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Hearing Research. 53 (1991) 123-130 9 1991 Elsevier Science Publishers B.V. 0378-5955/91/$03.50

HEARES

01555

A nicotinic acetylcholine receptor-like a-bungarotoxin-binding

site on outer

hair cells PK. Plinkert

(Received

1*2,H.P. Zenner

26 March

1990; accepted

’ and E. Heilbronn

28 November

1

1990)

Acetylcholine (ACh) appears to be the major neurotransmitter liberated from olivocochlear efferents terminating on outer hair cells (OHC). Recently, cholinergic receptor epitopes were visualized at the basal pole of the OHCs. To evaluate the ACh receptor type at OHC we performed binding studies with [‘2sI]-labelled a-bungarotoxin (a-bgtx), a close to irreversibly acting blocker of the nicotinic acetylcholine receptor (nAChR) of skeletal muscle and of electrocytes of Torpedo and Elecfrophoms. An irreversible and saturable binding (80 nM) of the radiolabelled compound to OHCs was observed. The number of a-bgtx sensitive binding sites present on each OHC was calculated to be about 2X10-” mol/OHC. which would amount to about 10’ binding sites/cell. Preincubation with the reversibly acting choline&c ligands. carbamylchohne (1 mM), nicotine (0.1 mM) and d-tubocurarine (I-100 PM) was found to inhibit a-bgtx binding to a varying degree. Atropine (0.05 mM), a muscarinic antagonist, had no influence on the binding of ru-bgtx to OHCs. [3H]-QNB, a specific marker and antagonist for muscarinic AChR, and [‘2SI]-k-toxin, known to react with neuronal and ganglionic nAChR, showed no specific binding to OHCs. The data indicate that a peripheral type nAChR is present on OHCs mediating ACh-induced modulation of the biomechanics of the cochlea by influencing OHC motihty. Inner ear; Efferent

innervation;

Hearing:

Cochlea:

Motility;

Cytoskeleton

Introduction

The organ of Corti is innervated by efferent nerve fibres originating from various nuclei of the superior olivary complex (Wan: et al., 1986; Spoendlin, 1986) and te~nating at the lower pole of outer hair cells (OHC). The fibers are postulated to control the biomechanics of the mammalian cochlea (Rhode, 1971; Davis, 1983). Several lines of evidence suggest that the neurotransmitter released from their efferent terminals is acetylcholine (ACh) (see Klinke, 1986, for review). The presence of enzymes necessary for the synthesis (choline acetyltransferase, CAT) and the hydrolysis (ACh-esterase, AChE) of this transmitter has been shown (Schuhknecht et al., 1956;

Correspondence to: Peter K. Plinkert, Department of Otolaryngology, University of Tiibingen, Silcherstr. 5, D-7400 Tubingen, F.R.G.

Jasser and Guth, 1973; Altschuler et al., 1985). Early in vivo-experiments suggested that effects caused by the electrical stimulation of the crossed olivocochlear bundle could be blocked by intracochlearly applied nicotinic or musca~nic antagonists, the latter, however, at very high concentrations (Fex, 1968; Galley et al., 19’73; Bobbin and Konishi, 1974). Fex and Adams (1978) described a reversible block produced by the intracochlear application of a-bgtx to the crossed olivo-cochlear bundle. This observation could have suggested the presence of a nAChR of the neuronal type (Loring and Zigmond, 1988; for review). Recently nAChR epitopes were visualized on OHCs by means of monoclonal antibodies directed towards the nAChR of electric organ of Torpedo c~~~~~~~~caand E~ectT~~ho~ electricus (Tzartos et al., 1981, 1986). It was shown, that extra- and intracellular nAChR-epitopes are located cuplike at the basal pole of isolated OHCs (Plinkert, 1989; Plinkert et al., 1990).

