Single particle cryo-EM structure of the outer hair cell motor protein prestin

The mammalian outer hair cell (OHC) protein prestin (Slc26a5) differs from other Slc26 family members due to its unique piezoelectric-like property that drives OHC electromotility, the putative mechanism for cochlear amplification. Here, we use cryo-electron microscopy to determine prestin’s structure at 3.6 Å resolution. Prestin is structurally similar to the anion transporter Slc26a9. It is captured in an inward-open state which may reflect prestin’s contracted state. Two well-separated transmembrane (TM) domains and two cytoplasmic sulfate transporter and anti-sigma factor antagonist (STAS) domains form a swapped dimer. The transmembrane domains consist of 14 transmembrane segments organized in two 7+7 inverted repeats, an architecture first observed in the bacterial symporter UraA. Mutation of prestin’s chloride binding site removes salicylate competition with anions while retaining the prestin characteristic displacement currents (Nonlinear Capacitance), undermining the extrinsic voltage sensor hypothesis for prestin function.

Phase tracking algorithms detect both real and imaginary components of outer hair cell nonlinear membrane capacitance that exhibits dielectric loss.

Outer hair cell (OHC) nonlinear membrane capacitance (NLC) represents voltage-dependent sensor charge movements within prestin (SLC26a5) that drive OHC electromotility. Dielectric loss, a shift in charge movement phase from purely capacitive to resistive, is likely indicative of prestin interaction with the viscous lipid bilayer and has been suggested to correspond to prestin power output. The frequency response of NLC in OHC membrane patches has been measured with phase tracking and complex capacitance methodologies. While the latter approach can directly determine the presence of dielectric loss by assessing charge movement both in and out of phase with driving voltage, the former has been suggested to fail in this regard. Here we show that standard phase tracking in the presence of dielectric loss does indeed register this loss. Such estimates of NLC correspond to the absolute magnitude of complex NLC, indicating that total charge movement regardless of phase is assessed, thereby validating past and present measures of NLC frequency response that limits its effectiveness at high frequencies. This observation has important implications for understanding the role of prestin in cochlear amplification.

MANF supports the inner hair cell synapse and the outer hair cell stereocilia bundle in the cochlea

Failure in the structural maintenance of the hair cell stereocilia bundle and ribbon synapse causes hearing loss. Here, we have studied how ER stress elicits hair cell pathology, using mouse models with inactivation of Manf (mesencephalic astrocyte-derived neurotrophic factor), encoding an ER-homeostasis-promoting protein. From hearing onset, Manf deficiency caused disarray of the outer hair cell stereocilia bundle and reduced cochlear sound amplification capability throughout the tonotopic axis. In high-frequency outer hair cells, the pathology ended in molecular changes in the stereocilia taper region and in strong stereocilia fusion. In high-frequency inner hair cells, Manf deficiency degraded ribbon synapses. The altered phenotype strongly depended on the mouse genetic background. Altogether, the failure in the ER homeostasis maintenance induced early-onset stereociliopathy and synaptopathy and accelerated the effect of genetic causes driving age-related hearing loss. Correspondingly, MANF mutation in a human patient induced severe sensorineural hearing loss from a young age onward. Thus, we present MANF as a novel protein and ER stress as a mechanism that regulate auditory hair cell maintenance in both mice and humans.

Effects of Calcitonin-Gene-Related-Peptide on Auditory Nerve Activity

Calcitonin-gene-related peptide (CGRP) is a lateral olivocochlear (LOC) efferent neurotransmitter. Depression of sound-driven auditory brainstem response amplitude in CGRP-null mice suggests the potential for endogenous CGRP release to upregulate spontaneous and/or sound-driven auditory nerve (AN) activity. We chronically infused CGRP into the guinea pig cochlea and evaluated changes in AN activity as well as outer hair cell (OHC) function. The amplitude of both round window noise (a measure of ensemble spontaneous activity) and the synchronous whole-nerve response to sound (compound action potential, CAP) were enhanced. Lack of change in both onset adaptation and steady state amplitude of sound-evoked distortion product otoacoustic emission (DPOAE) responses indicated CGRP had no effect on OHCs, suggesting the origin of the observed changes was neural. Combined with results from the CGRP-null mice, these results appear to confirm that endogenous CGRP enhances auditory nerve activity when released by the LOC neurons. However, infusion of the CGRP receptor antagonist CGRP (8–37) did not reliably influence spontaneous or sound-driven AN activity, or OHC function, results that contrast with the decreased ABR amplitude measured in CGRP-null mice.

