Ca 2+ -binding protein 2 (CaBP2) inhibits the inactivation of heterologously expressed voltage-gated Ca 2+ channels of type 1.3 (Ca V 1.3) and is defective in human autosomal-recessive deafness 93 (DFNB93). Here, we report a newly identified mutation in CABP2 that causes a moderate hearing impairment likely via nonsense-mediated decay of CABP2-mRNA. To study the mechanism of hearing impairment resulting from CABP2 loss of function, we disrupted Cabp2 in mice (Cabp2 LacZ/LacZ ). CaBP2 was expressed by cochlear hair cells, preferentially in inner hair cells (IHCs), and was lacking from the postsynaptic spiral ganglion neurons (SGNs). Cabp2 LacZ/LacZ mice displayed intact cochlear amplification but impaired auditory brainstem responses. Patch-clamp recordings from Cabp2 LacZ/LacZ IHCs revealed enhanced Ca 2+ -channel inactivation. The voltage dependence of activation and the number of Ca 2+ channels appeared normal in Cabp2 LacZ/LacZ mice, as were ribbon synapse counts. Recordings from single SGNs showed reduced spontaneous and sound-evoked firing rates. We propose that CaBP2 inhibits Ca V 1.3 Ca 2+ -channel inactivation, and thus sustains the availability of Ca V 1.3 Ca 2+ channels for synaptic sound encoding. Therefore, we conclude that human deafness DFNB93 is an auditory synaptopathy.H earing relies on faithful transmission of information at ribbon synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs; recently reviewed in refs. 1, 2). Ca 2+ channels at the IHC presynaptic active zone are key signaling elements because they couple the sound-evoked IHC receptor potential to the release of glutamate. IHC Ca 2+ -channel complexes are known to contain Ca V 1.3 α1 subunit (Cav1.3α1) (3-5), betasubunit 2 (Ca V β2) (6), and alpha2-delta subunit 2 (α2δ2) (7) to activate at around −60 mV (8-10), and are partially activated already at the IHC resting potential in vivo [thought to be between −55 and −45 mV (11, 12)], thereby mediating "spontaneous" glutamate release during silence (13).Compared with Ca V 1.3 channels studied in heterologous expression systems, Ca V 1.3 channels in IHCs show little inactivation, which has been attributed to inhibition of calmodulin-mediated Ca 2+ -dependent inactivation (CDI) (14-17) by Ca 2+ -binding proteins (CaBPs) (18,19) and/or the interaction of the distal and proximal regulatory domains of the Ca V 1.3α1 C terminus (20)(21)(22). This "noninactivating" phenotype of IHC Ca V 1.3 enables reliable excitation-secretion coupling during ongoing stimulation (23-25). In fact, postsynaptic spike rate adaptation during ongoing sound stimulation is thought to reflect primarily presynaptic vesicle pool depletion, with minor contributions of Ca V 1.3 inactivation or AMPA-receptor desensitization (23-26). CaBPs are calmodulin-like proteins that use three functional out of four helix-loop-helix domains (EF-hand) for Ca 2+ binding (27). They are thought to function primarily as signaling proteins (28) and differentially modulate calmodulin effectors (29,30). In addition, CaBPs m...