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The nAChRs of vertebrate skeletal muscle and of the closely related electroplax are receptor-ion channel glycoprotein macromolecules, spanning the entire membrane. Their ion channel is cationselective and formed by four homologous subunits (a, p, y, 6) with a molar stoichiometry of 2 : 1 : 1 : 1. Smaller variations in the subunit structure occur with the state of development and the animal species, e.g. an embryonic subunit contributes to developing nAChR (Takai et al., 1985). They have been well characterized from biochemical. molecular biological, physiological, pharmacological and pathological aspects (e.g. Maelicke. 1986: Patrick et al.. 1987; Poulter et al., 1989; Dani, 1989). They have been isolated using biospecific chromatography on reversibly acting (Ytoxin columns (Karlson et al., 1972; Olsen et al., 1972; Klett et al., 1973) and later by various other means. Their immunology has been studied and connected to the autoimmune disease myasthenia gravis (for review, see e.g. Heilbronn, 1985: Drachman, 1987). They have been cloned and expressed in e.g. oocytes (Raftery et al., 1980: Noda et al.. 1982, 1983) and their genes have been detected. The cY-subunits carry the binding sites for ACh and for snake a-toxins. Other nAChR-like proteins exist, in the central and peripheral nervous system of both vertebrates and invertebrates. e.g. autonomic ganglia and parts of the CNS, differing both structurally and pharmacologically from those of skeletal muscle and electroplax (Boulter et al., 1986). The neural crest cell line PC12 carries nAChR, yet a-bgtx binds to PC12 without inhibiting its response to ACh. Some anti-nAChR antibodies, however, block the ACh response but not the c*-bgtx binding (Patrick and Stallcup, 1977). The response of the neural receptor to ACh is instead blocked by another snake toxin, kappatoxin (Chappinelli, 1983). We report here experiments carried out with both radiolabelled cY-bgtx and labelled K-toxin, identifying and quantifying nAChR-like binding sites in OHCs that. judged from their binding characteristics, are similar to those of skeletal muscle or electric organ nAChR. Materials and Methods Microdissection of OHC The non-enzymatic isolation OHCs was performed according

of mammalian to the method

described by Zenner et al., (1985a). Pigmented guinea pigs either sex and with a positive Preyer’s reflex and a body weight of about 250 g were decapitated. The temporal bone was removed and cooled to 4” C. After removal of the lateral wall of the bony cochlea the tissue was transferred to a balanced salt solution (125 mM NaCl, 5 mM KCl. 1.2 mM Mg SO,. 1.2 mM KH,PO,, 1 mM CaCl,. 25 mM TRIS), which was adjusted to 300 mosmol/l (pH 7.4) using 4 M NaCl. The dissection procedure was performed under an inverted microscope. No collagenase was added in order not to modify any properties of the presumptive cholinergic receptors. A.wq~ for a-toxin binding to OHC cw-bungarotoxin used in the present study was from NEN Research Products and radiolabelled with I’57 using the modified Hunter and Greenwood method (1962). which results in a product iodinated only on Tyrs4 (Wang and Schmidt, 1980). [“‘I]-a-bgtx had a specific radioactivity of 15.6 pCi/pg. The isolated OHCs were incubated at room temperature for 1 hour with 40 ~1 radiolabelled a-bgtx (l-100 nM final cont.), centrifuged in an airdriven ultracentrifuge (Beckman airfuge) at 200000 X R (4” C) and the supernatant was discarded. The sediment was washed twice with buffer (pH 7.4: 300 osmol/l) to remove free toxin. The samples were counted in a scintillation counter (LKB Rackbeta 1214). Nonspecific binding was checked by preincubating OHCs with ‘cold’ a-bgtx (10 PM; room temperature: 1 h) prior to the addition of the radiolabelled compound. Data for specific a-bgtx binding were calculated from total minus unspecific a-toxin binding. Effects of other cholinergic ligands on a-toxin binding were tested by preincubating the OHCs for 10 min with the desired ligand concentration. Carbamylcholine chloride (Sigma: 10 mM), nicotine (Sigma: 0.1 mM), d-tubocurarine (Sigma; l100 PM) and atropine sulfate (Sigma; 0.05 mM) were used. [“51]-cu-bgtx (10 nM) was added for 10 min before centrifugation. washing and scintillation counting as described above. In further experiments the snake toxin Kbungarotoxin (We thank professor B. ContiTronconi for the gift of cold K-toxin) was radio-

