The Ca²⁺Channel Subunit β2 Regulates Ca²⁺Channel Abundance and Function in Inner Hair Cells and Is Required for Hearing

Abstract

Hearing relies on Ca2+ influx-triggered exocytosis in cochlear inner hair cells (IHCs). Here we studied the role of the Ca2+ channel subunit CaVβ2 in hearing. Of the CaVβ1–4 mRNAs, IHCs predominantly contained CaVβ2. Hearing was severely impaired in mice lacking CaVβ2 in extracardiac tissues (CaVβ2−/−). This involved deficits in cochlear amplification and sound encoding. Otoacoustic emissions were reduced or absent in CaVβ2−/− mice, which showed strongly elevated auditory thresholds in single neuron recordings and auditory brainstem response measurements. CaVβ2−/− IHCs showed greatly reduced exocytosis (by 68%). This was mostly attributable to a decreased number of membrane-standing CaV1.3 channels. Confocal Ca2+ imaging revealed presynaptic Ca2+ microdomains albeit with much lower amplitudes, indicating synaptic clustering of fewer CaV1.3 channels. The coupling of the remaining Ca2+ influx to IHC exocytosis appeared unaffected. Extracellular recordings of sound-evoked spiking in the cochlear nucleus and auditory nerve revealed reduced spike rates in the CaVβ2−/− mice. Still, sizable onset and adapted spike rates were found during suprathreshold stimulation in CaVβ2−/− mice. This indicated that residual synaptic sound encoding occurred, although the number of presynaptic CaV1.3 channels and exocytosis were reduced to one-third. The normal developmental upregulation, clustering, and gating of large-conductance Ca2+ activated potassium channels in IHCs were impaired in the absence of CaVβ2. Moreover, we found the developmental efferent innervation to persist in CaVβ2-deficient IHCs. In summary, CaVβ2 has an essential role in regulating the abundance and properties of CaV1.3 channels in IHCs and, thereby, is critical for IHC development and synaptic encoding of sound.

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

“…IHC Ca V 1.3 Ca 2+ channels stand out by reason of their hyperpolarized activation range and modest inactivation (10,18,19,22,45). These properties have been attributed to the molecular composition of the native Ca 2+ -channel complexes that, in addition to the specific pore-forming Ca V 1.3α1 splice variant (46), auxiliary Ca V β2 (6,47), and Ca V α 2 δ2 (7) subunit, also contain interactions partners, such as Rab3-interacting molecule (RIM) (48,49); harmonin (50, 51); calmodulin; and, notably, CaBPs (this study and refs. 18,19,34).…”

“…Tissue was washed three times for 15 min each time in detergent rinse solution containing 0.02% Igepal, 0.01% 7-deoxycholic acid sodium salt, and 2 mM MgCl 2 in 100 mM PBS (pH 7.3). For the staining reaction, samples were kept in X-Gal staining solution: 0.02% Igepal, 0.01% 7-deoxycholic acid, 5 mM K 3 Fe(CN) 6 , 5 mM K 4 Fe(CN) 6 , 2 mM MgCl 2 , and 1 mg/mL X-Gal in 100 mM PBS (pH 7.3) overnight at 37°C.…”

Auditory neuropathy — neural and synaptic mechanisms

“…To date, deaf ness associated with mutations in the genes for the auxiliary subunits of the hair cell Ca V 1.3 Ca 2+ channel has not been described. A mouse mutagenesis study revealed that the ß2subunit (Ca V ß2) of this channel is required for hair cell Ca 2+ influx and hearing 70 . The α2δ subunit(s) that are expressed by hair cells remain to be determined.…”

Unconventional molecular regulation of synaptic vesicle replenishment in cochlear inner hair cells

“…Munc13-1 +/2 (Augustin et al, 1999), Munc13-2-KO (Varoqueaux et al, 2002), Munc13-3-KO (Augustin et al, 2001) and CAPS1 +/2 -CAPS2-KO (Jockusch et al, 2007) as well as Munc13-4-KO (Jinx mice; Jackson Laboratories) and Baiap3-KO (Wojcik et al, 2013) were used alongside littermates or age-matched (3-10 weeks old) C57Bl/6 WT mice for testing ABR thresholds as described previously (Neef et al, 2009). …”

“…Immunohistochemistry was performed on acutely dissected apical organs of Corti whole-mount preparations as described previously (Neef et al, 2009). Specimens were imaged using Leica SP2 or SP5 laser-scanning confocal microscopes with a 1.4 NA 636 or 1006 oil-immersion objective, respectively.…”