-counter for [125I]a-BTX. For the tritiated ligands, pellets were washed once and then dissolved in 10% SDS before addition of 1.3 ml of scintillation fluid and counting in a liquid scintillation spectrometer. Addition of protease inhibitors (1 mM EDTA, 100pg/ml bacitracin and l/~g/ml pepstatin A, Sigma Chemical Co., St. Louis, MO) increased binding by -20% (checked with a-BTX). All radioligands were purchased from DuPont, New England Nuclear Corp. and unlabelled ligands from Sigma. Calculations were done using Fig. P and Kaleidograph computer software. For [125I]a-BTX (specific activity 116 Ci/mmol; final conc. 15-380 nM), samples were incubated for 60 min and then treated as described above. Specific binding was determined using 10pM unlabelled toxin. The dissociation rate constant k-l for the AChR-a-BTX complex was
65
determined taking samples at intervals between 0 and 96 h. For L-[3H]nicotine (80 Ci/mmol; 26-300 nM; no protease inhibitors), binding was allowed for 30 min whereafter samples were treated as above. For determination of specific binding, 195/tM cold L-nicotine was added. The kinetic constant for association was determined at a fixed radioligand concentration (104 nM; 0-60 min) and for dissociation by adding excess of cold nicotine (195/~M). For [3H]strychnine (24.5 Ci/mmol; 16-328 nM), binding was determined after a 30-min incubation (specific binding in the presence of 100/tM cold strychnine or 100~M to 10 mM glycine). The influence of membrane depolarization on strychnine binding was studied by adding [3H]strychnine (16.4 nM) and ACh (100/~M) or [K÷] (56.2 mM) simultaneously, or by adding [3H]strychnine 5 min before the above concentrations of ACh or [K+]. For [3H]3-QNB (43.5 Ci/mmol; 9.5-322 nM), binding was allowed for 60 min and specific binding determined using 10/zM cold 3-QNB or atropine sulphate. Dosedependent competitive binding was studied by adding the M3 antagonist 4-DAMP or the non-specific antagonist atropine [19] to OHC 5 min before [3H]3-QNB (23 riM). [125I]a-BTX bound specifically, saturably and reversibly to a single class of low affinity sites. Hill coefficient and other data are shown in Fig. 1 and Table 1. Association kinetics were too fast to be determined by the centrifugation technique. The dissociation rate constant k_l for the AChR-a-BTX complex was 2.2 x 10-4 min -1, tl/2 = 52.5 h (figure not shown). L-[3H]Nicotine was found to bind specifically, non-saturably and reversibly to a single class of low affinity sites (assuming saturation occurred upon addition of higher L-[3H]nicotine concentrations; Table 1; figure not shown). The rate constant of association kl was 6.2 x 10-4 min -l nM -1, with tl/2 of 11 min and the rate constant of dissociation (k_l) was 0.23 min -1 with a tl/2 of 3 min (figure not shown). Specific binding of [3H]strychnine is shown in Fig. 2, Table 1. Addition of [3H]strychnine (16.4 nM) simultaneously with ACh (100/~M) or 5 min before addition of ACh resuited in no significant change in specific binding 29.9 _+4.9 pM, i.e. compared to controls 22.4 _+4.6 pM. In contrast, OHC at depolarizing [K÷] (56.2 mM) showed a 3-4-fold increase in specific binding 108.8 _+32.2 pM. When cold glycine (100pM to 10 mM) was used instead of cold strychnine, specific binding of [3H]strychnine was not observed. No specific binding of [3H]3-QNB was observed at concentrations commonly used for binding of muscarinic ligands to their receptors, i.e. at pM ranges [9]. At several hundred times higher 3-QNB concentrations, specific binding to isolated OHC occurred to a single class of low affinity sites with a KD of 11.5 +- 5 nM (figure not shown). From competition binding of [3H]3QNB with two other muscarinic ligands, we obtained a
G. Lawoko et al. / Neuroscience Letters 195 (1995) 64-68
66
teristically, the latter binds a-BTX irreversibly and has a lower KD (0.5 nM, Torpedo nAChR [12]; 11.7 nM, rat skeletal muscle endplate nAChR [20]) than the OHC AChR. Binding studies in the absence of protease inhibitors showed only one affinity site (KD = 45 nM) for aBTX, but addition of protease inhibitors revealed proteolysis of toxin-binding sites (KD = 62 nM), as the number of binding sites was 20% higher after 1 h. Nicotine binding was non-saturable, and again only one single class of binding sites (Ko ~ 230 nM) was detected. The existence of high-affinity binding sites for a-BTX and nicotine, respectively, on the native receptor in situ seems plausible but they may have been lost by rapid receptor desensitization, as shown for example for the a-BTX-binding receptor from chick embryo ciliary ganglion neurons [25]. [3H]Strychnine, in the presence of excess cold strychnine, bound specifically to OHC. Such binding was not found with an excess of cold glycine, thus casting doubt on the ability of glycine to bind to the OHC AChR's extracellular surface. Strychnine may also bind to sites other than an extracellular one since binding of strychnine to isolated OHC increased in a high [K ÷] depolarizing medium. Binding may thus occur to a site accessible to strychnine only after depolarization, and/or after cellular uptake. A structural homology between the GIyR strychnine-