125

T method labelled with 125I by the chloramine (Lindstrom et al., 1981). The specific radioactivity of the mono-iodo-K-toxin used was 95 Ci/mmol. Isolated OHCs were incubated with radiolabelled K-toxin (50-150 nM) for 1 h at room temperature. Labelling of samples and counting was as described for a-bgtx (see above). Finally, the muscarinic antagonist [ 3H]-QNB (Amersham, UK) (1 nM) and atropine (100 nM) were used to search for the possible presence of a muscarinic AChR on OHCs. The radiolabelled [ 3H]-QNB had a specific radioactivity of 41.5 Ci/ mmol. 20

0

40

EgtZJ

60

80

100

nM

Fig. 1. Specific a-bgtx binding (l-100 nM) to OHCs (solid circles). Saturation level is reached with 80 nM. Values of the specific c+bgtx binding were calculated from total (solid triangle) minus unspecific (open squares) n-toxin binding. Incubation: 1 h; 21°C.

Results OHCs were isolated without addition of proteolytic enzymes in order to avoid alterations of cell surface associated binding sites. An inverted microscope was used to collect only OHCs which appeared free from fragments of nerve endings. An electronmicroscopic control of 29 light micro-

q Carbamyl. Nicotine n x 1000 4.5

a

Curare

q Atropine n Controls

x 1000 4.5,

4.0

4.0

1

C P M

3.5

3.5

3.0

3.0

C

2.5

2.5

b

n L!

Controls 7 1 10 100

P

2.0

M

2.0

1.5

1.5

1.0

1.0

0.5

0.5

0.0 i

-1

0.0

Cholinergic Ligands

d-Tubocurarine nM

Fig. 2. Binding properties of cY-bgtx to the nAChR-like binding site on OHCs. (a) Total inhibition of cY-bgtx binding (30 nM) with nicotine (0.1 mM) and curare (0.1 mM); carbamylcholine (1 mM) reduced a-toxin binding about 14%. The muscarinic antagonist atropine (0.05 mM) had no effect. (b) Concentration-dependent protection of a-toxin binding with d-tubocurarine (1.10 and 100 p;). The duration of each incubation step was 10 min at 21 o C; centifugation was performed at 4” C.

b Fig. 3. Electronmicroscopy of isolated OHCs from the guinea pig cochlea. The contamination with residues of the efferent nerve terminals at the basal cell pole was determined. In 52% (N = 29) OHCs presynaptic membrane fragments were absent (a), whereas 45% showed a minor contamination (b). Only 1 isolated sensory cell possessed multiple presynaptic membrane residues (c).

scopically nerve-free OHCs revealed the absence of any nerve ending fragments in 52% of the collected cells (Fig. 3a). 45% of the cells showed minor contaminations by small vesicle-like structures (Fig. 3b). while only 1 cell possessed nerve ending-like residues. In a first series of experiments the cells were incubated with [“‘I]-cy-bgtx in the absence or presence of an excess of unlabelled a-bgtx (1 h: 21”C, 10 pm). Binding saturation was found at 80 nM. Results are shown in Fig. 1. About 0.02 fM [ “‘I]-a-bgtx bind to each OHC, representing equal amounts of a presumptive nAChR. For a further biochemical characterization of the toxin binding component the isolated OHCs were preincubated with either the cholinergic agonists carbamylcholine (1 mM) and nicotine (0.1 mM) or the antagonist d-tubocurarine (l-100 PM). Results are shown in Fig. 2a/b. Either nicotine or dtubocurarine significantly reduced the binding of radiolabelled ol-bgtx, as could be expected if a skeletal muscle nAChR-like a-bgtx-binding site is present. Somewhat less protection of the cr-toxin binding site was observed after preincubation with carbamylcholine. In a further experiment the muscarinic receptor antagonist atropine (0.5 mM) was used and had no significant effect on the binding properties of [“‘I]-a-bgtx (Fig. 2a). In additional experiments OHCs were incubated with

[ ‘HI-QNB, a specific probe for muscarinic AChR or with [‘251]-k-toxin, a marker for neuronal and ganglionic nAChR. Neither probe showed any specific binding to OHC isolated from the guinea pig organ of Corti.

