Ca ²⁺ -binding protein 2 inhibits Ca ²⁺ -channel inactivation in mouse inner hair cells

Abstract

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...

CaBP1 regulates Cav1 L-type Ca2+ channels and their coupling to neurite growth and gene transcription in mouse spiral ganglion neurons

“…For example, alternative splicing and RNA editing can produce Ca v 1 channels in neurons with limited CDI (Bazzazi et al, 2013; Shen et al, 2006; Singh et al, 2008; Tan et al, 2011). It is noteworthy that knock-out of CaBP2 causes increased voltage-dependent inactivation rather than CDI of the Ca v 1.3 current in inner hair cells (Picher et al, 2017), despite evidence that CaBP2 strongly suppresses CDI of Ca v 1.3 channels in transfected HEK293T cells (Schrauwen et al, 2012). Clearly, the native environment in which Ca v channels are expressed may strongly influence the extent to which Ca v channels are modulated by CaBPs.…”

“…Of the multiple CaBP family members, CaBP2 is specifically enriched in inner and outer hair cells (Cui et al, 2007; Yang et al, 2016). However, CDI of Ca v 1.3 in inner hair cells of CaBP2 knock-out mice was not affected (Picher et al, 2017), despite the fact that CaBP2 strongly suppresses CDI of Ca v 1.3 channels in transfected HEK293 cells (Schrauwen et al, 2012). Thus, whether CaBPs regulate CDI of Ca v 1 channels in neuronal cell-types remains to be established.…”

RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

“…However, in IHCs, both CDI and VDI are extremely slow. Suppression of CDI is mediated by CaMlike Ca 2+ -binding proteins, such as CaBP2, which are abundantly expressed in IHCs [48,55,69] and inhibit CaMmediated CDI [15,68]. In contrast, the molecular basis for the uniquely slow VDI in IHCs is incompletely understood, although several factors are known to modulate VDI.…”

Balancing presynaptic release and endocytic membrane retrieval at hair cell ribbon synapses

“…Recent work revealed that the number, activation kinetics and open probability of these channels not only differ based on their tonotopic position along the cochlear axis , but also depend on their subcellular AZ localization , thereby suggesting the location‐dependent presence of yet‐to‐be‐identified regulatory proteins. In this context, Ca V 1.3 channels have been shown to be regulated by a number of modulatory proteins, including Gipc3 and CaBP2 (for a recent extensive review on this topic see Ref. ).…”