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rank order potency of atropine>4-DAMP (atropine IC50 = 6.1 nM; 4-DAMP IC50 = 6.5 nM; Fig. 3). The present findings confirm and extend our result [21] and those of others (see recent reviews by Eybalin [9] and Heilbronn et al. [13]), i.e. that the mammalian OHC carries a mainly nicotinic AChR which differs from the 'classical' peripheral nAChR in several ways. Charac-
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Summary of ligand binding parameters of [1251]a-BTX, L-[3H]nicotine, [3H]strychnine and [3H]3-QNB binding to the AChR on isolated OHC of the guinea pig cochlea
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Fig. 2. Binding of [3Hlstrychnine to OHC isolated from the guinea pig cochlea. (A) Saturation binding of [3H]strychnine to OHC. OHC were incubated with [3H]strychnine (16-328 nM) for 30 min at 21°C (n -> 3). Non-specific binding was determined in the presence of unlabelled strychnine (100MM). (B) Scatchard analysis of [3H]strychnine binding. Inset: Hill analysis of [3H]strychnine binding.
G. Lawoko et al. / Neuroscience Letters 195 (1995) 64-68
In conclusion, our present and previous work [21] shows the basically nicotinic nature of the OHC nAChR and provides new biochemical evidence confirming its unusual pharmacological properties. The observed depolarization-dependent strychnine binding and the significance of muscarinic ligand binding to OHC require further analysis.
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Fig. 3. Displacement of [3H]3-QNB from isolated OHC of the guinea pig cochlea. OHC were incubated with 23 nM [3H]3-QNB and increasing concentrations of 4-DAMP (0) and atropine (rl) for 60 min at 21°C. IC5o values were calculated by non-linear regression and were 6.1 nM and 6.5 nM, respectively. Data are mean values of three independent experiments and are expressed as percent specific binding of [3H]3-QNB compared to control. binding a subunit and a D r o s o p h i l a nAChR receptor protein, which both belong to the ligand-gated ion channel receptor superfamily, has been suggested [11]. In vitro mutagenesis and functional analysis of the GlyR ctl subunit have identified amino acids which form the two extracellular domains of the strychnine-binding site: Gly-160 and Tyr-161 in the first domain, and Lys-200 and Tyr-202 in the second domain [23]. A m i n o acid sequence comparison between GlyR a l subunit and nAChR a 9 subunit revealed that their tyrosine residues were conserved while Gly-160 was substituted for threonine, which also has an uncharged polar R group, in the a9, and the positively charged Lys-200 replaced by a negatively charged glutamic acid residue. Thus, the conserved tyrosine residues and Thr-160 may be part of an extracellular site binding strychnine to OHC AChR. Another interpretation of strychnine action on OHC could build on the observation that strychnine blocks Ca2+-dependent K ÷ channels [6]. Such channels are present in OHC [14]. The 3-4-fold rise now observed in specific strychnine binding to depolarized OHC may thus point to both extracellular binding sites on the AChR subtype and/or to an intracellular binding site on their Ca2+-dependent K + channels. Previous electrophysiological recordings have indicated inhibition of ACh-induced membrane currents by muscarinic antagonists on dissociated guinea pig OHC [10,17], yet binding of [3H]3-QNB at concentrations higher than those used to detect known mAChR subtypes (0.8 nM) ruled out the presence of muscarinic AChR on isolated OHC [21]. The present results support suggestions that interaction of high muscarinic ligand concentrations exist with OHC. We obtained a rank order of competitive antagonistic action of atropine > 4-DAMP with [3H]3-QNB, consistent with electrophysiological studies on dissociated OHC from guinea-pig cochlea [8,17]. The very high concentrations needed for [3H]3-QNB binding and the number of binding sites found, however, do not point to an agonist (ACh) site of a conventional muscarinic AChR (see Ref. [13] for review).