Discussion The aim of the present study was to further elucidate the nature of the earlier found nAChRlike epitopes present on OHCs (Plinkert, 1989). The whole-cell-preparation of OHCs (Zenner et al., 1985a) used has the advantage to exclude a contamination with additional cells because only OHCs are transferred to the incubation tubes, with a pipette. Furthermore. electronmicroscopy showed that contamination by other membrane residues occurred in less than 5% of OHCs. Each test tube contained about 1000 cells (+ / - 10%). We observed a specific and saturable binding of [‘*‘I]-a-bgtx to OH&. From the binding curve a number of roughly 10’ binding sites/cell could be calculated. The nAChR antagonist d-tubocurarine and the agonist nicotine, known to interact with nAChR at the neuromuscular junction (Prives, 1980) decreased significantly and in a concentration-dependent manner the specific binding of [“‘I]-a-bgtx, whereas a somewhat lower than ex-

127

petted protection was obtained with the cholinergic agonist carbamylcholine (1 mM). Possibly the OHC nAChR shows some small differences in its amino acid composition at its agonist binding site, as compared to nAChR from Torpedo electrocyte or skeletal muscle. The muscarinic AChR antagonist atropine had, as expected, no effect on the ol-bgtx binding to OHCs. Furthermore, no muscarinic AChR binding sites were detected with the specific marker [3H]-QNB. K-toxin, known to react with neuronal and ganglionic nAChR showed no significant binding to OHCs. In conclusion, the obtained ligand binding profile reminds of that obtained for the peripheral nAChRs of the neuromuscular junction of mammalia though very minor structural differences may exist (see carbachol) (Prives, 1980; McCarthy et al., 1986). This will be revealed when our pharmacological and immunochemical study is completed with molecular biological studies of the nAChR. Binding of two molecules of ACh to sarcolemma1 nAChR induces a change in the receptor conformation, resulting in a transient opening of the cation-channel of the glycoprotein macromolecule (Sakmann et al., 1984). Subsequent influx of cations into the cell causes ion channel opening and sarcolemma depolarization, penetrating the interior of the cell through the transverse tubules (TT) which are continuous with the outer membrane. Subsequently. excitation-contraction coupling occurs, in a still partially unknown manner (Inesi, 1985). The opening of Cal+-channels, allowing influx of Ca’+ from the extracellular space and the release of CaZt from the intracellular store, the sacroplasmic reticulum (SR), results in a reversible increase in the cytoplasmic Ca’+-conwhich finally initiates actomyocin centration, activation. Several different ways for the cytoplasmic Ca’+ increase necessary for excitationconctraction coupling at the T-SR junction in skeletal muscle have been discussed: 1) The calcium-induced calcium release hypothesis (Fabiato, 1985). In vitro experiments have shown that the SR calcium release channel may be regulated adenine nucleotides, Mg’+ and by Ca’+, calmodulin (Lai and Meissner, 1989). Ryanodine and caffeine affect SR Ca2+ release. The Ca-release channel was isolated and functionally recon-