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Glycine is an essential co-agonist of the excitatory N-methyl-D-aspartate (NMDA) receptor, a subtype of the ionotropic glutamate receptor family. The glycine binding site of this hetero-oligomeric ion channel protein is formed by two distinct extracellular regions, S1 and S2, of the NR1 subunit, whereas the homologous domains of the NR2 subunit mediate glutamate binding. Here, segments S1 and S2 of the NR1 polypeptide were fused via a linker peptide followed by N-and C-terminally tagging with Flag and His 6 epitopes, respectively. Infection of High Five insect cells with a recombinant baculovirus containing this glycine binding site construct resulted in efficient secretion of a soluble fusion protein of about 53 kDa. After affinity purification to near-homogeneity, the fusion protein bound the competitive glycine site antagonist [ 3 H]MDL105,519 with high affinity (K d ‫؍‬ 5.22 ؎ 0.13 nM) similar to that determined with rat brain membrane fractions. This high affinity binding could be competed by the glycine site antagonist 7-chlorokynurenic acid as well as the agonists glycine and D-serine but not by L-glutamate. This indicates that the S1 and S2 domains of the NR1 subunit are sufficient for the formation of a glycine binding site that displays pharmacological properties similar to those of the NMDA receptor in vivo.Excitatory neurotransmission in the mammalian brain is predominantly mediated by members of the glutamate receptor family of ligand-gated ion channels, which have been grouped into three distinct subfamilies, the ␣-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) 1 receptors, the kainate receptors, and the N-methyl-D-aspartate (NMDA) receptors (1). Among the glutamate receptors, the NMDA receptor has gained particular interest because of a number of unique properties that classify this ligand gated ion channel as a coincidence detecting molecule in synaptic plasticity and memory formation (2). NMDA receptors are highly permeable to Ca 2ϩ (3), they are regulated by a voltage-dependent Mg 2ϩ block (4), and simultaneous binding of both glutamate and the co-agonist glycine is required for efficient activation of NMDA receptors (5, 6). Prolonged stimulation resulting in high Ca 2ϩ influx causes neuronal cell death, which has been implicated in anoxic-ischemic injury and possibly other neurodegenerative disorders (7). Therefore, the binding site for the co-agonist glycine is a target for site-specific NMDA receptor antagonists, which are proved to be neuroprotective in animal stroke models (8).Structural analysis of the NMDA receptor was initiated by expression cloning of the NMDA receptor subunit NMDAR1 (NR1 or ). Subsequently, additional cDNAs including eight NR1 splice variants (NR1a to NR1h) and different isoforms of a second type of NMDA receptor subunits (NR2A-D, ⑀1-4) were identified (reviewed in Ref. 9). At the N terminus of both, the NR1 and NR2 subunits, a cleavable leader sequence is followed by a long extracellular domain and, in the second half of the polypeptides, four hydrophobic m...

Peripheral nerve transection and neuroma-in-continuity injuries are associated with permanent functional deficits, often despite successful end-organ reinnervation. Axonal misdirection with non-specific reinnervation, frustrated regeneration and axonal attrition are believed to be among the anatomical substrates that underlie the poor functional recovery associated with these devastating injuries. Yet, functional deficits associated with axonal misdirection in experimental neuroma-in-continuity injuries have not yet been studied. We hypothesized that experimental neuroma-in-continuity injuries would result in motor axon misdirection and attrition with proportional persistent functional deficits. The femoral nerve misdirection model was exploited to assess major motor pathway misdirection and axonal attrition over a spectrum of experimental nerve injuries, with neuroma-in-continuity injuries simulated by the combination of compression and traction forces in 42 male rats. Sciatic nerve injuries were employed in an additional 42 rats, to evaluate the contribution of axonal misdirection to locomotor deficits by a ladder rung task up to 12 weeks. Retrograde motor neuron labeling techniques were utilized to determine the degree of axonal misdirection and attrition. Characteristic histological neuroma-in-continuity features were demonstrated in the neuroma-in-continuity groups and poor functional recovery was seen despite successful nerve regeneration and muscle reinnervation. Good positive and negative correlations were observed respectively between axonal misdirection (p<.0001, r2=.67), motor neuron counts (attrition) (p<.0001, r2=.69) and final functional deficits. We demonstrate prominent motor axon misdirection and attrition in neuroma-in-continuity and transection injuries of mixed motor nerves that contribute to the long-term functional deficits. Although widely accepted in theory, to our knowledge, this is the first experimental evidence to convincingly demonstrate these correlations with data inclusive of the neuroma-in-continuity spectrum. This work emphasizes the need to focus on strategies that promote both robust and accurate nerve regeneration to optimize functional recovery. It also demonstrates that clinically relevant neuroma-in-continuity injuries can now also be subjected to experimental investigation.