[1] Ashmore, J.F., The cellular machinery of the cochlea, Exp. Physiol., 79 (1994) 113-134. [2] Anand,R., Fuchs, P.A., Cooper, J.F., Peng, X. and Lindstrom,J., Evidence that a-bungarotoxin-sensitiveacetylcholinereceptors in chick's cochlea contain a7 subunits, Soc. Neurosci. Abstr., 19 (1993) 465. [3] Bartolami, S., Ripoll, C. and Eybalin, M., Anticholinergiceffects of strychnine in the cochlea do not involve muscarinic receptors, NeuroReport, 4 (1993) 1003-1006. [4] Bartolami,S., Ripoll, C., Planche, M. and Pujol, R., Localisation of functionalmuscarinicreceptors in the rat cochlea: evidence for efferent presynaptic autoreceptors, Brain Res., 626 (1993) 200209. [5] Bobbin,R.P. and Thompson, M.H., Effects of putative transmitters on afferent cochlear transmission,Ann. Otol. Rhinol. Laryngol., 87 (1978) 185-190. [6] Cook, N.S. and Haylett, D.G., Effects of apamine, quinine and neuromuscularblockers on calcium-activatedpotassium channels in guinea-pig hepatocytes, J. Physiol. (London), 358 (1985) 373394. [7] Elgoyhen, A.B., Johnson, D., Boulter, J., Vetter, D. and Heinemann, S., Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells, Cell, 79 (1994) 705-715. [8] Erostegui, C., Norris, C.H. and Bobbin, R.P., In vitro characterization of a cholinergicreceptor on outer hair cells, Hear. Res., 74 (1994) 135-147. [9] Eybalin, M., Neurotransmitters and neuromodulators of the mammaliancochlea, Physiol. Rev., 73 (1993) 309-372. [10] Fuchs, P.A. and Murrow, B.W., A novel cholinergic receptor mediates inhibition of chick cochlear hair cells, Proc. R. Soc. London (Biol.), 248 (1992) 35-40. [11] Grenningloh,G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger,E.D. and Betz, H., The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors, Nature, 328 (1987) 215220. [12] Griesmann, G.E., McCormick, D.J., De Aizpurua, H.J. and Lennon, V.A., a-Bungarotoxin binds to human acetylcholinereceptor a-subunit peptide 185-199 in solution and solid phase but not to peptides 125-147 and 389-409, J. Neurochem., 54 (1990) 15411547. [13] Heilbronn, E., J~lebark, L. and Lawoko, G., Cholinergic and purinergic signalling in outer hair cells of mammalian cochlea, Neurochem. Int., (1995) in press. [14] Housley, G.D. and Ashmore, J.F., Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea, Proc. R. Soc. London (Biol.), 244 (1991) 161-167. [15] Housley, G.D., Batcher, S., Kraft, M. and Ryan, A.F., Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction, Hear. Res., 75 (1994) 4753. [16] Hulme, E.C., Receptor-Ligand Interactions - A Practical Approach, IRL Press, Oxford, 1992. [17] Kakehata, S., Nakagawa, T., Takasakao T. and Akaike, N., Cellular mechanism of acetylcholine-inducedresponse in dissociated outer hair cells of guinea-pig cochlea, J. Physiol. (London), 463 (1993) 227-244.
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G. Lawoko et al. / Neuroscience Letters 195 (1995) 64-68
[18] Kujawa, S.G., Glattke, T.J., Fallon, M. and Bobbin, R.P., A nicotinic-like receptor mediates suppression of distortion product otoacoustic emissions by contralateral sound, Hear. Res., 74 (1994) 122-134. [19] Mitchelson, F., Muscarinic receptor differentiation, Pharmacol. Ther., 37 (1988) 357--423. [20] Motomura, M., Niwa, M., Kataoka, Y., Ootsuru, I., Yoshimura, T., Tsujihata, M. and Nagataki, S., In vitro quantitative autoradiography of [125I]a-bungarotoxin binding at the motor endplates of experimental autoimmune myasthenia gravis, Neurosci. Lett., 143 (1992) 139-142. [21] Plinkert, P.K., Zenner, H.P. and Heilbronn, E., A nicotinic acetylcholine receptor-like a-bungarotoxin-binding site on outer hair cells, Hear. Res., 53 (1991) 123-130.