stituted into planar lipid bilayers and liposomes (Lai et al., 1988). 2) The ‘feet’ theory which suggests that this structure (the ryanodine sensitive channel?) at the T-SR connection directly responds to depolarization-induced, small transient intramembrane charge movement in the T-membrane (Schneider, 1981). It has also been discussed (Vergara et al., 1985; Volpe et al., 1986; Haggblad and Heilbronn, 1987) that the second messenger IP, may play a role in excitation-contraction coupling in skeletal muscle. Modulators or cotransmitters, e.g. ATP (for review see e.g. Haggblad et al., 1985) and calcitonin gene-related peptide (CGRP; Laufer and Changeux, 1987) triggering a receptor - G protein - phospholipase C cascade. increase the cytoplasmic IP, content of skeletal muscle (myotube in culture) and contraction. As shown by Heilbronn and Eriksson (submitted) membrane depolarization in chick myotubes per se also causes turnover of phosphoinositides such as phosphatidylinositol 4,5-biphosphate (PIP,) which are located to the inner leaflet of the cell membrane. An observed voltage-triggered IP? release (Novotny et al., 1983) supports this. It was further shown that increase of intracellular IP, triggers the release of Ca’+ from intracellular stores (sarcoplasmic reticulum, SR) (Adam0 et al., 1985). Eriksson and Heilbronn (1988) showed that increases in intracellular IPJ level (and DAG) trigger increases in cytoplasmic free Ca’+ levels from two sources: an intracellular one (SR?), followed by influx of extracellular Ca’+ through a dihydropyridine-sensitive Ca’+-channel. There is evidence. that the ACh-induced receptor activation on OHCs is followed by a cascade of events, similar to those at the neuromuscular junction: (I) a nAChR seems to be present; (II) an actomyosin skeleton is present (Flock and Cheung, 1977; Tilney et al.. 1980; Zenner, 1980, 1986a; Dreckhahn et al., 1983), and its activation induces active OHC movements (Zenner, 1986, 1988; (III) IP, has the ability to elicit motile responses (Schacht and Zenner, 1987). (IV) depolarization induces a contraction of the cylindrical hair cell body and a tilting movement of the cuticular plate (Zenner et al., 1985b, 1987, 1988; Brownell et al., 1985; Zenner. 1986: Ashmore, 1987); (V) ACh applied to the OHC membrane increases intracellular Ca2+-levels (Reuter et al., 1990): (VI) the

12X application of ACh to the OHC membrane is followed by a reversible cell contraction (Brownell et al.. 1985 Slepecky et al., 1988; Plinkert et al.. 1990). Transmitter-induced OHC-motility is discussed controversely. Bobbin et al. (1990) did not find OHC contractions after application of ACh to the outer cell membrane. This is possibly due to a desensitization of the postsynaptic cholinergic receptors. In our experiments we used an improved technique (digital image subtraction), which enabled us to observe ACh-induced contraction, usually not detectable on the videoscreen. Moreover transmitter-induced motility was specifically inhibited by d-tubocurarine (Plinkert et al.. 1990). In recent immunocytological experiments we localized postsynaptic transmembraneous nAChR-like epitopes present at the base of OHCs opposite to the presynaptic vesiculated terminals. The turn selective isolation of the auditory sensory cells exhibited a decreasing baso-apical receptor distribution (Zenner et al., 1989: Plinkert et al.. 1990). In addition postsynaptic GABA, receptors were also demonstrated at the lower pole of the sensory cells. These receptors were arranged in a tonotopical distribution, which was however inverse to the nAChR arrangement along the basilar membrane (Plinkert et al., 1989). Beside the two predominant efferent neurotransmitters ACh and GABA. Fex and Altschuler (1981. 1986) as well as Eybalin and Pujol (1984) found immunosensitivity to dynorphin. enkephalin and metenkephalin in olivocochlear efferents reaching auditory sensory cells. These compounds may act as cotransmitters. which are released into the synaptic cleft to influence the action of the major chemical messengers or modulators. The significance of the efferent olivocochlear innervation is seen in the modulation of active cochlear processes. The efferent control mechanisms may affect motile properties of the OHCs, which are postulated as the cellular basis of the active cochlear mechanisms (Neely and Kim. 1983; Zenner. 1986b: Ashmore, 1987: Zenner et al.. 1988).

Acknowledgement We thank Gerd Balke for performing the electronmicroscopic controls. This work was sup-

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