Deafness is genetically very heterogeneous and forms part of several syndromes. So far, delayed rectifier potassium channels have been linked to human deafness associated with prolongation of the QT interval on electrocardiograms and ventricular arrhythmia in Jervell and Lange-Nielsen syndrome. Ca(v)1.3 voltage-gated L-type calcium channels (LTCCs) translate sound-induced depolarization into neurotransmitter release in auditory hair cells and control diastolic depolarization in the mouse sinoatrial node (SAN). Human deafness has not previously been linked to defects in LTCCs. We used positional cloning to identify a mutation in CACNA1D, which encodes the pore-forming α1 subunit of Ca(v)1.3 LTCCs, in two consanguineous families with deafness. All deaf subjects showed pronounced SAN dysfunction at rest. The insertion of a glycine residue in a highly conserved, alternatively spliced region near the channel pore resulted in nonconducting calcium channels that had abnormal voltage-dependent gating. We describe a human channelopathy (termed SANDD syndrome, sinoatrial node dysfunction and deafness) with a cardiac and auditory phenotype that closely resembles that of Cacna1d(-/-) mice.

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca 2ϩ -and stimulus-dependent compensating CME in mature IHCs.

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