[22] Renee, R.A., Griffiths, J.M. and Wilkinson, M.L., In Basic Biochemical Methods, Wiley, New York, 1985, pp. 179-183. [23] Vandenberg, R.J., French, C.R., Barry, P.H., Shine, J. and Schofield, P.R., Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor a subunit form the strychnine-binding site, Proc. Natl. Acad. Sci. USA, 89 (1992) 1765-1769. [24] Zenner, H.P., Gitter, A.H., Zimmermann, U., Schmitt, U. and Fr6mter, E., Die isolierte Haarzelle, Laryngol. Rhinol. Otol., 64 (1985) 642-648. [25] Zhang, Z.-W., Vijayaraghavan, S. and Berg, D.K., Neuronal acetylcholine receptors that bind a-bungarotoxin with high affinity function as ligand-gated ion channels, Neuron, 12 (1994) 167177.
Neuroscience Letters 195 (1995) 64-68
N[UKgSCI[HC[ LETTERS
Ligand-binding properties of an unusual nicotinic acetylcholine receptor subtype on isolated outer hair cells from guinea pig cochlea G. Lawoko a,b, L. J~lebark,, E. H e i l b r o n n a,* aDepartment ~'Neurochemistry and Neurotoxicology, Stockholm University, S-106 91 Stockholm, Sweden bDepartment of Pharmacology, University ~Lund, Lund, Sweden
Received 30 March 1995;accepted 15 June 1995
Abstract Acetylcholine receptors on isolated guinea pig cochlear outer hair cells (OHC) were characterized by radioligand binding. Equilibrium binding of [125I]ct-bungarotoxin revealed a KD of 62 _+2 nM, Bmax = 7.2 -+ 1.8 × 10 7 binding sites/OHC, and a slowly reversible dissociation rate constant, k_1 = 2.2_+ 0.01 × 10-4 min-l. L-[3H]Nicotine bound reversibly (estimated KD~ 230 nM and Bmax ~ 5 X 10 7) with kinetic rate constants of association kj = 6.2 _+0.06 × 10 4 min-I nM-1 and dissociation k_1 = 0.23 ± 0.003 min-1. [3H]Strychnine bound to OHC with a KD of 35 +_6 nM and Bma x = 2.6 + 0.5 × 10 7, and binding increased 3-4 fold after membrane depolarization with 56.2 mM [K+], suggesting additional binding sites. Binding, seen only at > n M concentrations, of [3H]3quinuclidinyl benzilate (KD = 11.5 ± 5 nM; Bma x = 2.5 + 0.6 × 106) was competitively inhibited by the muscarinic antagonists atropine and 4-DAMP (IC50 of 6.1 ± 0.5 and 6.5 ± 0.4 nM). The OHC receptor is thus an atypical nicotinic acetylcholine receptor subtype with unusual pharmacological properties.
Keywords: Auditory
system; Outer hair cell; Efferent synapse; Acetylcholine receptor; Radioligand binding; Nicotinic ligands; Strychnine; Muscarinic ligands
Two types of mechanoreceptors in the mammalian inner ear convert sound from mechanical to electrical signals. Inner hair cells are the primary transducers of incoming acoustic information in the cochlea, i.e. deflection of hair cell stereocilia regulates neurotransmitter release to afferents of the eighth cranial nerve. Outer hair cells (OHC) are innervated by medial olivocochlear efferent neurons and form a modulatory feedback mechanism by virtue of their electromotile properties, thereby affecting frequency selectivity and sensitivity in the organ of Corti [1]. A number of data indicate that acetylcholine (ACh) is the main neurotransmitter at the efferent synapse [9]. The crossed olivocochlear bundle (an efferent inhibitory tract of nerve fibers which forms synaptic junctions at the base of the scala tympani of mammalia) has been shown to be blocked by both cholinergic and noncholinergic drugs introduced into the medial efferent system. Distortion product otoacoustic emissions * Corresponding author, Tel.: +46 8 164269; Fax: +46 8 161371; E-mail: [email protected].