Cochlear Implantation Outcomes in Children With CDH23 Mutations–Associated Hearing Loss

Objective Mutations in the cadherin 23 gene ( CDH23) have been reported to cause cochlear damage, but few studies have investigated the auditory and speech outcome of patients after cochlear implantation. Here, we describe the genetic, auditory, and postoperative outcomes of patients with CDH23 mutations who received cochlear implants. Study Design Retrospective case review. Setting Tertiary referral center. Methods Targeted deafness-related gene panels were sequenced in Chinese families with profound sensorineural hearing loss. The clinical features of subjects carrying potentially pathogenic CDH23 mutations were analyzed. Results Between 2017 and 2019, we identified 5 children with prelinguistically profound hearing loss at our center who harbored 6 variants of CDH23 that segregated with the disease. Of these, 4 variants were novel (c.2591G>T, c.4785G>C, c.5765A>G, and c.9280_9281insTT). All affected individuals had a loss of outer hair cell function, with an average residual hearing level of 3 to 10 dB SPL. Cochlear implantations were arranged for the patients at 11 to 36 months of age. All children made gains in their hearing, language, and speech performances 14 to 120 months after surgery. Their auditory outcomes improved during follow-up intervals. Conclusion This study revealed that children with congenital cochlear defects caused by CDH23 variants can acquire an acceptable auditory and speech outcome after cochlear implantation. Early genetic detection and prenatal counseling for rare deafness genes such as CDH23 remain a priority for the future.

Cryo-EM structures reveal the electromotility mechanism of prestin, the cochlear amplifier

Outer hair cell electromotility, driven by prestin, is essential for mammalian cochlear amplification. Here, we report the cryo-EM structures of thermostabilized human prestin (hPresTS), complexed with chloride, sulfate, or salicylate at 3.52–3.61 Å resolutions, revealing a crossed dimeric arrangement. The central positively-charged cavity allows flexible binding of various anion species, resulting in distinct modulations of nonlinear capacitance (NLC), playing an important role in electromotility. Comparisons of these hPresTS structures suggest rigid-body movement between the core and gate domains, and provide mechanistic insight into prestin inhibition by salicylate. Mutations at the dimeric interface severely diminished NLC, suggesting that stabilization of the gate domain facilitates core domain movement, thereby contributing to the expression of NLC. These findings advance our understanding of the molecular mechanism underlying mammalian cochlear amplification.

Stability and Biocompatibility Tests of Alginate as Cochlear Implant Coating Material

Inner ear trauma caused by cochlear implantation is a severe clinical problem. It was shown that an electrode alginate coating can reduce the insertion forces in vitro. The grade of the alginate viscosity can be adjusted by using different metal ions for cross-linking the salts of the alginic acid. The aim was to investigate the stability and inner ear biocompatibility of alginate using different in vitro established cross-linkers. Alginate beads were cross-linked in either calcium chloride (CaCl2) or barium chloride (BaCl2) solution. The beads were cultivated in artificial perilymph and stability and swelling were observed for 13 months. Ototoxicity was tested on cochlear whole mount explants from neonatal rats. Neomycin served as positive control to induce hair cell damage and explants without any addition served as negative control. The beads and explants were co-cultured for 48 hours and the hair cell survival was analysed microscopically. Neomycin treatment induced an extensive inner and outer hair cell loss. Neither CaCl2 nor BaCl2 cross-linked alginate beads caused any damage to the hair cells. Even though the same volume of alginate and cross-linkers were used, in CaCl2 cross-linked beads were initially almost double the size of in BaCl2 cross-linked beads. None of the cross-linked alginate beads had a significant volume change within 3 months being cultured in artificial perilymph. After 3 months the CaCl2 cross-linked beads swelled massively and dissolved within one week whereas BaCl2 cross-linked alginate beads remained unchanged until month 13 after culture start. Alginate beads gelled with both cross-linkers are biocompatible with the inner ear sensory epithelium. Both cross-linkers ensure a stable gelation of alginate but a swelling followed by degradation of the in CaCl2 cross-linked beads occurred after 3 months. For coatings, which need to be long term stable, BaCl2 should be chosen whereas CaCl2 may be more suitable for applications where limited stability is needed and the swelling is not affecting the surrounding tissue. Therefore, BaCl2 cross-linking of alginate may be the best choice for cochlear implant coating.