Cholinergic and purinergic signalling in outer hair cells of mammalian cochlea

Abstract

Outer (OHC) and inner (IHC) hair cells in the organ of Corti of the mammalian cochlea process sound. OHC and their efferent synapse are part of a feedback system assumed to control and modulate information carried by afferent neurons passing from IHC to the brain. Underlying mechanisms are not well understood. This paper discusses recent progress. In vivo and in vitro information is presented on structure, pharmacology, function and localization of the pre- and postsynaptic acetylcholine receptors (AChRs) at the efferent synapse. Recent data are given on a presynaptic M3 AChR subtype, probably an autoreceptor involved in transmitter release. Data from our lab on specific binding of [ 3H]3-quinuclidinyl benzilate ([ 3H]3-QNB) to non-enzymatically isolated guinea pig OHC reveal a K D several 100 x higher than that for any known muscarinic receptor subtype, including the above-mentioned presynaptic muscarinic AChR of the OHC efferent synapse. The extremely high concentrations of [ 3H]3-QNB needed for any binding at all to OHC thus rule out presynaptic membrane impurities as the cause of such binding, and also the presence of a typical mAChR subtype on OHC. The number of [ 3H]3-QNB binding sites (∼ 10 6/OHC) we found on OHC was 1 10 th of that we found for binding of nicotinic ligands to OHC, further making it questionable that an ACh-binding site on OHC binds [ 3H]3-QNB. Observations may instead point to the possibility of another binding site, e.g. an (allosteric) site involved with the as yet not understood ‘weak’ muscarinic properties of the OHC AChR. Further new data on the OHC AChR confirm reversible α-bungarotoxin, nicotine and d-tubocurarine binding. [ 3H]α-Bungarotoxin and [ 3H]-nicotine binding sites are estimated at ∼6 · 10 7 sites/OHC. Strychnine, a glycine receptor blocker suggested to interfere with cholinergic sites of the efferent OHC synapse, was found to bind to OHC (cold strychnine for unspecific binding). This binding, not seen in the presence of high [glycine], increased in the presence of depolarizing [K +], while ACh (100 μM) had no significant effect. Results suggest strychnine binding to the outside of OHC, but also to sites accessible only after cell depolarization, possibly to the hyperpolarizing Ca 2+-dependent K + channel. Recent molecular cloning of the OHC AChR indicates a novel α-subunit. An often observed ACh-activated Ca 2+-influx close to zero into OHC leaves an unanswered question. OHC also carry P 2-purinergic receptors (P 2Rs), a more rapid ionotropic P 2zR-like subtype and a quantitatively dominating slow metabotropic P 2yR subtype coupled to a G protein-phospholipase C cascade and not desensitized. Both contribute to increased cytoplasmic [Ca 2+], from respectively external and internal sources. Whether or not such receptors are part of efferent synaptic activity is unknown; their localization on the OHC plasma membrane is so far only indirect and synaptic vesicles of the efferent nerve endings have not yet been analyzed for their ATP content. Localization, function and interaction of [Ca 2+] increases triggered by, respectively, ATP and ACh are currently studied in this laboratory.