(DPOAEs), thought to reflect OHC activity and mediated by the efferent cholinergic system, were blocked by selective antagonists of nicotinic and muscarinic receptors [18] as well as by the glycine receptor (GIyR) antagonist strychnine, suggesting that these receptors may be present in the cochlea. Muscarinic AChR, although present at the efferent presynapse [4], have, however, not been demonstrated on OHC, thus block of transmitter release from the cholinergic presynapse was probably involved. The presence of GIyR at the efferent synapse was ruled out [5] as was the presence of strychnine binding sites on rat cochlea mAChR, since neither binding of the mAChR antagonist 3-quinuclidinyl benzilate (3-QNB) nor carbachol-activated inositol phosphate formation were inhibited by strychnine (1-50/~M) [3]. Results from molecular cloning of cochlear nAChR subunits have suggested that an a 5 (rat) [15] and/or a 7 (chick) [2] subunit is present. Furthermore, recent cloning of a novel ct subunit, a 9 [7], from olfactory epithelium, and subsequent amplification of rat cochlear cDNA (polymerase chain reaction) using a9-specific primers revealed ex-
0304-3940/95/$09.50 © 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0304-3940(95)11774-N
G. Lawoko et al. /Neuroscience Letters 195 (1995) 64-68
pression of this subunit gene in the inner ear. In situ hybridization analysis confirmed expression in the hair cell area. Previous binding studies in this laboratory have identified the guinea pig OHC AChR as primarily nicotinic, but not identical with the 'classical' AChR [21]. The present paper further characterizes this AChR and approximates the number of a-bungarotoxin (a-BTX), nicotine, strychnine and 3-QNB-binding sites/OHC. Binding of the muscarinic antagonist 3-QNB, observed at very high concentrations, was competitively reduced by other muscarinic antagonists (atropine and 4-diphenylacetoxy-Nmethylpiperidine methiodide; 4-DAMP) and the possibility of strychnine binding to sites other than extracellular nAChR domains is discussed. OHC were non-enzymatically isolated (to avoid receptor modification) from pigmented guinea pigs of either sex (Duncin Hartley, body weight 300-400 g) after anaesthesia with CO2 prior to decapitation [24]. In short, the temporal bones were taken out, stored in ice-cold buffer and the lateral wall of the bony cochlea removed under a dissection microscope. The organ of Corti with the modiolus bone was transferred to a balanced salt solution (125 mM NaCI, 5 mM KCI, 1.2 mM MgSO 4, 1.2 mM KH2PO4, 1.0mM CaCI 2, 25 mM Tris-HCl, pH 7.4, osmolarity 300___ 10mosm/l), also used for all binding assays. OHC were microdissected from all turns of the cochlea and separated from membranes and supporting cells (mostly Deiters cells) by gentle pipetting and transferring to a Petri dish. An assay utilizing release of cytosolic enzyme lactate dehydrogenase (LDH) [22] was used to check the integrity of isolated OHC; no LDH activity was detected outside the cells 1 h after incubation at 21°C. Binding assays (modified from Ref. [16]) were performed in Eppendorf tubes (silanized for a-BTX) at 21°C (pH 7.4), final incubation volume 500~1, containing 1600 _+ 200 OHC. After centrifugation (5 min, 4°C, 14 500 x g) the supernatant was discarded, pellets were washed three times with ice-cold buffer and radioactivity was counted, using a y-counter for [125I]a-BTX. For the tritiated ligands, pellets were washed once and then dissolved in 10% SDS before addition of 1.3 ml of scintillation fluid and counting in a liquid scintillation spectrometer. Addition of protease inhibitors (1 mM EDTA, 100pg/ml bacitracin and l/~g/ml pepstatin A, Sigma Chemical Co., St. Louis, MO) increased binding by -20% (checked with a-BTX). All radioligands were purchased from DuPont, New England Nuclear Corp. and unlabelled ligands from Sigma. Calculations were done using Fig. P and Kaleidograph computer software. For [125I]a-BTX (specific activity 116 Ci/mmol; final conc. 15-380 nM), samples were incubated for 60 min and then treated as described above. Specific binding was determined using 10pM unlabelled toxin. The dissociation rate constant k-l for the AChR-a-BTX complex was
65
