Ribbon synapses of cochlear inner hair cells (IHCs) operate with high rates of neurotransmission; yet, the molecular regulation of synaptic vesicle (SV) recycling at these synapses remains poorly understood. Here, we studied the role of endophilins‐A1‐3, endocytic adaptors with curvature‐sensing and curvature‐generating properties, in mouse IHCs. Single‐cell RT–PCR indicated the expression of endophilins‐A1‐3 in IHCs, and immunoblotting confirmed the presence of endophilin‐A1 and endophilin‐A2 in the cochlea. Patch‐clamp recordings from endophilin‐A‐deficient IHCs revealed a reduction of Ca2+ influx and exocytosis, which we attribute to a decreased abundance of presynaptic Ca2+ channels and impaired SV replenishment. Slow endocytic membrane retrieval, thought to reflect clathrin‐mediated endocytosis, was impaired. Otoferlin, essential for IHC exocytosis, co‐immunoprecipitated with purified endophilin‐A1 protein, suggestive of a molecular interaction that might aid exocytosis–endocytosis coupling. Electron microscopy revealed lower SV numbers, but an increased occurrence of coated structures and endosome‐like vacuoles at IHC active zones. In summary, endophilins regulate Ca2+ influx and promote SV recycling in IHCs, likely via coupling exocytosis to endocytosis, and contributing to membrane retrieval and SV reformation.

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Endophilins-A are conserved endocytic adaptors with membrane curvature-sensing and -inducing properties. We show here that, independently of their role in endocytosis, endophilin-A1 and endophilin-A2 regulate exocytosis of neurosecretory vesicles. The number and distribution of neurosecretory vesicles were not changed in chromaffin cells lacking endophilin-A, yet fast capacitance and amperometry measurements revealed reduced exocytosis, smaller vesicle pools and altered fusion kinetics. The levels and distributions of the main exocytic and endocytic factors were unchanged, and slow compensatory endocytosis was not robustly affected. Endophilin-A's role in exocytosis is mediated through its SH3domain, specifically via a direct interaction with intersectin-1, a coordinator of exocytic and endocytic traffic. Endophilin-A not able to bind intersectin-1, and intersectin-1 not able to bind endophilin-A, resulted in similar exocytic defects in chromaffin cells. Altogether, we report that two endocytic proteins, endophilin-A and intersectin-1, are enriched on neurosecretory vesicles and regulate exocytosis by coordinating neurosecretory vesicle priming and fusion.

The neuronal endocytic adaptor protein AP180 plays a dual role in inner hair cells of the murine cochlea: it is required for clearance of release sites and for membrane retrieval.

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, in mice 30% of the IHCs are electrochemically coupled in 'mini-syncytia'. This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers. The membrane capacitance, Ca 2+-current, and resting current increase with the number of dye-coupled IHCs. Dual voltage-clamp experiments substantiate low resistance electrical coupling. Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3D electron microscopy indicates instead that IHCs are coupled by membrane fusion sites. Consequently, depolarization of one IHC triggers presynaptic Ca 2+-influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.

Die Synthese von Dicarbonyl(π‐pentamethylcyclopentadienyl)‐(trifluormethylisocyanid)mangan (4) gelingt durch Umsetzung von Cp*Mn(CO)2(η2‐C8H14) (Cp*=(CH3)5C5) (3) mit Trifluormethylisocyanid (1). Cp*Mn(CO)2(CNCH3) (7) wird aus Cp*Mn‐(CO)2(THF) (5) und CH3NC (6) erhalten. Cp*Mn(CO)2(CS) (9) Bildet sich aus 3 und CS2 (8) in Gegenwart von Triphenylphosphan. Cp*Mn(CNCF3)3 (10) entsteht bei der Photolyse von Cp*Mn(CO)3 (2) mit überschüssigem 1. Alle (Pentamethylcyclopentadienyl)(trifluormethylisocyanid)mangan‐Komplexe zeigen niedrigliegende NC‐Valenzschwingungen und hochliegende CO‐Valenzschwingungen. Aus den Kraftkonstanten der CO‐Valenzschwingung läßt sich die relative π‐Akzeptorstärke des Trifluormethylisocyanid‐Liganden abschätzen, die sogar die des Thiocarbonylliganden übertrifft.

251ChemInform Abstract Starting with the tricarbonyl complex (I) the trifluoromethyl isocyanide complex (III) is synthesized via the derivative (II). Photolysis of (I) in the presence of excess isocyanide yields (IV). For comparison the compounds (V) and (VI) are also prepared as shown. All the trifluoromethyl isocyanide manganese complexes exhibit IR absorption bands at low wave numbers for the NC vibrations and at high wave numbers for the CO vibrations. The force constant of CO vibration indicates that the trifluoromethyl isocyanide ligand is an extremely strong π-accepting ligand which exceeds even the thiocarbonyl ligand in its π-accepting ability.

Inner hair cells (IHCs) are the primary receptors for hearing. They are housed in the cochlea and convey sound information to the brain via synapses with the auditory nerve. IHCs have been thought to be electrically and metabolically independent from each other. We report that, upon developmental maturation, 30% of the IHCs are electrochemically coupled in 'mini-syncytia'.This coupling permits transfer of fluorescently-labeled metabolites and macromolecular tracers.The membrane capacitance, Ca 2+ -current, and resting current increase with the number of dyecoupled IHCs. Dual voltage clamp experiments substantiate low resistance electrical coupling.Pharmacology and tracer permeability rule out coupling by gap junctions and purinoceptors. 3Delectron-microscopy indicates instead that IHCs are coupled by membrane fusion sites.Consequently, depolarization of one IHC triggers presynaptic Ca 2+ -influx at active zones in the entire mini-syncytium. Based on our findings and modeling, we propose that IHC-mini-syncytia enhance sensitivity and reliability of cochlear sound encoding.