determined taking samples at intervals between 0 and 96 h. For L-[3H]nicotine (80 Ci/mmol; 26-300 nM; no protease inhibitors), binding was allowed for 30 min whereafter samples were treated as above. For determination of specific binding, 195/tM cold L-nicotine was added. The kinetic constant for association was determined at a fixed radioligand concentration (104 nM; 0-60 min) and for dissociation by adding excess of cold nicotine (195/~M). For [3H]strychnine (24.5 Ci/mmol; 16-328 nM), binding was determined after a 30-min incubation (specific binding in the presence of 100/tM cold strychnine or 100~M to 10 mM glycine). The influence of membrane depolarization on strychnine binding was studied by adding [3H]strychnine (16.4 nM) and ACh (100/~M) or [K÷] (56.2 mM) simultaneously, or by adding [3H]strychnine 5 min before the above concentrations of ACh or [K+]. For [3H]3-QNB (43.5 Ci/mmol; 9.5-322 nM), binding was allowed for 60 min and specific binding determined using 10/zM cold 3-QNB or atropine sulphate. Dosedependent competitive binding was studied by adding the M3 antagonist 4-DAMP or the non-specific antagonist atropine [19] to OHC 5 min before [3H]3-QNB (23 riM). [125I]a-BTX bound specifically, saturably and reversibly to a single class of low affinity sites. Hill coefficient and other data are shown in Fig. 1 and Table 1. Association kinetics were too fast to be determined by the centrifugation technique. The dissociation rate constant k_l for the AChR-a-BTX complex was 2.2 x 10-4 min -1, tl/2 = 52.5 h (figure not shown). L-[3H]Nicotine was found to bind specifically, non-saturably and reversibly to a single class of low affinity sites (assuming saturation occurred upon addition of higher L-[3H]nicotine concentrations; Table 1; figure not shown). The rate constant of association kl was 6.2 x 10-4 min -l nM -1, with tl/2 of 11 min and the rate constant of dissociation (k_l) was 0.23 min -1 with a tl/2 of 3 min (figure not shown). Specific binding of [3H]strychnine is shown in Fig. 2, Table 1. Addition of [3H]strychnine (16.4 nM) simultaneously with ACh (100/~M) or 5 min before addition of ACh resuited in no significant change in specific binding 29.9 _+4.9 pM, i.e. compared to controls 22.4 _+4.6 pM. In contrast, OHC at depolarizing [K÷] (56.2 mM) showed a 3-4-fold increase in specific binding 108.8 _+32.2 pM. When cold glycine (100pM to 10 mM) was used instead of cold strychnine, specific binding of [3H]strychnine was not observed. No specific binding of [3H]3-QNB was observed at concentrations commonly used for binding of muscarinic ligands to their receptors, i.e. at pM ranges [9]. At several hundred times higher 3-QNB concentrations, specific binding to isolated OHC occurred to a single class of low affinity sites with a KD of 11.5 +- 5 nM (figure not shown). From competition binding of [3H]3QNB with two other muscarinic ligands, we obtained a
G. Lawoko et al. / Neuroscience Letters 195 (1995) 64-68
66
teristically, the latter binds a-BTX irreversibly and has a lower KD (0.5 nM, Torpedo nAChR [12]; 11.7 nM, rat skeletal muscle endplate nAChR [20]) than the OHC AChR. Binding studies in the absence of protease inhibitors showed only one affinity site (KD = 45 nM) for aBTX, but addition of protease inhibitors revealed proteolysis of toxin-binding sites (KD = 62 nM), as the number of binding sites was 20% higher after 1 h. Nicotine binding was non-saturable, and again only one single class of binding sites (Ko ~ 230 nM) was detected. The existence of high-affinity binding sites for a-BTX and nicotine, respectively, on the native receptor in situ seems plausible but they may have been lost by rapid receptor desensitization, as shown for example for the a-BTX-binding receptor from chick embryo ciliary ganglion neurons [25]. [3H]Strychnine, in the presence of excess cold strychnine, bound specifically to OHC. Such binding was not found with an excess of cold glycine, thus casting doubt on the ability of glycine to bind to the OHC AChR's extracellular surface. Strychnine may also bind to sites other than an extracellular one since binding of strychnine to isolated OHC increased in a high [K ÷] depolarizing medium. Binding may thus occur to a site accessible to strychnine only after depolarization, and/or after cellular uptake. A structural homology between the GIyR strychnine-
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rank order potency of atropine>4-DAMP (atropine IC50 = 6.1 nM; 4-DAMP IC50 = 6.5 nM; Fig. 3). The present findings confirm and extend our result [21] and those of others (see recent reviews by Eybalin [9] and Heilbronn et al. [13]), i.e. that the mammalian OHC carries a mainly nicotinic AChR which differs from the 'classical' peripheral nAChR in several ways. Charac-
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Summary of ligand binding parameters of [1251]a-BTX, L-[3H]nicotine, [3H]strychnine and [3H]3-QNB binding to the AChR on isolated OHC of the guinea pig cochlea
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Values are means of at least three separate experiments; K D values are given as means - SD.
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Fig. 2. Binding of [3Hlstrychnine to OHC isolated from the guinea pig cochlea. (A) Saturation binding of [3H]strychnine to OHC. OHC were incubated with [3H]strychnine (16-328 nM) for 30 min at 21°C (n -> 3). Non-specific binding was determined in the presence of unlabelled strychnine (100MM). (B) Scatchard analysis of [3H]strychnine binding. Inset: Hill analysis of [3H]strychnine binding.
G. Lawoko et al. / Neuroscience Letters 195 (1995) 64-68
In conclusion, our present and previous work [21] shows the basically nicotinic nature of the OHC nAChR and provides new biochemical evidence confirming its unusual pharmacological properties. The observed depolarization-dependent strychnine binding and the significance of muscarinic ligand binding to OHC require further analysis.
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Fig. 3. Displacement of [3H]3-QNB from isolated OHC of the guinea pig cochlea. OHC were incubated with 23 nM [3H]3-QNB and increasing concentrations of 4-DAMP (0) and atropine (rl) for 60 min at 21°C. IC5o values were calculated by non-linear regression and were 6.1 nM and 6.5 nM, respectively. Data are mean values of three independent experiments and are expressed as percent specific binding of [3H]3-QNB compared to control. binding a subunit and a D r o s o p h i l a nAChR receptor protein, which both belong to the ligand-gated ion channel receptor superfamily, has been suggested [11]. In vitro mutagenesis and functional analysis of the GlyR ctl subunit have identified amino acids which form the two extracellular domains of the strychnine-binding site: Gly-160 and Tyr-161 in the first domain, and Lys-200 and Tyr-202 in the second domain [23]. A m i n o acid sequence comparison between GlyR a l subunit and nAChR a 9 subunit revealed that their tyrosine residues were conserved while Gly-160 was substituted for threonine, which also has an uncharged polar R group, in the a9, and the positively charged Lys-200 replaced by a negatively charged glutamic acid residue. Thus, the conserved tyrosine residues and Thr-160 may be part of an extracellular site binding strychnine to OHC AChR. Another interpretation of strychnine action on OHC could build on the observation that strychnine blocks Ca2+-dependent K ÷ channels [6]. Such channels are present in OHC [14]. The 3-4-fold rise now observed in specific strychnine binding to depolarized OHC may thus point to both extracellular binding sites on the AChR subtype and/or to an intracellular binding site on their Ca2+-dependent K + channels. Previous electrophysiological recordings have indicated inhibition of ACh-induced membrane currents by muscarinic antagonists on dissociated guinea pig OHC [10,17], yet binding of [3H]3-QNB at concentrations higher than those used to detect known mAChR subtypes (0.8 nM) ruled out the presence of muscarinic AChR on isolated OHC [21]. The present results support suggestions that interaction of high muscarinic ligand concentrations exist with OHC. We obtained a rank order of competitive antagonistic action of atropine > 4-DAMP with [3H]3-QNB, consistent with electrophysiological studies on dissociated OHC from guinea-pig cochlea [8,17]. The very high concentrations needed for [3H]3-QNB binding and the number of binding sites found, however, do not point to an agonist (ACh) site of a conventional muscarinic AChR (see Ref. [13] for review).
[1] Ashmore, J.F., The cellular machinery of the cochlea, Exp. Physiol., 79 (1994) 113-134. [2] Anand,R., Fuchs, P.A., Cooper, J.F., Peng, X. and Lindstrom,J., Evidence that a-bungarotoxin-sensitiveacetylcholinereceptors in chick's cochlea contain a7 subunits, Soc. Neurosci. Abstr., 19 (1993) 465. [3] Bartolami, S., Ripoll, C. and Eybalin, M., Anticholinergiceffects of strychnine in the cochlea do not involve muscarinic receptors, NeuroReport, 4 (1993) 1003-1006. [4] Bartolami,S., Ripoll, C., Planche, M. and Pujol, R., Localisation of functionalmuscarinicreceptors in the rat cochlea: evidence for efferent presynaptic autoreceptors, Brain Res., 626 (1993) 200209. [5] Bobbin,R.P. and Thompson, M.H., Effects of putative transmitters on afferent cochlear transmission,Ann. Otol. Rhinol. Laryngol., 87 (1978) 185-190. [6] Cook, N.S. and Haylett, D.G., Effects of apamine, quinine and neuromuscularblockers on calcium-activatedpotassium channels in guinea-pig hepatocytes, J. Physiol. (London), 358 (1985) 373394. [7] Elgoyhen, A.B., Johnson, D., Boulter, J., Vetter, D. and Heinemann, S., Alpha 9: an acetylcholine receptor with novel pharmacological properties expressed in rat cochlear hair cells, Cell, 79 (1994) 705-715. [8] Erostegui, C., Norris, C.H. and Bobbin, R.P., In vitro characterization of a cholinergicreceptor on outer hair cells, Hear. Res., 74 (1994) 135-147. [9] Eybalin, M., Neurotransmitters and neuromodulators of the mammaliancochlea, Physiol. Rev., 73 (1993) 309-372. [10] Fuchs, P.A. and Murrow, B.W., A novel cholinergic receptor mediates inhibition of chick cochlear hair cells, Proc. R. Soc. London (Biol.), 248 (1992) 35-40. [11] Grenningloh,G., Rienitz, A., Schmitt, B., Methfessel, C., Zensen, M., Beyreuther, K., Gundelfinger,E.D. and Betz, H., The strychnine-binding subunit of the glycine receptor shows homology with nicotinic acetylcholine receptors, Nature, 328 (1987) 215220. [12] Griesmann, G.E., McCormick, D.J., De Aizpurua, H.J. and Lennon, V.A., a-Bungarotoxin binds to human acetylcholinereceptor a-subunit peptide 185-199 in solution and solid phase but not to peptides 125-147 and 389-409, J. Neurochem., 54 (1990) 15411547. [13] Heilbronn, E., J~lebark, L. and Lawoko, G., Cholinergic and purinergic signalling in outer hair cells of mammalian cochlea, Neurochem. Int., (1995) in press. [14] Housley, G.D. and Ashmore, J.F., Direct measurement of the action of acetylcholine on isolated outer hair cells of the guinea pig cochlea, Proc. R. Soc. London (Biol.), 244 (1991) 161-167. [15] Housley, G.D., Batcher, S., Kraft, M. and Ryan, A.F., Nicotinic acetylcholine receptor subunits expressed in rat cochlea detected by the polymerase chain reaction, Hear. Res., 75 (1994) 4753. [16] Hulme, E.C., Receptor-Ligand Interactions - A Practical Approach, IRL Press, Oxford, 1992. [17] Kakehata, S., Nakagawa, T., Takasakao T. and Akaike, N., Cellular mechanism of acetylcholine-inducedresponse in dissociated outer hair cells of guinea-pig cochlea, J. Physiol. (London), 463 (1993) 227-244.
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G. Lawoko et al. / Neuroscience Letters 195 (1995) 64-68
[18] Kujawa, S.G., Glattke, T.J., Fallon, M. and Bobbin, R.P., A nicotinic-like receptor mediates suppression of distortion product otoacoustic emissions by contralateral sound, Hear. Res., 74 (1994) 122-134. [19] Mitchelson, F., Muscarinic receptor differentiation, Pharmacol. Ther., 37 (1988) 357--423. [20] Motomura, M., Niwa, M., Kataoka, Y., Ootsuru, I., Yoshimura, T., Tsujihata, M. and Nagataki, S., In vitro quantitative autoradiography of [125I]a-bungarotoxin binding at the motor endplates of experimental autoimmune myasthenia gravis, Neurosci. Lett., 143 (1992) 139-142. [21] Plinkert, P.K., Zenner, H.P. and Heilbronn, E., A nicotinic acetylcholine receptor-like a-bungarotoxin-binding site on outer hair cells, Hear. Res., 53 (1991) 123-130.
[22] Renee, R.A., Griffiths, J.M. and Wilkinson, M.L., In Basic Biochemical Methods, Wiley, New York, 1985, pp. 179-183. [23] Vandenberg, R.J., French, C.R., Barry, P.H., Shine, J. and Schofield, P.R., Antagonism of ligand-gated ion channel receptors: two domains of the glycine receptor a subunit form the strychnine-binding site, Proc. Natl. Acad. Sci. USA, 89 (1992) 1765-1769. [24] Zenner, H.P., Gitter, A.H., Zimmermann, U., Schmitt, U. and Fr6mter, E., Die isolierte Haarzelle, Laryngol. Rhinol. Otol., 64 (1985) 642-648. [25] Zhang, Z.-W., Vijayaraghavan, S. and Berg, D.K., Neuronal acetylcholine receptors that bind a-bungarotoxin with high affinity function as ligand-gated ion channels, Neuron, 12 (1994) 167177.